TREA

METHODS AND COMPOSITIONS FOR MODULATING A GENOME

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Information

  • Patent Application
  • 20240035049
  • Publication Number
    20240035049
  • Date Filed
    July 19, 2023
    10 months ago
  • Date Published
    February 01, 2024
    4 months ago
Information
Abstract
Methods and compositions for modulating a target genome are disclosed.
Description
SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 14, 2022, is named V2065-700621FT_SL.xml and is 4,441,816 bytes in size.


BACKGROUND

Integration of a nucleic acid of interest into a genome occurs at low frequency and with little site specificity, in the absence of a specialized protein to promote the insertion event. Some existing approaches, like CRISPR/Cas9, are more suited for small edits that rely on host repair pathways, and are less effective at integrating longer sequences. Other existing approaches, like Cre/loxP, require a first step of inserting a loxP site into the genome and then a second step of inserting a sequence of interest into the loxP site. There is a need in the art for improved compositions (e.g., proteins and nucleic acids) and methods for inserting, altering, or deleting sequences of interest in a genome.


SUMMARY OF THE INVENTION

This disclosure relates to novel compositions, systems and methods for altering a genome at one or more locations in a host cell, tissue or subject, in vivo or in vitro. In particular, the invention features compositions, systems and methods for inserting, altering, or deleting sequences of interest in a host genome.


Features of the compositions or methods can include one or more of the following enumerated embodiments.


ENUMERATED EMBODIMENTS

1. A system for modifying DNA comprising:

    • (a) a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, e.g., a nickase domain; and
    • (b) a template RNA (or DNA encoding the template RNA) comprising (e.g., from 5′ to 3′) (i) optionally a sequence (e.g., a CRISPR spacer) that binds a target site (e.g., a second strand of a site in a target genome), (ii) optionally a sequence that binds the polypeptide, (iii) a heterologous object sequence, and (iv) a 3′ target homology domain.


2. A system for modifying DNA comprising:

    • (a) a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, e.g., a nickase domain; and
    • (b) a template RNA (or DNA encoding the template RNA) comprising (e.g., from 5′ to 3′) (i) optionally a sequence that binds a target site (e.g., a second strand of a site in a target genome), (ii) optionally a sequence that binds the polypeptide, (iii) a heterologous object sequence, and (iv) a 3′ target homology domain;
    • wherein:
    • (i) the polypeptide comprises a heterologous targeting domain (e.g., in the DBD or the endonuclease domain) that binds specifically to a sequence comprised in the target site; and/or (ii) the template RNA comprises a heterologous homology sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology to a sequence comprised in a target site.


3. A system for modifying DNA comprising:

    • (a) a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, e.g., a nickase domain; and
    • (b) a template RNA (or DNA encoding the template RNA) comprising (e.g., from 5′ to 3′) (i) optionally a sequence that binds a target site (e.g., a second strand of a site in a target genome), (ii) optionally a sequence that binds the polypeptide, (iii) a heterologous object sequence, and (iv) a 3′ target homology domain,
    • wherein the RT domain comprises a sequence of Table 1 or 3 or a sequence of a reverse transcriptase domain of Table 2 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.


4. A system for modifying DNA comprising:

    • (a) a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, e.g., a nickase domain; and
    • (b) a template RNA (or DNA encoding the template RNA) comprising (e.g., from 5′ to 3′) (i) optionally a sequence that binds a target site (e.g., a second strand of a site in a target genome), (ii) optionally a sequence that binds the polypeptide, (iii) a heterologous object sequence, and (iv) a 3′ target homology domain,
    • wherein the RT domain comprises a sequence of Table 1 or 3, or a sequence of a reverse transcriptase domain of Table 2,
    • wherein the RT domain further comprises a number of substitutions relative to the natural sequence, e.g., at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 substitutions.


5. A system for modifying DNA comprising:

    • (a) a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, e.g., a nickase domain; and
    • (b) a template RNA (or DNA encoding the template RNA) comprising (e.g., from 5′ to 3′) (i) optionally a sequence that binds a target site (e.g., a second strand of a site in a target genome), (ii) optionally a sequence that binds the polypeptide, (iii) a heterologous object sequence, and (iv) a 3′ target homology domain,
    • wherein the system is capable of producing an insertion into the target site of at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides.


6. A system for modifying DNA comprising:

    • (a) a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, e.g., a nickase domain; and
    • (b) a template RNA (or DNA encoding the template RNA) comprising (e.g., from 5′ to 3′) (i) optionally a sequence that binds a target site (e.g., a second strand of a site in a target genome), (ii) optionally a sequence that binds the polypeptide, (iii) a heterologous object sequence, and (iv) a 3′ target homology domain,
    • wherein the system is capable of producing an insertion into the target site of at least 1, 2, 3, 4, 5, 10, 20, 30, 40, or 44 nucleotides.


7. A system for modifying DNA comprising:

    • (a) a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, e.g., a nickase domain; and
    • (b) a template RNA (or DNA encoding the template RNA) comprising (e.g., from 5′ to 3′) (i) optionally a sequence that binds a target site (e.g., a second strand of a site in a target genome), (ii) optionally a sequence that binds the polypeptide, (iii) a heterologous object sequence, and (iv) a 3′ target homology domain,
    • wherein the heterologous object sequence is at least 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 120, 140, 160, 180, 200, 500, or 1,000 nts in length.


8. A system for modifying DNA comprising:

    • (a) a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, e.g., a nickase domain; and
    • (b) a template RNA (or DNA encoding the template RNA) comprising (e.g., from 5′ to 3′) (i) optionally a sequence that binds a target site (e.g., a second strand of a site in a target genome), (ii) optionally a sequence that binds the polypeptide, (iii) a heterologous object sequence, and (iv) a 3′ target homology domain,
    • wherein the heterologous object sequence is at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, or 73 nucleotides in length.


9. The system of any of the preceding embodiments, wherein one or more of: the RT domain is heterologous to the DBD; the DBD is heterologous to the endonuclease domain; or the RT domain is heterologous to the endonuclease domain.


10. A system for modifying DNA comprising:

    • (a) a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, e.g., a nickase domain; and
    • (b) a template RNA (or DNA encoding the template RNA) comprising (e.g., from 5′ to 3′) (i) optionally a sequence that binds a target site (e.g., a second strand of a site in a target genome), (ii) optionally a sequence that binds the polypeptide, (iii) a heterologous object sequence, and (iv) a 3′ target homology domain,
    • wherein the system is capable of producing a deletion into the target site of at least 81, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides.


11. A system for modifying DNA comprising:

    • (a) a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, e.g., a nickase domain; and
    • (b) a template RNA (or DNA encoding the template RNA) comprising (e.g., from 5′ to 3′) (i) optionally a sequence that binds a target site (e.g., a second strand of a site in a target genome), (ii) optionally a sequence that binds the polypeptide, (iii) a heterologous object sequence, and (iv) a 3′ target homology domain,
    • wherein the system is capable of producing a deletion into the target site of at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, or 80 nucleotides.


12. A system for modifying DNA comprising:

    • (a) a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, e.g., a nickase domain; and
    • (b) a template RNA (or DNA encoding the template RNA) comprising (e.g., from 5′ to 3′) (i) optionally a sequence that binds a target site (e.g., a second strand of a site in a target genome), (ii) optionally a sequence that binds the polypeptide, (iii) a heterologous object sequence, and (iv) a 3′ target homology domain,
    • wherein the system is capable of producing nucleotide substitutions, e.g., transitions and/or transversions, into the target site of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.


13. A system for modifying DNA comprising:

    • (a) a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, e.g., a nickase domain; and
    • (b) a template (or DNA encoding the template RNA) comprising (e.g., from 5′ to 3′) (i) optionally a sequence that binds a target site (e.g., a second strand of a site in a target genome), (ii) optionally a sequence that binds the polypeptide, (iii) a heterologous object sequence, and (iv) a 3′ target homology domain,
    • wherein (a)(ii) and/or (a)(iii) comprises a TAL domain; a zinc finger domain; or a CRISPR/Cas domain chosen from Table 4 or a functional variant (e.g., mutant) thereof.


14. A system for modifying DNA comprising:

    • (a) a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, e.g., a nickase domain; and
    • (b) a template RNA (or DNA encoding the template RNA) comprising (e.g., from 5′ to 3′) (i) optionally a sequence (e.g., a CRISPR spacer) that binds a target site (e.g., a second strand of a site in a target genome), (ii) optionally a sequence that binds the polypeptide, (iii) a heterologous object sequence, and (iv) a 3′ target homology domain,
    • wherein the endonuclease domain, e.g., nickase domain, cuts both the first strand and the second strand of the target site DNA, and wherein the cuts are separated from one another by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 30 nucleotides.


15. A system for modifying DNA comprising:

    • (a) a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, e.g., a nickase domain; and
    • (b) a template RNA (or DNA encoding the template RNA) comprising (e.g., from 5′ to 3′) (i) optionally a sequence that binds a target site (e.g., a second strand of a site in a target genome), (ii) a sequence that specifically binds the RT domain, (iii) a heterologous object sequence, and (iv) a 3′ target homology domain.


16. The system of any of the preceding embodiments, wherein the template RNA further comprises a sequence that binds (a)(ii) and/or (a)(iii).


17. A system for modifying DNA comprising:

    • (a) a first polypeptide or a nucleic acid encoding the first polypeptide, wherein the first polypeptide comprises (i) a reverse transcriptase (RT) domain and (ii) optionally a DNA-binding domain,
    • (b) a second polypeptide or a nucleic acid encoding the second polypeptide, wherein the second polypeptide comprises (i) a DNA-binding domain (DBD); (ii) an endonuclease domain, e.g., a nickase domain; and
    • (c) a template RNA (or DNA encoding the template RNA) comprising (e.g., from 5′ to 3′) (i) optionally a sequence that binds the second polypeptide (e.g., that binds (b)(i) and/or (b)(ii)), (ii) optionally a sequence that binds the first polypeptide (e.g., that specifically binds the RT domain), (iii) a heterologous object sequence, and (iv) a 3′ target homology domain.


18. A system for modifying DNA comprising:

    • (a) a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, and (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, e.g., a nickase domain;
    • (b) a first template RNA (or DNA encoding the RNA) comprising (e.g., from 5′ to 3′) (i) a sequence that binds the polypeptide (e.g., that binds (a)(ii) and/or (a)(iii)) and (ii) a sequence that binds a target site (e.g., a second strand of a site in a target genome), (e.g., wherein the first RNA comprises a gRNA);
    • (c) a second template RNA (or DNA encoding the RNA) comprising (e.g., from 5′ to 3′) (i) optionally a sequence that binds the polypeptide (e.g., that specifically binds the RT domain), (ii) a heterologous object sequence, and (iii) a 3′ target homology domain.


19. The system of any of the preceding embodiments, wherein the second template RNA comprises (i).


20. The system of any of the preceding embodiments, wherein the first template RNA comprises a first conjugating domain and the second template RNA comprises a second conjugating domain.


21. The system of any of the preceding embodiments, wherein the first and second conjugating domains are capable of hybridizing to one another, e.g., under stringent conditions, e.g., wherein the stringent conditions for hybridization includes hybridization in 4× sodium chloride/sodium citrate (SSC), at about 65° C., followed by a wash in 1×SSC, at about 65° C.


22. The system of any of the preceding embodiments, wherein the first and second conjugating domains may be joined covalently, e.g., by splint ligation, e.g., by the method described by Moore, M. J., & Query, C. C. Methods in Enzymology, 317, 109-123, 2000.


23. The system of any of the preceding embodiments, wherein association of the first conjugating domain and the second conjugating domain colocalizes the first template RNA and the second template RNA.


24. The system of any of the preceding embodiments, wherein the reverse transcriptase (RT) domain is from a retrotransposon, or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.


25. A system for modifying DNA comprising:

    • (a) a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain from a retrotransposon, or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, e.g., a nickase domain; and
    • (b) a template RNA (or DNA encoding the template RNA) comprising (e.g., from 5′ to 3′) (i) optionally a sequence (e.g., a CRISPR spacer) that binds a target site (e.g., a second strand of a site in a target genome), (ii) optionally a sequence that binds the polypeptide, (iii) a heterologous object sequence, and (iv) a 3′ target homology domain.


26. The system of any of the preceding embodiments, wherein the template RNA comprises (i).


27. The system of any of the preceding embodiments, wherein the template RNA comprises (ii).


28. The system of any of the preceding embodiments, wherein the template RNA comprises (i) and (ii).


29. The system of any of the preceding embodiments, wherein the reverse transcriptase domain comprises an amino acid sequence according to a reverse transcriptase domain of any of Table 30, Table 31, Table 41, Table 44, or Table 50, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or a functional fragment thereof.


30. A template RNA (or DNA encoding the template RNA) comprising a targeting domain (e.g., a heterologous targeting domain) that binds specifically to a sequence comprised in the target DNA molecule (e.g., a genomic DNA), a sequence that specifically binds an RT domain of a polypeptide, and a heterologous object sequence.


31. A template RNA (or DNA encoding the template RNA) comprising (e.g., from 5′ to 3′) (i) optionally a sequence that binds a target site (e.g., a second strand of a site in a target genome), (ii) optionally a sequence that binds an endonuclease and/or a DNA-binding domain of a polypeptide, (iii) a heterologous object sequence, and (iv) a 3′ target homology domain.


32. The template RNA of any of the preceding embodiments, wherein the template RNA comprises (i).


33. The template RNA of any of the preceding embodiments, wherein the template RNA comprises (ii).


34. A template RNA (or DNA encoding the template RNA) comprising (e.g., from 5′ to 3′) (i) a sequence that binds a target site (e.g., a second strand of a site in a target genome), (ii) a sequence that binds an endonuclease and/or a DNA-binding domain of a polypeptide, (iii) a heterologous object sequence, and (iv) a 3′ target homology domain,

    • wherein (i) comprises a nucleic acid sequence with complementarity to a sequence of a gene of any of Tables 9-12 or with no more than 1, 2, 3, 4, or 5 differences from said sequence having said complementarity.


35. A template RNA (or DNA encoding the template RNA) comprising (e.g., from 5′ to 3′) (i) a sequence that binds a target site (e.g., a second strand of a site in a target genome), (ii) a sequence that specifically binds an RT domain of a polypeptide, (iii) a heterologous object sequence, and (iv) a 3′ target homology domain.


36. The template RNA of any of the preceding embodiments, further comprising (v) a sequence that binds an endonuclease and/or a DNA-binding domain of a polypeptide (e.g., the same polypeptide comprising the RT domain).


37. The template RNA of any of the preceding embodiments, wherein the RT domain comprises a sequence selected of Table 1 or 3 or a sequence of a reverse transcriptase domain of Table 2 or a sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.


38. The template RNA of any of the preceding embodiments, wherein the RT domain comprises a sequence selected of Table 1 or 3 or a sequence of a reverse transcriptase domain of Table 2, wherein the RT domain further comprises a number of substitutions relative to the natural sequence, e.g., at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 substitutions.


39. The template RNA of any of the preceding embodiments, wherein the sequence of (ii) specifically binds the RT domain.


40. The template RNA of any of the preceding embodiments, wherein the sequence that specifically binds the RT domain is a sequence, e.g., a UTR sequence, of Table 1 or from a domain of Table 2, or a sequence having at least 70, 75, 80, 85, 90, 95, or 99% identity thereto.


41. A template RNA (or DNA encoding the template RNA) comprising from 5′ to 3′: (ii) a sequence that binds an endonuclease and/or a DNA-binding domain of a polypeptide, (i) a sequence that binds a target site (e.g., a second strand of a site in a target genome), (iii) a heterologous object sequence, and (iv) a 3′ target homology domain.


42. A template RNA (or DNA encoding the template RNA) comprising from 5′ to 3′: (iii) a heterologous object sequence, (iv) a 3′ target homology domain, (i) a sequence that binds a target site (e.g., a second strand of a site in a target genome), and (ii) a sequence that binds an endonuclease and/or a DNA-binding domain of a polypeptide.


43. The system or template RNA of any of the preceding embodiments, wherein the template RNA, first template RNA, or second template RNA comprises a sequence that specifically binds the RT domain.


44. The system or template RNA of any of the preceding embodiments, wherein the sequence that specifically binds the RT domain is disposed between (i) and (ii).


45. The system or template RNA of any of the preceding embodiments, wherein the sequence that specifically binds the RT domain is disposed between (ii) and (iii).


46. The system or template RNA of any of the preceding embodiments, wherein the sequence that specifically binds the RT domain is disposed between (iii) and (iv).


47. The system or template RNA of any of the preceding embodiments, wherein the sequence that specifically binds the RT domain is disposed between (iv) and (i).


48. The system or template RNA of any of the preceding embodiments, wherein the sequence that specifically binds the RT domain is disposed between (i) and (iii).


49. A system for modifying DNA, comprising:

    • (a) a first template RNA (or DNA encoding the first template RNA) comprising (i) sequence that binds an endonuclease domain, e.g., a nickase domain, and/or a DNA-binding domain (DBD) of a polypeptide, and (ii) a sequence that binds a target site (e.g., a second strand of a site in a target genome), (e.g., wherein the first RNA comprises a gRNA);
    • (b) a second template RNA (or DNA encoding the second template RNA) comprising (i) a sequence that specifically binds a reverse transcriptase (RT) domain of a polypeptide (e.g., the polypeptide of (a)), (ii) a heterologous object sequence, and (iii) 3′ target homology domain.


50. The system of any of the preceding embodiments, wherein the nucleic acid encoding the first template RNA and the nucleic acid encoding the second template RNA are two separate nucleic acids.


51. The system of any of the preceding embodiments, wherein the nucleic acid encoding the first template RNA and the nucleic acid encoding the second template RNA are part of the same nucleic acid molecule, e.g., are present on the same vector.


52. The system of any of the preceding embodiments, wherein the system is capable of producing an insertion into the target site of at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides.


53. The system of any of the preceding embodiments, wherein the heterologous object sequence is at least 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 120, 140, 160, 180, 200, 500, or 1,000 nts in length.


54. The system of any of the preceding embodiments, wherein the system is capable of producing a deletion into the target site of at least 81, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides.


55. The system of any of the preceding embodiments, wherein one or both of the template RNA and the RNA encoding the polypeptide of (a) comprises chemically modified mRNA, e.g., mRNA comprising a chemically modified base, e.g., mRNA comprising 5-methoxyuridine.


56. The system of any of the preceding embodiments, wherein one or both of the template RNA and the RNA encoding the polypeptide of (a) comprises chemically modified RNA, e.g., RNA comprising a chemically modified base, e.g., RNA comprising 2′-o-methyl phosphorothioate.


57. The system of any of the preceding embodiments, wherein one or both of the template RNA and the RNA encoding the polypeptide of (a) comprises chemically modified RNA, e.g., RNA comprising a chemically modified base, e.g., 2′-o-methyl phosphorothioate, at one or both of the 3, 4, or 5 bases at the 5′ or 3′ end of the RNA.


58. A polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain; wherein the DBD and/or the endonuclease domain comprise a heterologous targeting domain that binds specifically to a sequence comprised in a target DNA molecule (e.g., a genomic DNA).


59. A polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, e.g., a nickase domain, wherein the RT domain has a sequence of Table 1 or 3 or a sequence of a reverse transcriptase domain of Table 2, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.


60. A polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, e.g., a nickase domain, wherein the RT domain has a sequence of Table 1 or 3 or a sequence of a reverse transcriptase domain of Table 2, wherein the RT domain further comprises a number of substitutions relative to the natural sequence, e.g., at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 substitutions.


61. The polypeptide of any of the preceding embodiments, wherein the polypeptide is encoded by an mRNA, e.g., a chemically modified mRNA, e.g., an mRNA comprising a chemically modified base, e.g., an mRNA comprising 5-methoxyuridine.


62. The polypeptide of any of the preceding embodiments, wherein the polypeptide is encoded by an mRNA, e.g., a chemically modified mRNA, e.g., an mRNA comprising a chemically modified base, e.g., an mRNA comprising N1-Methyl-Psuedouridine.


63. A system for modifying DNA, comprising:

    • (a) a first polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises a reverse transcriptase (RT) domain, wherein the RT domain has a sequence of Table 1 or 3 or a sequence of a reverse transcriptase domain of Table 2, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto; and optionally a DNA-binding domain (DBD) (e.g., a first DBD); and
    • (b) a second polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a DBD (e.g., a second DBD); and (ii) an endonuclease domain, e.g., a nickase domain.


64. A system for modifying DNA, comprising:

    • (a) a first polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises a reverse transcriptase (RT) domain, wherein the RT domain has a sequence of Table 1 or 3 or a sequence of a reverse transcriptase domain of Table 2, wherein the RT domain further comprises a number of substitutions relative to the natural sequence, e.g., at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 substitutions; and optionally a DNA-binding domain (DBD) (e.g., a first DBD); and
    • (b) a second polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a DBD (e.g., a second DBD); and (ii) an endonuclease domain, e.g., a nickase domain.


65. The system of any of the preceding embodiments, wherein the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide are two separate nucleic acids.


66. The system of any of the preceding embodiments, wherein the nucleic acid encoding the first polypeptide and the nucleic acid encoding the second polypeptide are part of the same nucleic acid molecule, e.g., are present on the same vector.


67. A reaction mixture comprising:

    • a cell and any system, polypeptide, template RNA, or DNA encoding the same of any preceding embodiment.


68. A reaction mixture comprising:

    • a DNA comprising a target site and any system, polypeptide, template RNA, or DNA encoding the same of any preceding embodiment.


69. A kit comprising:

    • the system, polypeptide, template RNA, or DNA encoding the same of any preceding embodiment;
    • instructions for using the system, polypeptide, template RNA, or DNA encoding the same; and
    • one or both of a cell or a DNA comprising a target site.


70. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the DBD comprises a TAL domain.


71. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the DBD comprises a zinc finger domain.


72. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the DBD comprises a CRISPR/Cas domain.


73. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the endonuclease domain is a nickase domain.


74. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the endonuclease domain comprises a CRISPR/Cas domain.


75. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the CRISPR/Cas domain comprises a domain or polypeptide from Table 4, or a functional variant (e.g., mutant) thereof.


76. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the CRISPR/Cas domain comprises a domain or polypeptide from genus/species from Table 4.


77. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the endonuclease domain comprises a type IIs nuclease (e.g., FokI), a Holliday Junction resolvase, or a double-stranded DNA nuclease comprising an alteration that abrogates its ability to cut one strand (e.g., transforming the double-stranded DNA nuclease into a nickase).


78. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the RT domain comprises a reverse transcriptase or functional fragment or variant thereof chosen from Table 1 or 3 or a sequence of a reverse transcriptase domain of Table 2.


79. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the RT domain comprises one or more mutations (e.g., an insertion, deletion, or substitution) relative to a naturally occurring RT domain or an RT domain or functional fragment chosen from Table 1 or 3 or a sequence of a reverse transcriptase domain of Table 2, or sequence listing SEQ ID NO: 1-67 from WO2018089860A1, incorporated herein by reference.


80. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the one or more mutations are chosen from D200N, L603W, T330P, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, W313F, L435G, N454K, H594Q, L671P, E69K or D653N in the RT domain of murine leukemia virus reverse transcriptase or a corresponding mutation at a corresponding position of another RT domain.


81. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the one or more mutations are chosen from WO2018089860A1, incorporated herein by reference (e.g., a C952S, and/or C956S, and/or C952S, C956S (double mutant), and/or C969S, and/or H970Y, and/or R979Q, and/or R976Q, and/or R1071S, and/or R328A, and/or R329A, and/or Q336A, and/or R328A, R329A, Q336A (triple mutant), and/or G426A, and/or D428A, and/or G426A, D428A (double mutant) mutation, and/or any combination thereof, positions relative to WO2018089860A1 SEQ ID NO: 52), in the RT domain of R2Bm retrotransposase or a corresponding mutation at a corresponding position of another RT domain.


82. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the DBD and/or the endonuclease domain (e.g., a CRISPR/Cas domain) comprises a domain or polypeptide from Table 4, or a functional variant (e.g., mutant) thereof.


83. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the DBD and/or the endonuclease domain (e.g., CRISPR/Cas domain) comprises a domain or polypeptide from Table 4.


84. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the RT domain and the DBD and/or the endonuclease domain (e.g., CRISPR/Cas domain) are fused via a peptide linker, e.g., a linker of Table 42.


85. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the linker is about 6-18, 8-16, 10-14, or 12 amino acids in length.


86. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the linker is comprises glycine and serine, e.g., wherein the linker comprises solely glycine and serine residues, e.g., wherein the linker comprises a sequence of GSSGSS (SEQ ID NO: 1736).


87. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the linker comprises a sequence according Table 42, e.g, linked 10 as disclosed in Table 42 to or a sequence having no more then 1, 2, or 3 substitutions relative thereto.


88. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the CRISPR/Cas domain comprises Cas9, e.g., wild-type Cas9 or nickase Cas9.


89. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the RT domain is positioned C-terminal of the DBD in the polypeptide.


90. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the RT domain is positioned C-terminal of the nickase domain in the polypeptide.


91. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the RT domain is positioned N-terminal of the DBD in the polypeptide.


92. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the RT domain is positioned N-terminal of the nickase domain in the polypeptide.


93. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the polypeptide comprises a linker, e.g., positioned between the RT domain and the DBD or the RT domain and the nickase domain.


94. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the linker is between 2-50, e.g., 2-30, amino acids in length.


95. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the linker is a flexible linker, e.g., comprising Gly and/or Ser residues.


96. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the 3′ target homology domain is complementary to a sequence adjacent to a site to be modified by the system, or comprises no more than 1, 2, 3, 4, or 5 mismatches to a sequence complementary to the sequence adjacent to a site to be modified by the system.


97. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the 3′ target homology domain is more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides long, (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long).


98. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the 3′ target homology domain is no more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides long.


99. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the heterologous object sequence is complementary to a site to be modified by the system except at the position or positions to be modified.


100. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the heterologous object sequence is complementary to a site to be modified by the system except at positions encoding a sequence to be inserted to the site.


101. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the heterologous object sequence is complementary to a site to be modified by the system except the heterologous object sequence does not comprise nucleotides encoding a sequence to be deleted at the site.


102. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the heterologous object sequence is more than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long, (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long).


103. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the heterologous object sequence is no more than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long.


104. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the heterologous object sequence substitutes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides for non-target site nucleotides.


105. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the heterologous object sequence inserts at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides, or at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 kilobases into the target site.


106. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the heterologous object sequence deletes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 81, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides.


107. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the heterologous object sequence is separated from the sequence that binds the polypeptide (e.g., that binds the endonuclease domain and/or DBD domain) by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 nucleotides.


108. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the sequence that binds the polypeptide (e.g., that binds the endonuclease domain and/or DBD domain) is at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, or 130 nucleotides long (and optionally no more than 150, 140, 130, 120, 110, 100, 90, 85, or 80 nucleotides long).


109. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the sequence that binds the polypeptide binds the endonuclease domain and/or DBD domain.


110. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the sequence that binds the polypeptide comprises a sequence according to one or both of a predicted 5′ UTR and a predicted 3′ UTR of Table 3 or Table 41, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or functional fragment thereof.


111. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the sequence that binds the polypeptide (e.g., that binds the endonuclease domain and/or DBD domain) comprises a gRNA.


112. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the sequence that binds a target site (e.g., a second strand of a site in a target genome) is at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, or 130 nucleotides long (and optionally no more than 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 nucleotides long), e.g., is 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides long.


113. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the sequence that binds a target site is complementary to the second strand of the target site, or comprises no more than 1, 2, 3, 4, or 5 mismatches to a sequence complementary to the second strand of the target site.


114. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the sequence that binds a target site (e.g., a second strand of a site in a target genome) is separated from the sequence that binds the polypeptide (e.g., that binds the endonuclease domain and/or DBD domain) by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 nucleotides.


115. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, further comprising a second strand-targeting gRNA that directs the endonuclease domain (e.g., nickase) domain to nick the second strand (e.g., in the target genome).


116. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the template RNA further comprises the second strand-targeting gRNA.


117. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the second strand-targeting gRNA is disposed on a separate nucleic acid from the template RNA.


118. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the gRNA directs the endonuclease domain (e.g., nickase) domain to nick the second strand (e.g., in the target genome) at a site that is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 nucleotides 5′ or 3′ of the target site modification (e.g., the nick on the first strand).


119. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the gRNA specifically binds the edited strand.


120. The system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the polypeptide comprises a heterologous targeting domain that binds specifically to a sequence comprised in the target DNA molecule (e.g., a genomic DNA).


121. The system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the heterologous targeting domain binds to a different nucleic acid sequence than the unmodified polypeptide.


122. The system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the polypeptide does not comprise a functional endogenous targeting domain (e.g., wherein the polypeptide does not comprise an endogenous targeting domain).


123. The system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the heterologous targeting domain comprises a zinc finger (e.g., a zinc finger that binds specifically to the sequence comprised in the target DNA molecule).


124. The system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the heterologous targeting domain comprises a Cas domain (e.g., a Cas9 domain, or a mutant or variant thereof, e.g., a Cas9 domain that binds specifically to the sequence comprised in the target DNA molecule).


125. The system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the Cas domain is associated with a guide RNA (gRNA).


126. The system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the heterologous targeting domain comprises an endonuclease domain (e.g., a heterologous endonuclease domain).


127. The system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the endonuclease domain comprises a Cas domain (e.g., a Cas9 or a mutant or variant thereof).


128. The system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the Cas domain is associated with a guide RNA (gRNA).


129. The system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the endonuclease domain comprises a Fok1 domain.


130. The system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the template nucleic acid molecule comprises at least one (e.g., one or two) heterologous homology sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology to a sequence comprised in a target DNA molecule (e.g., a genomic DNA).


131. The system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein one of the at least one heterologous homology sequences is positioned at or within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides of the 5′ end of the template nucleic acid molecule.


132. The system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein one of the at least one heterologous homology sequences is positioned at or within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides of the 3′ end of the template nucleic acid molecule.


133. The system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the heterologous homology sequence binds within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nick site (e.g., produced by a nickase, e.g., an endonuclease domain, e.g., as described herein) in the target DNA molecule.


134. The system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the heterologous homology sequence has less than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% sequence identity with a nucleic acid sequence complementary to an endogenous homology sequence of an unmodified form of the template RNA.


135. The system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the heterologous homology sequence has having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology to a sequence of the target DNA molecule that is different the sequence bound by an endogenous homology sequence (e.g., replaced by the heterologous homology sequence).


136. The system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the heterologous homology sequence comprises a sequence (e.g., at its 3′ end) having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology to a sequence positioned 5′ to a nick site of the target DNA molecule (e.g., a site nicked by a nickase, e.g., an endonuclease domain as described herein).


137. The system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the heterologous homology sequence comprises a sequence (e.g., at its 5′ end) suitable for priming target-primed reverse transcription (TPRT) initiation.


138. The system, method, kit, template RNA, or reaction mixture of any of any of the preceding embodiments, wherein the heterologous homology sequence has at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology to a sequence positioned within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides of (e.g., 3′ relative to) a target insertion site, e.g., for a heterologous object sequence (e.g., as described herein), in the target DNA molecule.


139. The system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the template nucleic acid molecule comprises a guide RNA (gRNA), e.g., as described herein.


140. The system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the template nucleic acid molecule comprises a gRNA spacer sequence (e.g., at or within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides of its 5′ end).


141. The system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein an RNA of the system (e.g., template RNA, the RNA encoding the polypeptide of (a), or an RNA expressed from a heterologous object sequence integrated into a target DNA) comprises a microRNA binding site, e.g., in a 3′ UTR.


142. The system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments wherein the microRNA binding site is recognized by a miRNA that is present in a non-target cell type, but that is not present (or is present at a reduced level relative to the non-target cell) in a target cell type.


143. The system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the miRNA is miR-142, and/or wherein the non-target cell is a Kupffer cell or a blood cell, e.g., an immune cell.


144. The system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the miRNA is miR-182 or miR-183, and/or wherein the non-target cell is a dorsal root ganglion neuron.


145. The system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the system comprises a first miRNA binding site that is recognized by a first miRNA (e.g., miR-142) and the system further comprises a second miRNA binding site that is recognized by a second miRNA (e.g., miR-182 or miR-183), wherein the first miRNA binding site and the second miRNA binding site are situated on the same RNA or on different RNAs of the system.


146. The system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the template RNA comprises at least 2, 3, or 4 miRNA binding sites, e.g., wherein the miRNA binding sites are recognized by the same or different miRNAs.


147. The system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the RNA encoding the polypeptide of (a) comprises at least 2, 3, or 4 miRNA binding sites, e.g., wherein the miRNA binding sites are recognized by the same or different miRNAs.


148. The system, method, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the RNA expressed from a heterologous object sequence integrated into a target DNA comprises at least 2, 3, or 4 miRNA binding sites, e.g., wherein the miRNA binding sites are recognized by the same or different miRNAs.


149. A system comprising:

    • an mRNA encoding the polypeptide or system of any of the preceding embodiments, and
    • a template RNA of any preceding embodiment.


150. The system of any of the preceding embodiments, wherein the mRNA encoding the polypeptide or system of any preceding embodiment and the template RNA of any preceding embodiment are disposed on different nucleic acid molecules.


151. A system comprising an RNA molecule comprising:

    • a template RNA (or RNA encoding the template RNA) of any preceding embodiment,
    • and a sequence encoding the system or polypeptide of any preceding embodiment.


152. The system of any of the preceding embodiments, wherein the RNA molecule comprises an internal ribosome entry site, e.g., operably linked to the sequence encoding the system or polypeptide.


153. The system of any of the preceding embodiments, wherein the RNA molecule comprises a cleavage site, e.g., situated between the template RNA (or RNA encoding the template RNA) and the sequence encoding the system or polypeptide.


154. The system or polypeptide of any of the preceding embodiments, wherein the polypeptide comprises a split intein, e.g., two or more (e.g., all) of the RT domain, DBD, endonuclease (e.g., nickase) domain, or combinations thereof are translated as separate proteins which combine into a single polypeptide by protein splicing.


155. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the system comprises one or more circular RNA molecules (circRNAs).


156. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the circRNA encodes the Gene Writer polypeptide.


157. The system of any of the preceding embodiments, wherein the circRNA comprises a template RNA.


158. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein circRNA is delivered to a host cell.


159. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the circRNA is capable of being linearized, e.g., in a host cell, e.g., in the nucleus of the host cell.


160. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the circRNA comprises a cleavage site.


161. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the circRNA further comprises a second cleavage site.


162. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the cleavage site can be cleaved by a ribozyme, e.g., a ribozyme comprised in the circRNA (e.g., by autocleavage).


163. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the circRNA comprises a ribozyme sequence.


164. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the ribozyme sequence is capable of autocleavage, e.g., in a host cell, e.g., in the nucleus of the host cell.


165. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the ribozyme is an inducible ribozyme.


166. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the ribozyme is a protein-responsive ribozyme, e.g., a ribozyme responsive to a nuclear protein, e.g., a genome-interacting protein, e.g., an epigenetic modifier, e.g., EZH2.


167. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the ribozyme is a nucleic acid-responsive ribozyme.


168. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the catalytic activity (e.g., autocatalytic activity) of the ribozyme is activated in the presence of a target nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA, miRNA, ncRNA, lncRNA, tRNA, snRNA, or mtRNA).


169. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the ribozyme is responsive to a target protein (e.g., an MS2 coat protein).


170. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the target protein localized to the cytoplasm or localized to the nucleus (e.g., an epigenetic modifier or a transcription factor).


171. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the ribozyme comprises the ribozyme sequence of a B2 or ALU retrotransposon, or a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.


172. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the ribozyme comprises the sequence of a tobacco ringspot virus hammerhead ribozyme, or a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.


173. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the ribozyme comprises the sequence of a hepatitis delta virus (HDV) ribozyme, or a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.


174. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the ribozyme is activated by a moiety expressed in a target cell or target tissue.


175. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the ribozyme is activated by a moiety expressed in a target subcellular compartment (e.g., a nucleus, nucleolus, cytoplasm, or mitochondria).


176. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the ribozyme is comprised in a circular RNA or a linear RNA.


177. A system comprising a first circular RNA encoding the polypeptide of a Gene Writing system; and a second circular RNA comprising the template RNA of a Gene Writing system.


178. The system of any of the preceding embodiments, wherein the nucleic encoding the polypeptide of (a) comprises a coding sequence that is codon-optimized for expression in human cells.


179. The system of any of the preceding embodiments, wherein the template RNA comprises a coding sequence that is codon-optimized for expression in human cells.


180. A lipid nanoparticle (LNP) comprising the system, template RNA, polypeptide (or RNA encoding the same), or DNA encoding the system, template RNA, or polypeptide, of any preceding embodiment.


181. A system comprising a first lipid nanoparticle comprising the polypeptide (or DNA or RNA encoding the same) of a Gene Writing system (e.g., as described herein); and a second lipid nanoparticle comprising a nucleic acid molecule of a Gene Writing System (e.g., as described herein).


182. The system, kit, polypeptide, or reaction mixture of any preceding embodiments, wherein the system, nucleic acid molecule, polypeptide, and/or DNA encoding the same, is formulated as a lipid nanoparticle (LNP).


183. The LNP of any of the preceding embodiments, comprising a cationic lipid.


184. The LNP of any of the preceding embodiments, wherein the cationic lipid having a following structure:




embedded image


185. The LNP of any of the preceding embodiments, further comprising one or more neutral lipid, e.g., DSPC, DPPC, DMPC, DOPC, POPC, DOPE, SM, a steroid, e.g., cholesterol, and/or one or more polymer conjugated lipid, e.g., a pegylated lipid, e.g., PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer or a PEG dialkyoxypropylcarbamate.


186. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the system, polypeptide, and/or DNA encoding the same, is formulated as a lipid nanoparticle (LNP).


187. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the lipid nanoparticle (or a formulation comprising a plurality of the lipid nanoparticles) lacks reactive impurities (e.g., aldehydes), or comprises less than a preselected level of reactive impurities (e.g., aldehydes).


188. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the lipid nanoparticle (or a formulation comprising a plurality of the lipid nanoparticles) lacks aldehydes, or comprises less than a preselected level of aldehydes.


189. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the lipid nanoparticle is comprised in a formulation comprising a plurality of the lipid nanoparticles.


190. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the lipid nanoparticle formulation is produced using one or more lipid reagents comprising less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content.


191. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the lipid nanoparticle formulation is produced using one or more lipid reagents comprising less than 3% total reactive impurity (e.g., aldehyde) content.


192. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the lipid nanoparticle formulation is produced using one or more lipid reagents comprising less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species.


193. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the lipid nanoparticle formulation is produced using one or more lipid reagent comprising less than 0.3% of any single reactive impurity (e.g., aldehyde) species.


194. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the lipid nanoparticle formulation is produced using one or more lipid reagents comprising less than 0.1% of any single reactive impurity (e.g., aldehyde) species.


195. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the lipid nanoparticle formulation comprises less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content.


196. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the lipid nanoparticle formulation comprises less than 3% total reactive impurity (e.g., aldehyde) content.


197. The system, kit, polypeptide, or reaction mixture of anany of the preceding embodiments, wherein the lipid nanoparticle formulation comprises less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species.


198. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the lipid nanoparticle formulation comprises less than 0.3% of any single reactive impurity (e.g., aldehyde) species.


199. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the lipid nanoparticle formulation comprises less than 0.1% of any single reactive impurity (e.g., aldehyde) species.


200. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein one or more, or optionally all, of the lipid reagents used for a lipid nanoparticle as described herein or a formulation thereof comprise less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content.


201. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein one or more, or optionally all, of the lipid reagents used for a lipid nanoparticle as described herein or a formulation thereof comprise less than 3% total reactive impurity (e.g., aldehyde) content.


202. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein one or more, or optionally all, of the lipid reagents used for a lipid nanoparticle as described herein or a formulation thereof comprise less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species.


203. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein one or more, or optionally all, of the lipid reagents used for a lipid nanoparticle as described herein or a formulation thereof comprise less than 0.3% of any single reactive impurity (e.g., aldehyde) species.


204. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein one or more, or optionally all, of the lipid reagents used for a lipid nanoparticle as described herein or a formulation thereof comprise less than 0.1% of any single reactive impurity (e.g., aldehyde) species.


205. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the total aldehyde content and/or quantity of any single reactive impurity (e.g., aldehyde) species is determined by liquid chromatography (LC), e.g., coupled with tandem mass spectrometry (MS/MS), e.g., according to the method described in Example 26.


206. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the total aldehyde content and/or quantity of reactive impurity (e.g., aldehyde) species is determined by detecting one or more chemical modifications of a nucleic acid molecule (e.g., as described herein) associated with the presence of reactive impurities (e.g., aldehydes), e.g., in the lipid reagents.


207. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the total aldehyde content and/or quantity of aldehyde species is determined by detecting one or more chemical modifications of a nucleotide or nucleoside (e.g., a ribonucleotide or ribonucleoside, e.g., comprised in or isolated from a nucleic acid molecule, e.g., as described herein) associated with the presence of reactive impurities (e.g., aldehydes), e.g., in the lipid reagents, e.g., as described in Example 41.


208. The system, kit, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the chemical modifications of a nucleic acid molecule, nucleotide, or nucleoside are detected by determining the presence of one or more modified nucleotides or nucleosides, e.g., using LC-MS/MS analysis, e.g., as described in Example 41.


209. A lipid nanoparticle (LNP) comprising the system, polypeptide (or RNA encoding the same), nucleic acid molecule, or DNA encoding the system or polypeptide, of any preceding embodiment.


210. A system comprising a first lipid nanoparticle comprising the polypeptide (or DNA or RNA encoding the same) of a Gene Writing system (e.g., as described herein); and a second lipid nanoparticle comprising a nucleic acid molecule of a Gene Writing System (e.g., as described herein).


211. The system, kit, polypeptide, or reaction mixture of any preceding embodiment, wherein the system, nucleic acid molecule, polypeptide, and/or DNA encoding the same, is formulated as a lipid nanoparticle (LNP).


212. A system comprising:

    • a first lipid nanoparticle comprising the polypeptide (or DNA or RNA encoding the same) of a system or polypeptide of any preceding embodiment; and
    • a second lipid nanoparticle comprising the template RNA (or DNA encoding the same) of a system or template RNA of any preceding embodiment.


213. A virus, viral-like particle, fusosome, or virosome comprising the system, template RNA, polypeptide (or RNA encoding the same), or DNA encoding the system, template RNA, or polypeptide, of any preceding embodiment.


214. A system comprising:

    • a first virus, viral-like particle, fusosome, or virosome comprising the polypeptide (or DNA or RNA encoding the same) of a system or polypeptide of any preceding embodiment; and
    • a second virus, viral-like particle, or virosome comprising the template RNA (or DNA encoding the same) of a system or template RNA of any preceding embodiment.


215. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein at least 80, 85, 90, 95, 96, 97, 98, or 99% of the template RNA present is greater than 100, 125, 150, 175, or 200 nucleotides long, or at least 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 kilobases long (and optionally less than 15, 10, 5, or 20 kilobases long, or less than 500, 400, 300, or 200 nucleotides long).


216. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein at least 80, 85, 90, 95, 96, 97, 98, or 99% of the template RNA present contains a polyA tail (e.g., a polyA tail that is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides in length (SEQ ID NO: 3663)).


217. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein at least 80, 85, 90, 95, 96, 97, 98, or 99% of the template RNA present contains:

    • a 5′ cap, e.g.: a 7-methylguanosine cap (e.g., a 0-Me-m7G cap); a hypermethylated cap analog; an NAD+-derived cap analog (e.g., as described in Kiledjian, Trends in Cell Biology 28, 454-464 (2018)); or a modified, e.g., biotinylated, cap analog (e.g., as described in Bednarek et al., Phil Trans R Soc B 373, 20180167 (2018)), and/or
    • a 3′ feature selected from one or more of: a polyA tail; a 16-nucleotide long stem-loop structure flanked by unpaired 5 nucleotides (e.g., as described by Mannironi et al., Nucleic Acid Research 17, 9113-9126 (1989)); a triple-helical structure (e.g., as described by Brown et al., PNAS 109, 19202-19207 (2012)); a tRNA, Y RNA, or vault RNA structure (e.g., as described by Labno et al., Biochemica et Biophysica Acta 1863, 3125-3147 (2016)); incorporation of one or more deoxyribonucleotide triphosphates (dNTPs), 2′O-Methylated NTPs, or phosphorothioate-NTPs; a single nucleotide chemical modification (e.g., oxidation of the 3′ terminal ribose to a reactive aldehyde followed by conjugation of the aldehyde-reactive modified nucleotide); or chemical ligation to another nucleic acid molecule.


218. The system, kit, template RNA, or reaction mixture of aany of the preceding embodiments, wherein the template RNA comprises one or more modified nucleotides, e.g., selected from dihydrouridine, inosine, 7-methylguanosine, 5-methylcytidine (5mC), 5′ Phosphate ribothymidine, 2′-O-methyl ribothymidine, 2′-O-ethyl ribothymidine, 2′-fluoro ribothymidine, C-5 propynyl-deoxycytidine (pdC), C-5 propynyl-deoxyuridine (pdU), C-5 propynyl-cytidine (pC), C-5 propynyl-uridine (pU), 5-methyl cytidine, 5-methyl uridine, 5-methyl deoxycytidine, 5-methyl deoxyuridine methoxy, 2,6-diaminopurine, 5′-Dimethoxytrityl-N4-ethyl-2′-deoxycytidine, C-5 propynyl-f-cytidine (pfC), C-5 propynyl-f-uridine (pfUJ), 5-methyl f-cytidine, 5-methyl f-uridine, C-5 propynyl-m-cytidine (pmC), C-5 propynyl-f-uridine (pmU), 5-methyl m-cytidine, 5-methyl m-uridine, LNA (locked nucleic acid), MGB (minor groove binder) pseudouridine (Ψ), 1-N-methylpseudouridine (1-Me-Ψ), or 5-methoxyuridine (5-MO-U).


219. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein at least 80, 85, 90, 95, 96, 97, 98, or 99% of the template RNA present contains one or more modified nucleotides.


220. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein at least 80, 85, 90, 95, 96, 97, 98, or 99% of the template RNA remains intact (e.g., greater than 100, 125, 150, 175, or 200 nucleotides long, or at least 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 kilobases long) after a stability test.


221. The system, kit, or reaction mixture of any of the preceding embodiments, wherein at least 1% of target sites are modified after the system is assayed for potency.


222. The system, kit, template RNA, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the system, polypeptide, template RNA, and/or DNA encoding the same, is formulated as a lipid nanoparticle (LNP).


223. The system, kit, template RNA, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the DNA encoding the system, polypeptide, and/or template RNA are packaged into a virus, viral-like particle, virosome, liposome, vesicle, exosome, or LNP.


224 The system, kit, template RNA, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the DNA encoding the system, template RNA, or polypeptide is packaged into an adeno-associated virus (AAV).


225. The system, kit, template RNA, polypeptide, or reaction mixture of any of the preceding embodiments, wherein the system, template RNA, polypeptide, lipid nanoparticle (LNP), virus, viral-like particle, or virosome is free or substantially free of pyrogen, virus, fungus, bacterial pathogen, and/or host cell protein contamination.


226. A virus, viral-like particle, or virosome comprising:

    • the system, template RNA, or polypeptide of any of the preceding embodiments, or DNA encoding any of the same, and
    • an adeno-associated virus (AAV) capsid protein.


227. The system, kit, template RNA, polypeptide, virus, viral-like particle, or virosome of any of the preceding embodiments, wherein the system, template RNA, and/or polypeptide is active in a target tissue and less active (e.g., not active) in a non-target tissue.


228. The system, kit, template RNA, polypeptide, virus, viral-like particle, or virosome of any of the preceding embodiments, further comprising one or more first tissue-specific expression-control sequences specific to the target tissue, wherein the one or more first tissue-specific expression-control sequences specific to the target tissue are in operative association with the template RNA, the polypeptide or nucleic acid encoding the same, or both.


229. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the endonuclease domain, e.g., nickase domain, nicks the first strand of the target site DNA and nicks the second strand at a site a distance from the first nick.


230. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the nicks are made in an outward orientation.


231. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the nicks are made in an outward orientation.


232. The system, kit, template RNA, or reaction mixture of any of any of the preceding embodiments,

    • wherein the sequence that binds a target site specifies the location of the nick to the first strand,
    • wherein the system further comprises an additional nucleic acid comprising a sequence that binds a site a distance from the target site, and wherein the sequence that binds a site a distance from the target site specifies the location of the nick to the second strand.


233. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the additional nucleic acid further comprises a sequence that binds the polypeptide (e.g., that binds the endonuclease domain and/or DBD), e.g., wherein the additional nucleic acid comprises a gRNA.


234. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the sequence that binds a site a distance from the target site (e.g., binds to the first strand of a site in a target genome) is at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, or 130 nucleotides long (and optionally no more than 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 nucleotides long), e.g., is 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides long.


235. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the sequence that binds a site a distance from the target site is complementary to the first strand of the target site, or comprises no more than 1, 2, 3, 4, or 5 mismatches to the first strand of the target site.


236. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the DBD and/or endonuclease domain comprise a CRISPR/Cas domain.


237. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the CRISPR/Cas domain and the template RNA bind to the target site, and wherein the first strand of the target site comprises a first PAM site.


238. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the CRISPR/Cas domain and the additional nucleic acid bind to the site a distance from the target site, and wherein the second strand of the site a distance from the target site comprises a second PAM site.


239. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the first PAM site and second PAM site are positioned between the location of the nick to the first strand and the location of the nick to the second strand.


240. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the location of the nick to the first strand and the location of the nick to the second strand are positioned between the first PAM site and second PAM site.


241. The system, kit, template RNA, or reaction mixture of anany of the preceding embodiments, further comprising an additional polypeptide comprising an additional DNA-binding domain (DBD) and an additional endonuclease domain, e.g., an additional nickase domain.


242. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the additional endonuclease domain, e.g., the additional nickase domain, comprises an endonuclease or nickase domain described herein, e.g., a CRISPR/Cas domain, a type IIs nuclease (e.g., FokI), a Holliday Junction resolvase, a meganuclease, or a double-stranded DNA nuclease comprising an alteration that abrogates its ability to nick one strand (e.g., transforming the double-stranded DNA nuclease into a nickase).


243. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the additional DBD binds a site a distance from the target site.


244. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the endonuclease domain of (a) or (b) nicks the first strand and the additional endonuclease domain (e.g., additional nickase domain) nicks the second strand.


245. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the nicks are made in an outward orientation.


246. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the nicks are made in an inward orientation.


247. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the DBD and optionally the template RNA (e.g., the sequence that binds the polypeptide) specifies the location of the nick to the first strand, and the additional DBD specifies the location of the nick to the second strand.


248. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the polypeptide (e.g., the DBD) comprises a TAL effector molecule.


249. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the polypeptide (e.g., the DBD) comprises a zinc finger molecule.


250. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the polypeptide (e.g., the DBD) comprises a CRISPR/Cas domain.


251. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the additional polypeptide (e.g., the additional DBD) comprises a TAL effector molecule.


252. The system, kit, template RNA, or reaction mixture of anany of the preceding embodiments, wherein the additional polypeptide (e.g., the additional DBD) comprises a zinc finger molecule.


253. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the additional polypeptide (e.g., the additional DBD) comprises a CRISPR/Cas domain.


254. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the polypeptide and the additional polypeptide bind to sites on the target DNA between the location of the nick to the first strand and the location of the nick to the second.


255. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the location of the nick to the first strand and the location of the nick to the second strand are between the sites where the polypeptide and the additional polypeptide bind to the target DNA.


256. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein, on the target DNA, the location of the nick to the second strand is positioned on the opposite side of the binding sites of the polypeptide and additional polypeptide relative to the location of the nick to the first strand.


257. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein, on the target DNA, the location of the nick to the second strand is positioned on the same side of the binding sites of the polypeptide and additional polypeptide relative to the location of the nick to the first strand.


258. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the CRISPR/Cas domain of the polypeptide and the template RNA bind to the target site, and wherein the first strand of the target site comprises a PAM site.


259. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the PAM site and the site at a distance from the target site are positioned between the location of the nick to the first strand and the location of the nick to the second strand.


260. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the location of the nick to the first strand and the location of the nick to the second strand are positioned between the PAM site and the site at a distance from the target site.


261. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, further comprising an additional nucleic acid (e.g., a gRNA) comprising a sequence that binds a site a distance from the target site, and wherein the sequence that binds a site a distance from the target site specifies the location of the nick to the second strand.


262. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the additional nucleic acid further comprises a sequence that binds the additional polypeptide (e.g., the CRISPR/Cas domain), e.g., wherein the additional nucleic acid comprises a gRNA.


263. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the sequence that binds a site a distance from the target site (e.g., to the first strand of a site in a target genome) is at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, or 130 nucleotides long (and optionally no more than 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 nucleotides long), e.g., is 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides long.


264. The system, kit, template RNA, or reaction mixture of anany of the preceding embodiments, wherein the sequence that binds a site a distance from the target site is complementary to the first strand of the target site, or comprises no more than 1, 2, 3, 4, or 5 mismatches to the first strand of the target site.


265. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the site a distance from the target site comprises a PAM site.


266. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the PAM site and the target site are positioned between the location of the nick to the first strand and the location of the nick to the second strand.


267. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the location of the nick to the second strand (e.g., relative to the nick to the first strand) is such that DNA polymerization by the RT domain proceeds toward the location of the nick to the second strand.


268. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the location of the nick to the second strand (e.g., relative to the nick to the first strand) is such that DNA polymerization by the RT domain proceeds away from the location of the nick to the second strand.


269. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the first nick and the second nick are at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides apart.


270. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the first nick and the second nick are no more than 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or 250 nucleotides apart.


271. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the first nick and the second nick are 20-200, 30-200, 40-200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200, 110-200, 120-200, 130-200, 140-200, 150-200, 160-200, 170-200, 180-200, 190-200, 20-190, 30-190, 40-190, 50-190, 60-190, 70-190, 80-190, 90-190, 100-190, 110-190, 120-190, 130-190, 140-190, 150-190, 160-190, 170-190, 180-190,20-180, 30-180, 40-180, 50-180, 60-180, 70-180, 80-180, 90-180, 100-180, 110-180, 120-180, 130-180, 140-180, 150-180, 160-180, 170-180, 20-170, 30-170, 40-170, 50-170, 60-170, 70-170, 80-170, 90-170, 100-170, 110-170, 120-170, 130-170, 140-170, 150-170, 160-170, 20-160, 30-160, 40-160, 50-160, 60-160, 70-160, 80-160, 90-160, 100-160, 110-160, 120-160, 130-160, 140-160, 150-160, 20-150, 30-150, 40-150, 50-150, 60-150, 70-150, 80-150, 90-150, 100-150, 110-150, 120-150, 130-150, 140-150, 20-140, 30-140, 40-140, 50-140, 60-140, 70-140, 80-140, 90-140, 100-140, 110-140, 120-140, 130-140, 20-130, 30-130, 40-130, 50-130, 60-130, 70-130, 80-130, 90-130, 100-130, 110-130, 120-130, 20-120, 30-120, 40-120, 50-120, 60-120, 70-120, 80-120, 90-120, 100-120, 110-120, 20-110, 30-110, 40-110, 50-110, 60-110, 70-110, 80-110, 90-110, 100-110, 20-100, 30-100, 40-100, 50-100, 60-100, 70-100, 80-100, 90-100, 20-90, 30-90, 40-90, 50-90, 60-90, 70-90, 80-90, 20-80, 30-80, 40-80, 50-80, 60-80, 70-80, 20-70, 30-70, 40-70, 50-70, 60-70, 20-60, 30-60, 40-60, 50-60, 20-50, 30-50, 40-50, 20-40, 30-40, or 20-30 nucleotides apart.


272. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the system produces fewer double-stranded breaks (e.g., at least 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% fewer) when modifying DNA than an otherwise similar system wherein one or more of a PAM site, target site, or site a distance from the target site is not situated between the location of the first strand nick and the location of the second strand nick.


273. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the system produces fewer double-stranded breaks (e.g., at least 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% fewer) when modifying DNA than an otherwise similar system wherein the polypeptide and the additional polypeptide bind to sites on the target DNA not between the location of the nick to the first strand and the location of the nick to the second.


274. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the system produces fewer double-stranded breaks (e.g., at least 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% fewer) when modifying DNA than an otherwise similar system wherein, on the target DNA, the location of the nick to the second strand and the location of the nick to the first strand are located between the binding sites of the polypeptide and additional polypeptide.


275. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the system produces fewer double-stranded breaks (e.g., at least 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% fewer) when modifying DNA than an otherwise similar system wherein the location of the nick to the second strand (e.g., relative to the nick to the first strand) is such that the RT domain initiates reverse transcription away from the location of the nick to the second strand.


276. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the system produces fewer deletions not encoded by the heterologous object sequence (e.g., at least 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% fewer) when modifying DNA than an otherwise similar system wherein one or more of a PAM site, target site, or site a distance from the target site is not situated between the location of the first strand nick and the location of the second strand nick, e.g., as measured by PacBio long read sequencing, e.g., as described in Example 29.


277. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the system produces fewer deletions (e.g., at least 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% fewer) when modifying DNA than an otherwise similar system wherein the polypeptide and the additional polypeptide bind to sites on the target DNA not between the location of the nick to the first strand and the location of the nick to the second, e.g., as measured by PacBio long read sequencing, e.g., as described in Example 29.


278. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the system produces fewer deletions not encoded by the heterologous object sequence (e.g., at least 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% fewer) when modifying DNA than an otherwise similar system wherein, on the target DNA, the location of the nick to the second strand and the location of the nick to the first strand are located between the binding sites of the polypeptide and additional polypeptide, e.g., as measured by PacBio long read sequencing, e.g., as described in Example 29.


279. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the system produces fewer deletions not encoded by the heterologous object sequence (e.g., at least 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% fewer) when modifying DNA than an otherwise similar system wherein the location of the nick to the second strand (e.g., relative to the nick to the first strand) is such that the RT domain initiates reverse transcription away from the location of the nick to the second strand, e.g., as measured by PacBio long read sequencing, e.g., as described in Example 29.


280. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the system produces fewer insertions not encoded by the heterologous object sequence (e.g., at least 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% fewer) when modifying DNA than an otherwise similar system wherein one or more of a PAM site, target site, or site a distance from the target site is not situated between the location of the first strand nick and the location of the second strand nick, e.g., as measured by PacBio long read sequencing, e.g., as described in Example 29.


281. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the system produces fewer insertions not encoded by the heterologous object sequence (e.g., at least 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% fewer) when modifying DNA than an otherwise similar system wherein the polypeptide and the additional polypeptide bind to sites on the target DNA not between the location of the nick to the first strand and the location of the nick to the second, e.g., as measured by PacBio long read sequencing, e.g., as described in Example 29.


282. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the system produces fewer insertions not encoded by the heterologous object sequence (e.g., at least 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% fewer) when modifying DNA than an otherwise similar system wherein, on the target DNA, the location of the nick to the second strand and the location of the nick to the first strand are located between the binding sites of the polypeptide and additional polypeptide, e.g., as measured by PacBio long read sequencing, e.g., as described in Example 29.


283. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the system produces fewer insertions not encoded by the heterologous object sequence (e.g., at least 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% fewer) when modifying DNA than an otherwise similar system wherein the location of the nick to the second strand (e.g., relative to the nick to the first strand) is such that the RT domain initiates reverse transcription away from the location of the nick to the second strand, e.g., as measured by PacBio long read sequencing, e.g., as described in Example 29.


284. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the system produces more desired Gene Writing modifications (e.g., at least 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% more) when modifying DNA than an otherwise similar system wherein one or more of a PAM site, target site, or site a distance from the target site is not situated between the location of the first strand nick and the location of the second strand nick, e.g., as measured by PacBio long read sequencing, e.g., as described in Example 29.


285. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the system produces more desired Gene Writing modifications (e.g., at least 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% more) when modifying DNA than an otherwise similar system wherein the polypeptide and the additional polypeptide bind to sites on the target DNA not between the location of the nick to the first strand and the location of the nick to the second, e.g., as measured by PacBio long read sequencing, e.g., as described in Example 29.


286. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the system produces more desired Gene Writing modifications (e.g., at least 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% more) when modifying DNA than an otherwise similar system wherein, on the target DNA, the location of the nick to the second strand and the location of the nick to the first strand are located between the binding sites of the polypeptide and additional polypeptide, e.g., as measured by PacBio long read sequencing, e.g., as described in Example 29.


287. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the system produces more desired Gene Writing modifications (e.g., at least 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% more) when modifying DNA than an otherwise similar system wherein the location of the nick to the second strand (e.g., relative to the nick to the first strand) is such that the RT domain initiates reverse transcription away from the location of the nick to the second strand, e.g., as measured by PacBio long read sequencing, e.g., as described in Example 29.


288. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the first nick and the second nick are at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 350, 400, 450, or 500 nucleotides apart, e.g., at least 100 nucleotides apart, (and optionally no more than 500, 400, 300, 200, 190, 180, 170, 160, 150, 140, 130, or 120 nucleotides apart).


289. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the first nick and the second nick are 100-200, 110-200, 120-200, 130-200, 140-200, 150-200, 160-200, 170-200, 180-200, 190-200, 100-190, 110-190, 120-190, 130-190, 140-190, 150-190, 160-190, 170-190, 180-190, 100-180, 110-180, 120-180, 130-180, 140-180, 150-180, 160-180, 170-180, 100-170, 110-170, 120-170, 130-170, 140-170, 150-170, 160-170, 100-160, 110-160, 120-160, 130-160, 140-160, 150-160, 100-150, 110-150, 120-150, 130-150, 140-150, 100-140, 110-140, 120-140, 130-140, 100-130, 110-130, 120-130, 100-120, 110-120, or 100-110 nucleotides apart.


290. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the system produces fewer insertions not encoded by the heterologous object sequence (e.g., at least 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% fewer) when modifying DNA than an otherwise similar system wherein the location of the nick to the second strand is less than 100 nucleotides away from the location of the nick to the first strand (and optionally at least 20, 30, 40, 50, 60, 70, 80, or 90 nucleotides away), e.g., as measured by PacBio long read sequencing, e.g., as described in Example 29.


291. The system, kit, template RNA, or reaction mixture of any of the preceding embodiments, wherein the system produces fewer deletions not encoded by the heterologous object sequence (e.g., at least 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% fewer) when modifying DNA than an otherwise similar system wherein the location of the nick to the second strand is less than 100 nucleotides away from the location of the nick to the first strand (and optionally at least 20, 30, 40, 50, 60, 70, 80, or 90 nucleotides away), e.g., as measured by PacBio long read sequencing, e.g., as described in Example 29.


292. Any above-numbered system, which does not comprise DNA, or which does not comprise more than 10%, 5%, 4%, 3%, 2%, or 1% DNA by mass or by molar amount.


293. A method of making a system for modifying DNA (e.g., as described herein), the method comprising:

    • (a) providing a template nucleic acid (e.g., a template RNA or DNA) comprising a heterologous homology sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology to a sequence comprised in a target DNA molecule, and/or
    • (b) providing a polypeptide of the system (e.g., comprising a DNA-binding domain (DBD) and/or an endonuclease domain) comprising a heterologous targeting domain that binds specifically to a sequence comprised in the target DNA molecule.


294. The method of any of the preceding embodiments, wherein:

    • (a) comprises introducing into the template nucleic acid (e.g., a template RNA or DNA) a heterologous homology sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology to the sequence comprised in a target DNA molecule, and/or
    • (b) comprises introducing into the polypeptide of the system (e.g., comprising a DNA-binding domain (DBD) and/or an endonuclease domain) the heterologous targeting domain that binds specifically to a sequence comprised in the target DNA molecule.


295. The method of any of the preceding embodiments, wherein the introducing of (a) comprises inserting the homology sequence into the template nucleic acid.


296. The method of any of the preceding embodiments, wherein the introducing of (a) comprises replacing a segment of the template nucleic acid with the homology sequence.


297. The method of any of the preceding embodiments, wherein the introducing of (a) comprises mutating one or more nucleotides (e.g., at least 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 nucleotides) of the template nucleic acid, thereby producing a segment of the template nucleic acid having the sequence of the homology sequence.


298. The method of any of the preceding embodiments, wherein the introducing of (b) comprises inserting the amino acid sequence of the targeting domain into the amino acid sequence of the polypeptide.


299. The method of any of the preceding embodiments, wherein the introducing of (b) comprises inserting a nucleic acid sequence encoding the targeting domain into a coding sequence of the polypeptide comprised in a nucleic acid molecule.


300. The method of any of the preceding embodiments, wherein the introducing of (b) comprises replacing at least a portion of the polypeptide with the targeting domain.


301. The method of any of the preceding embodiments, wherein the introducing of (a) comprises mutating one or more amino acids (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, or more amino acids) of the polypeptide.


302. A method for modifying a target site in genomic DNA in a cell, the method comprising contacting the cell with:

    • (a) a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, e.g., a nickase domain; and
    • (b) a template RNA (or DNA encoding the template RNA) comprising (e.g., from 5′ to 3′) (i) optionally a sequence that binds the target site (e.g., a second strand of a site in a target genome), (ii) optionally a sequence that binds the polypeptide, (iii) a heterologous object sequence, and (iv) a 3′ target homology domain, wherein:
    • (i) the polypeptide comprises a heterologous targeting domain (e.g., in the DBD or the endonuclease domain) that binds specifically to a sequence comprised in or adjacent to the target site of the genomic DNA; and/or
    • (ii) the template RNA comprises a heterologous homology sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homology to a sequence comprised in or adjacent to the target site of the genomic DNA;
      • thereby modifying the target site in genomic DNA in a cell.


303. A method for manufacturing an template RNA, comprising:

    • (a) providing an template RNA of any preceding embodiment, and
    • (b) assaying one or more of:
      • (i) the length of the template RNA, e.g., whether the template RNA has a length that is above a reference length or within a reference length range, e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the template RNA present is greater than 100, 125, 150, 175, or 200 nucleotides long;
      • (ii) the presence, absence, and/or length of a polyA tail on the template RNA, e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the template RNA present contains a polyA tail (e.g., a polyA tail that is at least 5, 10, 20, or 30 nucleotides in length (SEQ ID NO: 3664));
      • (iii) the presence, absence, and/or type of a 5′ cap on the template RNA, e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the template RNA present contains a 5′ cap, e.g., whether that cap is a 7-methylguanosine cap, e.g., a O-Me-m7G cap;
      • (iv) the presence, absence, and/or type of one or more modified nucleotides (e.g., selected from dihydrouridine, inosine, 7-methylguanosine, 5-methylcytidine (5mC), 5′ Phosphate ribothymidine, 2′-O-methyl ribothymidine, 2′-O-ethyl ribothymidine, 2′-fluoro ribothymidine, C-5 propynyl-deoxycytidine (pdC), C-5 propynyl-deoxyuridine (pdU), C-5 propynyl-cytidine (pC), C-5 propynyl-uridine (pU), 5-methyl cytidine, 5-methyl uridine, 5-methyl deoxycytidine, 5-methyl deoxyuridine methoxy, 2,6-diaminopurine, 5′-Dimethoxytrityl-N4-ethyl-2′-deoxycytidine, C-5 propynyl-f-cytidine (pfC), C-5 propynyl-f-uridine (pfUJ), 5-methyl f-cytidine, 5-methyl f-uridine, C-5 propynyl-m-cytidine (pmC), C-5 propynyl-f-uridine (pmU), 5-methyl m-cytidine, 5-methyl m-uridine, LNA (locked nucleic acid), MGB (minor groove binder) pseudouridine (Ψ), 1-N-methylpseudouridine (1-Me-Ψ), or 5-methoxyuridine (5-MO-U)) in the template RNA, e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the template RNA present contains one or more modified nucleotides;
      • (v) the stability of the template RNA (e.g., over time and/or under a pre-selected condition), e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the template RNA remains intact (e.g., greater than 100, 125, 150, 175, or 200 nucleotides long) after a stability test;
      • (vi) the potency of the template RNA in a system for modifying DNA, e.g., whether at least 1% of target sites are modified after a system comprising the template RNA is assayed for potency; or
      • (vii) the presence, absence, and/or level of one or more of a pyrogen, virus, fungus, bacterial pathogen, or host cell protein, e.g., whether the template RNA is free or substantially free of pyrogen, virus, fungus, bacterial pathogen, or host cell protein contamination.


304. A method for manufacturing a system for modifying DNA, comprising:

    • (a) providing a system for modifying DNA of any preceding embodiment, and
    • (b) assaying one or more of:
      • (i) the length of the template RNA, e.g., whether the template RNA has a length that is above a reference length or within a reference length range, e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the template RNA present is greater than 100, 125, 150, 175, or 200 nucleotides long;
      • (ii) the presence, absence, and/or length of a polyA tail on the template RNA, e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the template RNA present contains a polyA tail (e.g., a polyA tail that is at least 5, 10, 20, or 30 nucleotides in length (SEQ ID NO: 3664));
      • (iii) the presence, absence, and/or type of a 5′ cap on the template RNA, e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the template RNA present contains a 5′ cap, e.g., whether that cap is a 7-methylguanosine cap, e.g., a O-Me-m7G cap;
      • (iv) the presence, absence, and/or type of one or more modified nucleotides (e.g., selected from pseudouridine, dihydrouridine, inosine, 7-methylguanosine, 1-N-methylpseudouridine (1-Me-Ψ), 5-methoxyuridine (5-MO-U), 5-methylcytidine (5mC), or a locked nucleotide in the template RNA, e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the template RNA present contains one or more modified nucleotides;
      • (v) the stability of the template RNA (e.g., over time and/or under a pre-selected condition), e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the template RNA remains intact (e.g., greater than 100, 125, 150, 175, or 200 nucleotides long) after a stability test; (vi) the potency of the template RNA in a system for modifying DNA, e.g., whether at least 1% of target sites are modified after a system comprising the template RNA is assayed for potency;
      • (vii) the length of the polypeptide, first polypeptide, or second polypeptide, e.g., whether the polypeptide, first polypeptide, or second polypeptide has a length that is above a reference length or within a reference length range, e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the polypeptide, first polypeptide, or second polypeptide present is greater than 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, or 2000 amino acids long (and optionally, no larger than 2500, 2000, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, or 600 amino acids long);
      • (viii) the presence, absence, and/or type of post-translational modification on the polypeptide, first polypeptide, or second polypeptide, e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the polypeptide, first polypeptide, or second polypeptide contains phosphorylation, methylation, acetylation, myristoylation, palmitoylation, isoprenylation, glipyatyon, or lipoylation;
      • (ix) the presence, absence, and/or type of one or more artificial, synthetic, or non-canonical amino acids (e.g., selected from ornithine, β-alanine, GABA, δ-Aminolevulinic acid, PABA, a D-amino acid (e.g., D-alanine or D-glutamate), aminoisobutyric acid, dehydroalanine, cystathionine, lanthionine, Djenkolic acid, Diaminopimelic acid, Homoalanine, Norvaline, Norleucine, Homonorleucine, homoserine, O-methyl-homoserine and O-ethyl-homoserine, ethionine, selenocysteine, selenohomocysteine, selenomethionine, selenoethionine, tellurocysteine, or telluromethionine) in the polypeptide, first polypeptide, or second polypeptide, e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the polypeptide, first polypeptide, or second polypeptide present contains one or more artificial, synthetic, or non-canonical amino acids;
      • (x) the stability of the polypeptide, first polypeptide, or second polypeptide (e.g., over time and/or under a pre-selected condition), e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the polypeptide, first polypeptide, or second polypeptide remains intact (e.g., greater than 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, or 2000 amino acids long (and optionally, no larger than 2500, 2000, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, or 600 amino acids long)) after a stability test;
      • (xi) the potency of the polypeptide, first polypeptide, or second polypeptide in a system for modifying DNA, e.g., whether at least 1% of target sites are modified after a system comprising the polypeptide, first polypeptide, or second polypeptide is assayed for potency; or
      • (xii) the presence, absence, and/or level of one or more of a pyrogen, virus, fungus, bacterial pathogen, or host cell protein, e.g., whether the system is free or substantially free of pyrogen, virus, fungus, bacterial pathogen, or host cell protein contamination.


305. A method for modifying a target site in genomic DNA in a cell, the method comprising: contacting the cell with:

    • (a) a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, e.g., a nickase domain; and
    • (b) a template RNA (or DNA encoding the template RNA) comprising (e.g., from 5′ to 3′) (i) optionally a sequence that binds the target site (e.g., a second strand of a site in a target genome), (ii) optionally a sequence that binds the polypeptide, (iii) a heterologous object sequence, and (iv) a 3′ target homology domain,
    • thereby modifying the target site in genomic DNA in a cell.


306. A method for modifying a target site in genomic DNA in a cell, the method comprising: contacting the cell with a system, polypeptide, template RNA, or DNA encoding the same of any preceding embodiment, thereby modifying the target site in genomic DNA in a cell.


307. The method of any of the preceding embodiments, wherein a system, polypeptide, template RNA, or DNA are delivered to the target site by electroporation, e.g., nucleofection.


308. The method of any of the preceding embodiments, which does not comprise contacting the cell with DNA, e.g., or which comprises contacting the cell with a composition that not comprise more than 10%, 5%, 4%, 3%, 2%, or 1% DNA by mass or by molar amount.


309. The method of any of the preceding embodiments, which does not comprise contacting the cell with protein, e.g., or which comprises contacting the cell with a composition that not comprise more than 10%, 5%, 4%, 3%, 2%, or 1% protein by mass or by molar amount.


310. The method of any of the preceding embodiments, which comprises contacting a target cell or population of target cells with at least two template RNAs and/or at least two GeneWriter polypeptides, such that at least two target sites (a first target site and a second target site) are modified in a target cell.


311. The method of any of the preceding embodiments, wherein the first target site and the second site are each independently edited at a frequency of at least 5%, 10%, or 15% of copies of the site in a cell population.


312. The method of any of the preceding embodiments, wherein the first target site and the second site are each independently edited at a frequency of at least 50%, 60%, 70%, or 80% of the level of editing obtained in an otherwise similar cell population contacted with an otherwise similar system targeting only one of the target sites.


313. The method of any of the preceding embodiments, wherein the resulting cell population comprises no more than 5%, 10%, or 20% unwanted indels compared to the unwanted indels obtained in an otherwise similar cell population contacted with an otherwise similar system targeting only one of the target sites.


314. The method of any of the preceding embodiments, wherein the cell is a primary cell.


315. The method of any of the preceding embodiments, wherein the cell is a T cell.


316. A method for modifying a target site in genomic DNA in a cell, the method comprising: contacting the cell, e.g., by nucleofection or lipid particle delivery, with:

    • (a) a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, e.g., a nickase domain; and
    • (b) a template RNA (or DNA encoding the template RNA) comprising (e.g., from 5′ to 3′) (i) optionally a sequence that binds the target site (e.g., a second strand of a site in a target genome), (ii) optionally a sequence that binds the polypeptide, (iii) a heterologous object sequence, and (iv) a 3′ target homology domain,
    • thereby modifying the target site in genomic DNA in a cell,
    • wherein the cell is euploid, is not immortalized, is part of a tissue, is part of an organism, is a primary cell, is non-dividing, is haploid (e.g., a germline cell), is a non-cancerous polyploid cell, or is from a subject having a genetic disease.


317. The method of any of the preceding embodiments, wherein the template RNA comprises (i).


318. The method of any of the preceding embodiments, wherein the template RNA comprises (ii).


319. The method of any of the preceding embodiments, wherein the template RNA comprises (i) and (ii).


320. A method for treating a subject having a disease or condition associated with a genetic defect, the method comprising:

    • administering to the subject:
      • (a) a polypeptide or a nucleic acid encoding the polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase (RT) domain, (ii) a DNA-binding domain (DBD); and (iii) an endonuclease domain, e.g., a nickase domain; and
      • (b) a template RNA (or DNA encoding the template RNA) comprising (e.g., from 5′ to 3′) (i) optionally a sequence that binds the target site (e.g., a second strand of a site in a target genome), (ii) optionally a sequence that binds the polypeptide, (iii) a heterologous object sequence, and (iv) a 3′ target homology domain,
    • thereby treating the subject having a disease or condition associated with a genetic defect.


321. The method of any of the preceding embodiments, wherein the template RNA comprises (i).


322. The method of any of the preceding embodiments, wherein the template RNA comprises (ii).


323. The method of any of the preceding embodiments, wherein the template RNA comprises (i) and (ii).


324. A method for treating a subject having a disease or condition associated with a genetic defect, the method comprising:

    • administering to the subject a system, polypeptide, template RNA, or DNA encoding the same of any preceding embodiment,
    • thereby treating the subject having a disease or condition associated with a genetic defect.


325. The method of any of the preceding embodiments, wherein the disease or condition associated with a genetic defect is an indication listed in any of Tables 9-12, and/or wherein the genetic defect is a defect in a gene listed in any of Tables 9-12.


326. The method of any of the preceding embodiments, wherein the subject is a human patient.


Definitions

Domain: The term “domain” as used herein refers to a structure of a biomolecule that contributes to a specified function of the biomolecule. A domain may comprise a contiguous region (e.g., a contiguous sequence) or distinct, non-contiguous regions (e.g., non-contiguous sequences) of a biomolecule. Examples of protein domains include, but are not limited to, an endonuclease domain, a DNA binding domain, a reverse transcription domain; an example of a domain of a nucleic acid is a regulatory domain, such as a transcription factor binding domain.


Exogenous: As used herein, the term exogenous, when used with reference to a biomolecule (such as a nucleic acid sequence or polypeptide) means that the biomolecule was introduced into a host genome, cell or organism by the hand of man. For example, a nucleic acid that is as added into an existing genome, cell, tissue or subject using recombinant DNA techniques or other methods is exogenous to the existing nucleic acid sequence, cell, tissue or subject.


First/Second Strand: As used herein, first strand and second strand, as used to describe the individual DNA strands of target DNA, distinguish the two DNA strands based upon which strand the reverse transcriptase domain initiates polymerization, e.g., based upon where target primed synthesis initiates. The first strand refers to the strand of the target DNA upon which the reverse transcriptase domain initiates polymerization, e.g., where target primed synthesis initiates. The second strand refers to the other strand of the target DNA. First and second strand designations do not describe the target site DNA strands in other respects; for example, in some embodiments the first and second strands are nicked by a polypeptide described herein, but the designations ‘first’ and ‘second’ strand have no bearing on the order in which such nicks occur.


Genomic safe harbor site (GSH site): A genomic safe harbor site is a site in a host genome that is able to accommodate the integration of new genetic material, e.g., such that the inserted genetic element does not cause significant alterations of the host genome posing a risk to the host cell or organism. A GSH site generally meets 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the following criteria: (i) is located >300 kb from a cancer-related gene; (ii) is >300 kb from a miRNA/other functional small RNA; (iii) is >50 kb from a 5′ gene end; (iv) is >50 kb from a replication origin; (v) is >50 kb away from any ultraconservered element; (vi) has low transcriptional activity (i.e. no mRNA +/−25 kb); (vii) is not in copy number variable region; (viii) is in open chromatin; and/or (ix) is unique, with 1 copy in the human genome. Examples of GSH sites in the human genome that meet some or all of these criteria include (i) the adeno-associated virus site 1 (AAVS1), a naturally occurring site of integration of AAV virus on chromosome 19; (ii) the chemokine (C-C motif) receptor 5 (CCR5) gene, a chemokine receptor gene known as an HIV-1 coreceptor; (iii) the human ortholog of the mouse Rosa26 locus; (iv) the rDNA locus. Additional GSH sites are known and described, e.g., in Pellenz et al. epub Aug. 20, 2018 (doi.org/10.1101/396390).


Heterologous: The term heterologous, when used to describe a first element in reference to a second element means that the first element and second element do not exist in nature disposed as described. For example, a heterologous polypeptide, nucleic acid molecule, construct or sequence refers to (a) a polypeptide, nucleic acid molecule or portion of a polypeptide or nucleic acid molecule sequence that is not native to a cell in which it is expressed, (b) a polypeptide or nucleic acid molecule or portion of a polypeptide or nucleic acid molecule that has been altered or mutated relative to its native state, or (c) a polypeptide or nucleic acid molecule with an altered expression as compared to the native expression levels under similar conditions. For example, a heterologous regulatory sequence (e.g., promoter, enhancer) may be used to regulate expression of a gene or a nucleic acid molecule in a way that is different than the gene or a nucleic acid molecule is normally expressed in nature. In another example, a heterologous domain of a polypeptide or nucleic acid sequence (e.g., a DNA binding domain of a polypeptide or nucleic acid encoding a DNA binding domain of a polypeptide) may be disposed relative to other domains or may be a different sequence or from a different source, relative to other domains or portions of a polypeptide or its encoding nucleic acid. In certain embodiments, a heterologous nucleic acid molecule may exist in a native host cell genome, but may have an altered expression level or have a different sequence or both. In other embodiments, heterologous nucleic acid molecules may not be endogenous to a host cell or host genome but instead may have been introduced into a host cell by transformation (e.g., transfection, electroporation), wherein the added molecule may integrate into the host genome or can exist as extra-chromosomal genetic material either transiently (e.g., mRNA) or semi-stably for more than one generation (e.g., episomal viral vector, plasmid or other self-replicating vector).


Inverted Terminal Repeats: The term “inverted terminal repeats” or “ITRs” as used herein refers to AAV viral cis-elements named so because of their symmetry. These elements promote efficient multiplication of an AAV genome. It is hypothesized that the minimal elements for ITR function are a Rep-binding site (RBS; 5′-GCGCGCTCGCTCGCTC-3′ (SEQ ID NO: 1538) for AAV2) and a terminal resolution site (TRS; 5′-AGTTGG-3′ for AAV2) plus a variable palindromic sequence allowing for hairpin formation. According to the present invention, an ITR comprises at least these three elements (RBS, TRS and sequences allowing the formation of an hairpin). In addition, in the present invention, the term “ITR” refers to ITRs of known natural AAV serotypes (e.g. ITR of a serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 AAV), to chimeric ITRs formed by the fusion of ITR elements derived from different serotypes, and to functional variant thereof. By functional variant of an ITR, it is referred to a sequence presenting a sequence identity of at least 80%, 85%, 90%, preferably of at least 95% with a known ITR, allowing multiplication of the sequence that includes said ITR in the presence of Rep proteins.


Mutation or Mutated: The term “mutated” when applied to nucleic acid sequences means that nucleotides in a nucleic acid sequence may be inserted, deleted or changed compared to a reference (e.g., native) nucleic acid sequence. A single alteration may be made at a locus (a point mutation) or multiple nucleotides may be inserted, deleted or changed at a single locus. In addition, one or more alterations may be made at any number of loci within a nucleic acid sequence. A nucleic acid sequence may be mutated by any method known in the art.


Nucleic acid molecule: Nucleic acid molecule refers to both RNA and DNA molecules including, without limitation, cDNA, genomic DNA and mRNA, and also includes synthetic nucleic acid molecules, such as those that are chemically synthesized or recombinantly produced, such as RNA templates, as described herein. The nucleic acid molecule can be double-stranded or single-stranded, circular or linear. If single-stranded, the nucleic acid molecule can be the sense strand or the antisense strand. Unless otherwise indicated, and as an example for all sequences described herein under the general format “SEQ. ID NO:,” “nucleic acid comprising SEQ. ID NO:1” refers to a nucleic acid, at least a portion which has either (i) the sequence of SEQ. ID NO:1, or (ii) a sequence complimentary to SEQ. ID NO:1. The choice between the two is dictated by the context in which SEQ. ID NO:1 is used. For instance, if the nucleic acid is used as a probe, the choice between the two is dictated by the requirement that the probe be complimentary to the desired target. Nucleic acid sequences of the present disclosure may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more naturally occurring nucleotides with an analog, inter-nucleotide modifications such as uncharged linkages (for example, methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (for example, phosphorothioates, phosphorodithioates, etc.), pendant moieties, (for example, polypeptides), intercalators (for example, acridine, psoralen, etc.), chelators, alkylators, and modified linkages (for example, alpha anomeric nucleic acids, etc.). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of a molecule. Other modifications can include, for example, analogs in which the ribose ring contains a bridging moiety or other structure such as modifications found in “locked” nucleic acids. In various embodiments, the nucleic acids are in operative association with additional genetic elements, such as tissue-specific expression-control sequence(s) (e.g., tissue-specific promoters and tissue-specific microRNA recognition sequences), as well as additional elements, such as inverted repeats (e.g., inverted terminal repeats, such as elements from or derived from viruses, e.g., AAV ITRs) and tandem repeats, inverted repeats/direct repeats (e.g., transposon inverted repeats, e.g., transposon inverted repeats also containing direct repeats, e.g., inverted repeats also containing direct repeats), homology regions (segments with various degrees of homology to a target DNA), UTRs (5′, 3′, or both 5′ and 3′ UTRs), and various combinations of the foregoing. The nucleic acid elements of the systems provided by the invention can be provided in a variety of topologies, including single-stranded, double-stranded, circular, linear, linear with open ends, linear with closed ends, and particular versions of these, such as doggybone DNA (dbDNA), close-ended DNA (ceDNA).


Gene expression unit: a gene expression unit is a nucleic acid sequence comprising at least one regulatory nucleic acid sequence operably linked to at least one effector sequence. A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if the promoter or enhancer affects the transcription or expression of the coding sequence. Operably linked DNA sequences may be contiguous or non-contiguous. Where necessary to join two protein-coding regions, operably linked sequences may be in the same reading frame.


Host: The terms host genome or host cell, as used herein, refer to a cell and/or its genome into which protein and/or genetic material has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell and/or genome, but to the progeny of such a cell and/or the genome of the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. A host genome or host cell may be an isolated cell or cell line grown in culture, or genomic material isolated from such a cell or cell line, or may be a host cell or host genome which composing living tissue or an organism. In some instances, a host cell may be an animal cell or a plant cell, e.g., as described herein. In certain instances, a host cell may be a bovine cell, horse cell, pig cell, goat cell, sheep cell, chicken cell, or turkey cell. In certain instances, a host cell may be a corn cell, soy cell, wheat cell, or rice cell.


Operative association: As used herein, “operative association” describes a functional relationship between two nucleic acid sequences, such as a 1) promoter and 2) a heterologous object sequence, and means, in such example, the promoter and heterologous object sequence (e.g., a gene of interest) are oriented such that, under suitable conditions, the promoter drives expression of the heterologous object sequence. For instance, the template nucleic acid may be single-stranded, e.g., either the (+) or (−) orientation but an operative association between promoter and heterologous object sequence means whether or not the template nucleic acid will transcribe in a particular state, when it is in the suitable state (e.g., is in the (+) orientation, in the presence of required catalytic factors, and NTPs, etc.), it does accurately transcribe. Operative association applies analogously to other pairs of nucleic acids, including other tissue-specific expression control sequences (such as enhancers, repressors and microRNA recognition sequences), IR/DR, ITRs, UTRs, or homology regions and heterologous object sequences or sequences encoding a transposase.


Pseudoknot: A “pseudoknot sequence” sequence, as used herein, refers to a nucleic acid (e.g., RNA) having a sequence with suitable self-complementarity to form a pseudoknot structure, e.g., having: a first segment, a second segment between the first segment and a third segment, wherein the third segment is complementary to the first segment, and a fourth segment, wherein the fourth segment is complementary to the second segment. The pseudoknot may optionally have additional secondary structure, e.g., a stem loop disposed in the second segment, a stem-loop disposed between the second segment and third segment, sequence before the first segment, or sequence after the fourth segment. The pseudoknot may have additional sequence between the first and second segments, between the second and third segments, or between the third and fourth segments. In some embodiments, the segments are arranged, from 5′ to 3′: first, second, third, and fourth. In some embodiments, the first and third segments comprise five base pairs of perfect complementarity. In some embodiments, the second and fourth segments comprise 10 base pairs, optionally with one or more (e.g., two) bulges. In some embodiments, the second segment comprises one or more unpaired nucleotides, e.g., forming a loop. In some embodiments, the third segment comprises one or more unpaired nucleotides, e.g., forming a loop.


Stem-loop sequence: As used herein, a “stem-loop sequence” refers to a nucleic acid sequence (e.g., RNA sequence) with sufficient self-complementarity to form a stem-loop, e.g., having a stem comprising at least two (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) base pairs, and a loop with at least three (e.g., four) base pairs. The stem may comprise mismatches or bulges.


Tissue-specific expression-control sequence(s): As used herein, a “tissue-specific expression-control sequence” means nucleic acid elements that increase or decrease the level of a transcript comprising the heterologous object sequence in the target tissue in a tissue-specific manner, e.g., preferentially in an on-target tissue(s), relative to an off-target tissue(s). In some embodiments, a tissue-specific expression-control sequence preferentially drives or represses transcription, activity, or the half-life of a transcript comprising the heterologous object sequence in the target tissue in a tissue-specific manner, e.g., preferentially in an on-target tissue(s), relative to an off-target tissue(s). Exemplary tissue-specific expression-control sequences include tissue-specific promoters, repressors, enhancers, or combinations thereof, as well as tissue-specific microRNA recognition sequences. Tissue specificity refers to on-target (tissue(s) where expression or activity of the template nucleic acid is desired or tolerable) and off-target (tissue(s) where expression or activity of the template nucleic acid is not desired or is not tolerable). For example, a tissue-specific promoter (such as a promoter in a template nucleic acid or controlling expression of a transposase) drives expression preferentially in on-target tissues, relative to off-target tissues. In contrast, a micro-RNA that binds the tissue-specific microRNA recognition sequences (either on a nucleic acid encoding the transposase or on the template nucleic acid, or both) is preferentially expressed in off-target tissues, relative to on-target tissues, thereby reducing expression of a template nucleic acid (or transposase) in off-target tissues. Accordingly, a promoter and a microRNA recognition sequence that are specific for the same tissue, such as the target tissue, have contrasting functions (promote and repress, respectively, with concordant expression levels, i.e., high levels of the microRNA in off-target tissues and low levels in on-target tissues, while promoters drive high expression in on-target tissues and low expression in off-target tissues) with regard to the transcription, activity, or half-life of an associated sequence in that tissue.


The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.





BRIEF DESCRIPTION OF THE DRAWINGS


FIG. 1 is a schematic of the Gene Writing™ genome editing system.



FIG. 2 is a schematic of the structure of the Gene Writer™ genome editor polypeptide.



FIG. 3 is a schematic of the structure of exemplary Gene Writer™ template RNAs.



FIGS. 4A and 4B are a series of diagrams showing examples of configurations of Gene Writers using domains derived from a variety of sources. Gene Writers as described herein may or may not comprise all domains depicted. For example, a GeneWrite may, in some instances, lack an RNA-binding domain, or may have single domains that fulfill the functions of multiple domains, e.g., a Cas9 domain for DNA binding and endonuclease activity. Exemplary domains that can be included in a GeneWriter polypeptide include DNA binding domains (e.g., comprising a DNA binding domain, e.g., of a Table herein; a zinc finger; a TAL domain; Cas9; dCas9; nickase Cas9; a transcription factor, or a meganuclease), RNA binding domains (e.g., comprising an RNA binding domain of B-box protein, MS2 coat protein, dCas, or an element of a sequence of a Table herein), reverse transcriptase domains (e.g., comprising a reverse transcriptase domain of an element of a sequence of a Table herein; other retrotransposases (e.g., as listed in a Table herein); a peptide containing a reverse transctipase domain (e.g., as listed in a Table herein)), and/or an endonuclease domain (e.g., comprising an endonuclease domain of an element of a Table herein; Cas9; nickase Cas9; a restriction enzyme (e.g., a type II restriction enzyme, e.g., FokI); a meganuclease; a Holliday junction resolvase; an RLE retrotranspase; an APE retrotransposase; or a GIY-YIG retrotransposase). Exemplary GeneWriter polypeptides comprising exemplary combinations of such domains are shown in the bottom panel.



FIG. 5 is a diagram showing the modules of an exemplary GeneWriter RNA template. Individual modules of the exemplary template can be combined, re-arranged, and/or omitted, e.g., to produce a Gene Writer template. A=5′ homology arm; B=Ribozyme; C=5′ UTR; D=heterologous object sequence; E=3′ UTR; F=3′ homology arm.



FIG. 6 is a table listing the modules of an exemplary Gene Writer RNA template. Individual modules can be combined, re-arranged, and/or omitted, e.g., to produce a Gene Writer template. A=5′ homology arm; B=Ribozyme; C=5′ UTR; D=heterologous object sequence; E=3′ UTR; F=3′ homology arm.



FIGS. 7A and 7B are diagrams showing an exemplary second strand nicking process. (FIG. 7A) A Cas9 nickase is fused to a Gene Writer protein. The Gene Writer protein introduces a nick in a DNA strand through its EN domain (shown as *), and the fused Cas9 nickase introduces a nicks on either top or bottom DNA strands (shown as X). (FIG. 7B) A Gene Writer is targeted to DNA through its DNA biding domain and introduces a DNA nick with its EN domain (*). A Cas9 nickase is then used the generate a second nick (X) at the top or bottom strand, upstream or downstream of the EN introduced nick.



FIGS. 8A and 8B. The linker region at the C-terminus of the DNA-binding domain of R2Tg can be truncated and modified. Deletions in the Natural Linker from the myb domain at A or B to positions 1 or 2 along with replacement by 3GS (SEQ ID NO: 1024) or XTEN synthetic linkers were constructed (FIG. 8A). Integration efficiency was measured in HEK293T cells by ddPCR (FIG. 8B).



FIG. 9. Landing pads designed for testing target site mutations of R2Tg Gene Writer.



FIG. 10A. ddPCR assay measuring percentage of integrations from all lentiviral integrated landing pads per cell.



FIG. 10B. Amplicon-sequencing and NGS analysis of indels present at landing pads sites.



FIG. 11. AAVS1 ZFP replacement of DNA binding domain of a Retrotransposase Gene Writer. This Figure discloses “3GS Linker” as SEQ ID NO: 1024.



FIG. 12. Cas9 or Cas9 nickase replacement of DNA binding domain of Retrotransposase GeneWriters with or without active EN domain (*=mutant) FIG. 13. AAVS1 ZFP fusion to a Retrotransposase Gene Writer with or without functional DNA binding domain.



FIGS. 14A and 14B. Schematic of nickaseCas9-GeneWriter fusions. (FIG. 14A) Schematic of nickaseCas9 fused to Gene Writer protein. (FIG. 14B) Schematic of 3′ extended gRNA.



FIGS. 15A and 15B. Schematic of nickaseCas9-GeneWriter fusions. (FIG. 15A) Schematic of nickaseCas9 fused to Gene Writer protein. (FIG. 15B) Schematic of donor transgene flanked by UTRs and homology to the cut site.



FIGS. 16A-16C. Schematic of constructs. (FIG. 16A) Schematic of Gene Writer protein. (FIG. 16B) Schematic of donor transgene flanked by UTRs and homology to the cut site. (FIG. 16C) Schematic of Cas9 constructs used.



FIGS. 17A and 17B. The schematics for mRNA encoding Gene Writer (FIG. 17A). The native untranslated regions (UTRs) were replaced by 5′ and 3′ UTRs optimized for the protein expression (shown as 5′ UTRexp and 3′ UTRexp). The Gene Writer protein expression was assayed by HiBit assay by probing HiBit tag expression (FIG. 17B). This Figure discloses “3GS” as SEQ ID NO: 1024.



FIG. 18. Genome integration induced by Gene Writer protein with its native UTRs and UTRs optimized for the protein expression. The Gene Writing activity with non-native UTRs is stimulated by the presence of the RNA template bearing the retrotransposon native UTRs.



FIG. 19. Delivery of Gene Writer system using mRNA encoding the polypeptide and plasmid DNA encoding the RNA template for retrotransposition.



FIG. 20. Diagrams of example 5′UTR engineering strategies. HA=homology arm; K=Kozak sequence; pA=poly A signal; AMa=A. maritima; Rx=other species of retrotransposon.



FIG. 21. Possible location of an intron (or introns) within the RNA template. Introns are shown by curved lines. 5′HA: 5′ homology arm; 3′ HA: 3′ homology arm; 5′ UTR: Retrotransposon-specific 5′UTR; 3′ UTR: Retrotransposon-specific 3′ UTR; GOI gene of interest. Orange blocks correspond to the sequence designed to be expressed from the genomic location harboring its own cell specific promoter, poly(A) signal and UTRs for the protein expression (5′ and 3′ UTRexp). The sequence can be oriented in the sense (shown above) or the antisense orientation related to retrotransposon UTRs and homology arms. The intron can be located within GOI, or within UTRexp.



FIG. 22. Genome integration in HEK293T cells as reported by 3′ ddPCR assay. The Gene Writer mRNA at 0.5 μg/well was co-transfected with the RNA templates with or without enzymatically added cap 1 and the poly(A) tail. The Gene Writer mRNA to RNA transgene ratio was 1:1.



FIG. 23. Genome integration detected by 3′ ddPCR induced by expression of Gene Writer mRNA produced with either unmodified (GO) or modified nucleotides (pseudouridine (Ψ), 1-N-methylpseudouridine (1-Me-Ψ), 5-methoxyuridine (5-MO-U) or 5-methylcytidine (5mC)). 1 ug of Gene Writer mRNA per well was used. The non-modified RNA template was used. The Gene Writer RNA to the RNA template were co-transfected in 1:8 molar ratio.



FIG. 24. Construct diagram of driver and transgene plasmids. Homology arms (HA) and stuffer sequences are variable in this set of experiments.



FIGS. 25A-25C. (FIG. 25A) Timeline of experiment. (FIG. 25B) Schematic of R2Tg and transgene construct configurations. (FIG. 25C) Western Blot against Rad51 shows loss of Rad51 protein expression at day 3.



FIGS. 26A and 26B. U2OS cells were treated with a non targeting control siRNA (ctrl) or siRNA against Rad51, along with R2Tg Wt or control RT and EN mutants. ddPCR at the 3′ (FIG. 26A) or 5′ (FIG. 26B) junction was used to assess integration efficiency on day 3.



FIGS. 27A and 27B. (FIG. 27A) Sequence map of Ribozyme of R2 element from Taeniopygia guttata (R2Tg) in context of modules of Gene Writer transgene molecule RNA. The Ribozyme features are denoted as: P, based paired region; P′, based pair region complement strand; L, loop at end of P region; J, nucleotides joining base paired regions. Figure discloses SEQ ID NO: 1734. (FIG. 27B) Prediction of ribozyme secondary structure of R2Tg. Shaded box indicates a predicted catalytic position that could be used to inactivate the ribozyme. Figure discloses SEQ ID NO: 1734.



FIG. 28. Sequence map of Ribozyme of R2 element from Taeniopygia guttata (R2Tg) in context of modules of Gene Writer transgene molecule RNA. The Ribozyme features are denoted as: P, based paired region; P′, based pair region complement strand; L, loop at end of P region; J, nucleotides joining base paired regions. Figure discloses SEQ ID NO: 1734.



FIG. 29. Prediction of ribozyme secondary structure of R2 element from Taeniopygia guttata. Figure discloses SEQ ID NO: 1734.



FIG. 30. Gene Writing system for treating an exemplary repeat expansion disorder. Figure discloses SEQ ID NOS 1645, 1599, 1645, 1635-1636, 1645 and 1686-1688, respectively, in order of appearance.



FIG. 31. An illustration of two orientations of second strand nicking in an exemplary Gene Writing system.



FIGS. 32A and 32B. An illustration of the orientation and position of second strand nicking in an exemplary Gene Writing system and their effect on editing.



FIG. 33. Shows generation and expression of Cas9-RT fusion proteins. To assess expression of novel Gene Writer polypeptides in human cells, U2OS cells were transfected with Cas-RT expression plasmids harboring various RT domains from Tables 1 and 30 fused to a wild-type (WT) or Cas9(N863A) nickase. Cell lysates were collected on day 2 post-transfection and analyzed by Western blot using a primary antibody against Cas9. A primary antibody against GADPH was included as a loading control.



FIG. 34. Shows improving expression of Cas-RT fusions through choice of linker sequence. To assess how linkers can alter the expression of novel Gene Writer polypeptides in human cells, U2OS cells were transfected with Cas-RT expression plasmids harboring various linkers from Table 42 fusing the Cas9(N863A) nickase to the RT domain of an RNA-binding domain mutated R2Bm retrotransposase. Cell lysates were collected and analyzed by Western blot using a primary antibody against Cas9. A primary antibody against vinculin (left) or GADPH (right) was included as a loading control. Cas9 controls on the left represent titration of a Cas9 expression plasmid. Empty arrows indicate the original linker tested, while the filled arrow represents a linker (Linker 10) found to substantially improve expression of the fusion polypeptide. Sample numbers correspond to linker sequence identifiers in Table 42.



FIG. 35. Shows Cas/gRNA DNA targeting activity is preserved in Cas-RT fusions. Various RT domains were fused to Cas9(WT) and electroporated into U2OS cells. Genomic DNA was harvested and analyzed for mutational signatures by next generation sequencing. Mutations in the RNA or DNA-binding domains (RBD or DBD) of R2 retrotransposase domains is indicated, where relevant. Indel frequency is used here as a proxy for Cas activity preservation in the context of the RT fusion.



FIGS. 36A and 36B Disclose application of mutations improving reverse transcriptase domains. Conserved reverse transcriptase domains from the retrovirus genera Betaretrovirus, Deltaretrovirus, Gammaretrovirus, Epsilonretrovirus, and Spumavirus were aligned and compared to mutations previously shown to improve RT activity (Anzalone et al Nat Biotechnol 38(7):824-844 (2020); Baranauskas et al Protein Eng Des Sel 25(10):657-668 (2012); Arezi and Hogrefe Nucleic Acids Res 37(2):473-481 (2009)). FIG. 36A shows a set of 3 core mutations was identified and applied to RTs from these genera as indicated in. FIG. 36B discloses additional mutations were applied with first priority from the set of T306K/W313F, or alternately from L139P/E607K where neither of the first set were deemed transferrable. Selected mutations are shown in Table 45. FIGS. 36A and 36B disclose SEQ ID NOS 3610, 3623, 3637, 3611, 3624, 3638, 3611, 3624, 3639, 3612, 3625, 3640, 3613, 3626, 3641, 3611, 3627, 3642, 3614, 3628, 3643, 3615, 3629, 3644, 3616, 3630, 3645, 3617, 3630, 3645, 3618, 3631, 3646, 3619, 3632, 3647, 3620, 3633, 3648, 3621, 3634, 3649, 3622, 3635, 3650, 3622, 3636, 3651, 3652, 2060, 2738, 3653, 2086, 2758, 3653, 2086, 2759, 3654, 2087, 2773, 3655, 2088, 2775, 3653, 2086, 2863, 3656, 2103, 3046, 3657, 2104, 3080, 3658, 2120, 3081, 3658, 2175, 3081, 3659, 2221, 3082, 3660, 2279, 3102, 3661, 2525, 3103, 3662, 2704, 3122, 1850, 2736, 3125, 1905, 2737, and 2123, respectively, in order of appearance.



FIG. 37. U2OS cells were nucleofected with various Cas-RT fusion vectors in which the RT domain was selected from a database of monomeric retroviral reverse transcriptase domains. Editing of a HEK3 locus using a Template described in Table 43 was assessed by amplicon sequencing and analysis of precise editing vs indel signatures. Data are represented here as Activity Ratios, which are calculated as the ratio of the frequency of reads with the precisely intended edit (CTT insertion at the target nick site) to the frequency of reads with any other mutations (indels). Three Template RNA configurations assayed resulted in similar outcomes, so the results for a single template (Template P2 from Table 43) are shown.



FIG. 38. shows targeting multiple loci simultaneously results in efficient Gene Writing activity. HEK293 cells were nucleofected with Gene Writing systems comprising different compositions of Template plasmids to enable targeting of: 1) HEK3 alone, 2) HBB alone, or 3) both HBB and the HEK3 locus. Percent of editing is indicated for each locus upon delivery of one or both locus-specific Template RNA expression plasmids. Filled bars represent Perfect Writing events, while unfilled bars represent the frequency of indels. Target-locus-specific editing was seen when delivering either Template independently, and highly efficient and specific edits were seen at both loci when co-delivering the Templates.



FIG. 39. Shows effect of length on Gene Writing activity. HEK293T cells were nucleofected with all-RNA Gene Writing systems comprising various Template RNAs (Table 48) to test editing efficiency of the DNA-free approach at the HEK3 locus. Template 4, which encoded the same edit as Template 1, but with an addition of 20 nt at the 3′ end of the RT template, showed an approximately 3.1-fold drop in precise Writing activity and an approximately 2.4-fold drop in the ratio of precise corrections to indels.



FIG. 40. Shows effect of all-RNA delivery of Gene Writer using different mRNA compositions. Nucleofection of various Cas9-RT(MMLV) mRNAs (Table 49) into HEK293T using Template 1 (Table 48A). No strong effects were observed here in varying capping and UTR compositions.



FIG. 41. HEK293T cells were nucleofected with a Gene Writing system using a set Template (Template 1, Table 48) for editing the HEK3 locus and two different Cas-RT constructs. Sequence analysis indicated that both Cas-RT fusions made edits in a very precise and efficient manner. In both systems, there was an increase in efficiency under conditions including the optional secondary nick. These data show successful cloning and Precise Writing by the PERV RT domain in the context of these Cas-RT fusions.



FIG. 42. Shows the effect of all-RNA delivery of Gene Writer employing modified nucleotides. mRNA molecules encoding the Cas-RT(MMLV) polypeptide were varied in composition to determine effects (Table 49). Here, Template 1 is used to edit the HEK3 locus after incorporating modified nucleotides in the mRNA component. Gene Writing activity with a 5moU-modified mRNA component was found to both high and precise.



FIGS. 43A-43C show the effect of all-RNA delivery of Gene Writer using different mRNA compositions delivered into the cell via lipid particles. FIG. 43A shows all-RNA lipofection of various Cas9-RT(MMLV) mRNAs into HEK293T was performed using Template 1 (Table 48) and delivering via Lipofectamine 3000. FIG. 43B shows all-RNA lipofection of various Cas9-RT(MMLV) mRNAs into HEK293T was performed using Template 1 (Table 48) and delivering via MessengerMax reagent. These data indicated higher precise editing efficiencies with the MessengerMax reagent. FIG. 43C shows assay of two Templates differing in total length using MessengerMax reagent. No major changes in efficiency of editing were found to be associated with the template change in this experiment. Where included head-to-head, the addition of the second-nick gRNA resulted in an increase in efficiency of the system.



FIG. 44. shows all-RNA delivery of Cas-RT using lipid-based systems. The Cas9-RT(MMLV) and Cas9-RT(PERV) were delivered into HEK293T cells with Template 1 (Table 48) using MessengerMax lipid reagent. Here, activity for both enzymes was around 5% Precise Writing.



FIGS. 45A and 45B show expression of all-RNA Gene Writer system in primary human CD4+ T cells. FIG. 45A shows Gene Writer protein expression from mRNAs with varying doses delivered into primary human CD4+ T cells at day 1 post-nucleofection. Gene Writer was detected by an antibody targeting a Cas9 part of the polypeptide. GAPDH, a housekeeping gene, was detected by an antibody against GAPDH. Increasing expression levels were observed with increasing doses of nucleofected mRNA encoding the polypeptide were delivered, e.g., 0, 2.5, 5, and 10 μg Gene Writer mRNAs. Data for the detection of protein expression shown comprised 2 replicate. FIG. 45B shows Cell viability after nucleofection of 6 Template RNAs. Viability of primary CD4+ T cells after RNA delivery of the Gene Rewriter system at day 3 post nucleofection. Cell viability was assessed by flow cytometry after live/dead staining of harvested T cells (mean±s.d., n=2 replicates). [Gate: Live cells in a singlet population of cell population selected by FSC/SSC size plot]



FIGS. 46A and 46B show Gene Writing in primary human CD4+ T cells. FIG. 46A shows precise editing of the HEK3 genomic locus by a Gene Writer system in primary human CD4+ T cells, without addition of second-nick gRNA. FIG. 46B shows precise editing of the HEK3 genomic locus by a Gene Writer system in primary human CD4+ T cells. Genomic DNA was extracted from cells at day 3 post-nucleofection. Genome editing of HEK3 was examined by PCR-based amplicon-sequencing assay. DNA amplicons containing the expected genomic alteration were identified as Precise Write events, whereas amplicons with unintended editing (e.g. insertion, deletion) were counted as Indels. The percentage of each was calculated based on total reads per condition (mean±s.d., n=2 replicates).



FIGS. 47A and 47B show use of a second-nick gRNA for Gene Writing in primary human CD4+ T cells. The data generated in FIG. 46 are shown here for a direct comparison of potential effects of second-nick gRNA on efficiency. FIG. 47A shows in this experiment, the addition of a second-nick gRNA did not result in an enhanced precise writing signal. FIG. 47B shows rather, the use of a second-nick gRNA may have increased the frequency of indels. Thus, in some embodiments, a second nick gRNA sequence may be absent from a system described herein. Precise editing of HEK3 genomic site by the Gene Writer system in primary human CD4+ T cells, without (FIG. 47A) or with addition of second-nick gRNA (FIG. 47B). Genomic DNA was extracted from cells at day 3 post-nucleofection. Genome editing of HEK3 was examined by PCR-based amplicon-sequencing assay. DNA amplicons containing the expected genomic alteration by Gene Writer system were identified as Precise Write events, whereas amplicons with unintended editing (e.g. insertion, deletion) were counted as Indels. The percentage of each was calculated based on total reads per condition (mean±s.d., n=2 replicates).



FIG. 48. shows screening construct design for retrotransposon-mediated integration in human cells. A driver plasmid comprising a retrotransposase (Driver) expression cassette is transfected together with a template plasmid comprising a retrotransposon-dependent reporter cassette. Whereas expression from the template plasmid results in a non-functional GFP because of an interrupting antisense intron, transcription of the template molecule from the template plasmid results in the generation of an RNA with the intron removed by splicing that can then be reverse transcribed and integrated by the system. Expression of the reporter cassette will thus only occur from the integrated reporter cassette (Integrated gDNA, bottom) and not from the template plasmid. HA=homology arm, where applicable; CMV=mammalian CMV promoter; HiBit=HiBit tag for quantification of protein expression; T7=T7 RNA polymerase promoter; UTR=untranslated sequence, e.g., native retrotransposon UTRs; pA=poly A signal; SD-SA is used to indicate the splice-donor and splice-acceptor sites of an antisense intron in the GFP coding sequence.



FIG. 49. Screening of candidate retrotransposons identifies 25 candidates working to integrate a trans payload in human cells. A total of 163 retrotransposon systems were assayed for activity in human cells as described in Example 39. Integration as measured by ddPCR is shown as copies/genome for each retrotransposon driver/template system. The height of each bar indicates the average value of two replicates.



FIGS. 50A and 50B show luciferase activity assay for primary cells. LNPs formulated as according to Example 44 were analyzed for delivery of cargo to primary human (A) and mouse (B) hepatocytes, as according to Example 45. The luciferase assay revealed dose-responsive luciferase activity from cell lysates, indicating successful delivery of RNA to the cells and expression of Firefly luciferase from the mRNA cargo.



FIG. 51 discloses LNP-mediated delivery of RNA cargo to the murine liver. Firefly luciferase mRNA-containing LNPs were formulated and delivered to mice by iv, and liver samples were harvested and assayed for luciferase activity at 6, 24, and 48 hours post administration. Reporter activity by the various formulations followed the ranking LIPIDV005>LIPIDV004>LIPIDV003. RNA expression was transient and enzyme levels returned near vehicle background by 48 hours. Post-administration.





DETAILED DESCRIPTION

This disclosure relates to compositions, systems and methods for targeting, editing, modifying or manipulating a DNA sequence (e.g., inserting a heterologous object sequence into a target site of a mammalian genome) at one or more locations in a DNA sequence in a cell, tissue or subject, e.g., in vivo or in vitro. The heterologous object DNA sequence may include, e.g., a substitution, a deletion, an insertion, e.g., a coding sequence, a regulatory sequence, or a gene expression unit.


More specifically, the disclosure provides reverse transcriptase-based systems for altering a genomic DNA sequence of interest, e.g., by inserting, deleting, or substituting one or more nucleotides into/from the sequence of interest. This disclosure is based, in part, on a bioinformatic analysis to identify reverse transcriptase sequences, for example in retrotransposons from a variety of organisms (see Table 1 or 3).


The disclosure provides, in part, Gene Writer™ genome editors comprising a polypeptide component and a template nucleic acid (e.g., template RNA) component. In some embodiments, a Gene Writer™ genome editor can be used to introduce an alteration into a target site in a genome. In some embodiments, the polypeptide component comprises a writing domain (e.g., a reverse transcriptase domain), a DNA-binding domain, and an endonuclease domain (e.g., nickase domain). In some embodiments, the template nucleic acid (e.g., template RNA) comprises a sequence that binds a target site in the genome (e.g., that binds to a second strand of the target site), a sequence that binds the polypeptide component, a heterologous object sequence, and a 3′ target homology domain. Without wishing to be bound by theory, it is thought that the template nucleic acid (e.g., template RNA) binds to the second strand of a target site in the genome, and binds to the polypeptide component (e.g., localizing the polypeptide component to the target site in the genome). It is thought that the endonuclease (e.g., nickase) of the polypeptide component cuts the target site (e.g., the first strand of the target site), e.g., allowing the 3′homology domain to bind to a sequence adjacent to the site to be altered on the first strand of the target site. It is thought that the writing domain (e.g., reverse transcriptase domain) of the polypeptide component uses the 3′ target homology domain as a primer and the heterologous object sequence as a template to, e.g., polymerize a sequence complementary to the heterologous object sequence. Without wishing to be bound by theory, it is thought that selection of an appropriate heterologous object sequence can result in substitution, deletion, or insertion of one or more nucleotides at the target site.


In embodiments, the disclosure provides a nucleic acid molecule or a system for retargeting, e.g., of a Gene Writer polypeptide or nucleic acid molecule, or of a system as described herein. Retargeting (e.g., of a Gene Writer polypeptide or nucleic acid molecule, or of a system as described herein) generally comprises: (i) directing the polypeptide to bind and cleave at the target site; and/or (ii) designing the template RNA to have complementarity to the target sequence. In some embodiments, the template RNA has complementarity to the target sequence 5′ of the first-strand nick, e.g., such that the 3′ end of the template RNA anneals and the 5′ end of the target site serves as the primer, e.g., for target-primed reverse transcription (TPRT). In some embodiments, the endonuclease domain of the polypeptide and the 5′ end of the RNA template are also modified as described.


Gene Writer™ Genome Editors

Gene Writer™ genome editors are systems that are capable of modifying a host cell's genome and can be applied for the mutation, deletion, or other modification of a genomic target sequence, including the insertion of heterologous payloads. In some embodiments, these systems take inspiration from a group of naturally evolved mobile genetic elements known as retrotransposons. Gene Writer™ polypeptides can also comprise RT domains derived from sources other than retrotransposons, e.g., from viruses.


Non-long terminal repeat (LTR) retrotransposons are a type of mobile genetic elements that are widespread in eukaryotic genomes. They include two classes: the apurinic/apyrimidinic endonuclease (APE)-type and the restriction enzyme-like endonuclease (RLE)-type. The APE class retrotransposons are comprised of two functional domains: an endonuclease/DNA binding domain, and a reverse transcriptase domain. The RLE class are comprised of three functional domains: a DNA binding domain, a reverse transcription domain, and an endonuclease domain. The reverse transcriptase domain of non-LTR retrotransposon functions by binding an RNA sequence template and reverse transcribing it into the host genome's target DNA. The RNA sequence template has a 3′ untranslated region which is specifically bound to the transposase, and a variable 5′ region generally having Open Reading Frame(s) (“ORF”) encoding transposase proteins. The RNA sequence template may also comprise a 5′ untranslated region which specifically binds the retrotransposase.


In some embodiments, as described herein, the elements of such non-LTR retrotransposons can be functionally modularized and/or modified to target, edit, modify or manipulate a target DNA sequence, e.g., to insert an object (e.g., heterologous) nucleic acid sequence into a target genome, e.g., a mammalian genome, by reverse transcription. Such modularized and modified nucleic acids, polypeptide compositions and systems are described herein and are referred to as Gene Writer™ gene editors. A Gene Writer™ gene editor system comprises: (A) a polypeptide or a nucleic acid encoding a polypeptide, wherein the polypeptide comprises (i) a reverse transcriptase domain, and either (x) an endonuclease domain that contains DNA binding functionality or (y) an endonuclease domain and separate DNA binding domain; and (B) a template RNA comprising (i) a sequence that binds the polypeptide and (ii) a heterologous insert sequence. For example, the Gene Writer™ genome editor protein may comprise a DNA-binding domain, a reverse transcriptase domain, and an endonuclease domain. In some embodiments, the DNA-binding function may involve an RNA component that directs the protein to a DNA sequence, e.g, a gRNA. In other embodiments, the Gene Writer™ genome editor protein may comprise a reverse transcriptase domain and an endonuclease domain. In certain embodiments, the elements of the Gene Writer™ gene editor polypeptide can be derived from sequences of non-LTR retrotransposons, e.g., APE-type or RLE-type retrotransposons or portions or domains thereof. In some embodiments the RLE-type non-LTR retrotransposon is from the R2, NeSL, HERO, R4, or CRE clade. In some embodiments the Gene Writer™ genome editor is derived from R4 element X4_Line, which is found in the human genome. In some embodiments the APE-type non-LTR retrotransposon is from the R1, or Tx1 clade. In some embodiments the Gene Writer™ genome editor is derived from Tx1 element Mare6, which is found in the human genome. The RNA template element of a Gene Writer™ gene editor system is typically heterologous to the polypeptide element and provides an object sequence to be inserted (reverse transcribed) into the host genome. In some embodiments the Gene Writer™ genome editor protein is capable of target primed reverse transcription. In some embodiments, the Gene Writer genome editor protein is capable of second strand synthesis. Table 50 shows exemplary Gene Writer proteins and associated sequences from a variety of retrotransposases, identified using data mining. Column 1 indicates the family to which the retrotransposon belongs. Column 2 lists the element name. Column 3 indicates an accession number, if any. Column 4 lists an organism in which the retrotransposase is found. Column 5 lists the predicted 5′ untranslated region, and column 6 lists the predicted 3′ untranslated region; both are segments that are predicted to allow the template RNA to bind the retrotransposase of column 7. (It is understood that columns 5-6 show the DNA sequence, and that an RNA sequence according to any of columns 5-6 would typically include uracil rather than thymidine.) Column 7 lists the predicted retrotransposase amino acid sequence. Column 8 lists the predicted RT domain present based on sequence analysis, column 9 lists the start codon position, and column 10 lists the stop codon position.










Lengthy table referenced here




US20240035049A1-20240201-T00001


Please refer to the end of the specification for access instructions.






In some embodiments the Gene Writer™ genome editor is combined with a second polypeptide. In some embodiments the second polypeptide is derived from an APE-type non-LTR retrotransposon. In some embodiments the second polypeptide has a zinc knuckle-like motif. In some embodiments the second polypeptide is a homolog of Gag proteins.


Inspired by the success of retrotransposons in nature, it is further discussed here that the natural function of a retrotransposon can be recapitulated using functional parts derived from completely independent systems. For example, a functional Gene Writer™ can be made up of unrelated DNA binding, reverse transcription, and endonuclease domains. This modular structure allows combining of functional domains, e.g., dCas9 (DNA binding), MMLV reverse transcriptase (reverse transcription), FokI (endonuclease). In some embodiments, multiple functional domains may arise from a single protein, e.g., Cas9 nickase (DNA binding, endonuclease), R2 retrotransposon (DNA binding, reverse transcription, endonuclease).


In some embodiments, a Gene Writer™ system is capable of producing an insertion into the target site of at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides (and optionally no more than 500, 400, 300, 200, or 100 nucleotides). In some embodiments, a Gene Writer™ system is capable of producing an insertion into the target site of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides (and optionally no more than 500, 400, 300, 200, or 100 nucleotides). In some embodiments, a Gene Writer™ system is capable of producing an insertion into the target site of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 kilobases (and optionally no more than 1, 5, 10, or 20 kilobases). In some embodiments, a Gene Writer™ system is capable of producing a deletion of at least 81, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides (and optionally no more than 500, 400, 300, or 200 nucleotides). In some embodiments, a Gene Writer™ system is capable of producing a deletion of at least 81, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides (and optionally no more than 500, 400, 300, or 200 nucleotides). In some embodiments, a Gene Writer™ system is capable of producing a deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides (and optionally no more than 500, 400, 300, or 200 nucleotides). In some embodiments, a Gene Writer™ system is capable of producing a deletion of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 kilobases (and optionally no more than 1, 5, 10, or 20 kilobases). In some embodiments, a Gene Writer system is capable of producing a substitution into the target site of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more nucleotides. In some embodiments, the substitution is a transition mutation. In some embodiments, the substitution is a transversion mutation. In some embodiments, the substitution converts an adenine to a thymine, an adenine to a guanine, an adenine to a cytosine, a guanine to a thymine, a guanine to a cytosine, a guanine to an adenine, a thymine to a cytosine, a thymine to an adenine, a thymine to a guanine, a cytosine to an adenine, a cytosine to a guanine, or a cytosine to a thymine.


Polypeptide Component of Gene Writer™ Gene Editor System

Domains and Functions:


In some embodiments, the Gene Writer™ polypeptide possesses the functions of DNA target site binding, template nucleic acid (e.g., RNA) binding, DNA target site cleavage, and template nucleic acid (e.g., RNA) writing, e.g., reverse transcription. In some embodiments, each functions is contained within a distinct domain. In some embodiments, a function may be attributed to two or more domains (e.g., two or more domains, together, exhibit the functionality). In some embodiments, two or more domains may have the same or similar function (e.g., two or more domains each independently have DNA-binding functionality, e.g., for two different DNA sequences). In other embodiments, one or more domains may be capable of enabling one or more functions, e.g., a Cas9 domain enabling both DNA binding and target site cleavage. In some embodiments, the domains are all located within a single polypeptide. In some embodiments, a first domain is in one polypeptide and a second domain is in a second polypeptide. For example, in some embodiments, the Gene Writer™ polypeptide may be split between a first polypeptide and a second polypeptide, e.g., wherein the first polypeptide comprises a reverse transcriptase (RT) domain and wherein the second polypeptide comprises a DNA-binding domain and an endonuclease domain, e.g., a nickase domain. As a further example, in some embodiments, the first polypeptide and the second polypeptide each comprise a DNA binding domain (e.g., a first DNA binding domain and a second DNA binding domain). In some embodiments, the first and second polypeptide may be brought together post-translationally via a split-intein.


Writing Domain:


In certain aspects of the present invention, the writing domain of the Gene Writer™ system possesses reverse transcriptase activity and is also referred to as a reverse transcriptase domain (a RT domain). In some embodiments, the RT domain comprises an RT catalytic portion and and RNA-binding region (e.g., a region that binds the template RNA).


In certain aspects of the present invention, the writing domain is based on a reverse transcriptase domain of an APE-type or RLE-type non-LTR retrotransposon. A wild-type reverse transcriptase domain of an APE-type or RLE-type non-LTR retrotransposon can be used in a Gene Writer™ system or can be modified (e.g., by insertion, deletion, or substitution of one or more residues) to alter the reverse transcriptase activity for target DNA sequences. In some embodiments the reverse transcriptase is altered from its natural sequence to have altered codon usage, e.g. improved for human cells. In some embodiments the reverse transcriptase domain is a heterologous reverse transcriptase from a different retrovirus, LTR-retrotransposon, or non-LTR retrotransposon. In certain embodiments, a Gene Writer™ system includes a polypeptide that comprises a reverse transcriptase domain of an RLE-type non-LTR retrotransposon from the R2, NeSL, HERO, R4, or CRE clade, or of an APE-type non-LTR retrotransposon from the R1, or Tx1 clade. In certain embodiments, a Gene Writer™ system includes a polypeptide that comprises a reverse transcriptase domain of a non-LTR retrotransposon, LTR retrotransposon, group II intron, diversity-generating element, retron, telomerase, retroplasmid, retrovirus, or an engineered polymerase listed in Table 1 or Table 3. In some embodiments, a Gene Writer™ system includes a polypeptide that comprises a reverse transcriptase domain listed in Table 2. In embodiments, the amino acid sequence of the reverse transcriptase domain of a Gene Writer™ system is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to the amino acid sequence of a reverse transcriptase domain of a non-LTR retrotransposon, LTR retrotransposon, group II intron, diversity-generating element, retron, telomerase, retroplasmid, retrovirus, or an engineered polymerase whose DNA sequence is referenced in Table 1 or Table 3, or of a peptide comprising an RT domain referenced in Table 2. In some embodiments, the RT domain has a sequence selected from Table 1 or 3, or a sequence of a peptide comprising an RT domain selected from Table 2, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, the RT domain comprising a Gene Writer polypeptide has been mutated from its original amino acid sequence, e.g., has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 substitutions. In some embodiments, the RT domain is derived from the RT of a retrovirus, e.g., HIV-1 RT, Moloney Murine Leukemia Virus (MMLV) RT, avian myeloblastosis virus (AMV) RT, Rous Sarcoma Virus (RSV) RT. In some embodiments, the RT domain is derived from the RT of a Group II intron, e.g., the group II intron maturase RT from Eubacterium rectale (MarathonRT) (Zhao et al. RNA 24:22018), the RT domain from LtrA, the RT TGIRT (or trt). In some embodiments, the RT domain is derived from the RT of a retron, e.g., the reverse transcriptase from Ec86 (RT86). In some embodiments, the RT domain is derived from a diversity-generating retroelement, e.g., from the RT of Brt. In some embodiments, the RT domain is derived from the RT of a retroplasmid, e.g., the RT from the Mauriceville plasmid. In some embodiments, the RT domain is derived from a non-LTR retrotransposon, e.g., the RT from R2Bm, the RT from R2Tg, the RT from LINE-1, the RT from Penelope or a Penelope-like element (PLE). In some embodiments, the RT domain is derived from an LTR retrotransposon, e.g., the reverse transcriptase from Ty1. In some embodiments, the RT domain is derived from a telomerase, e.g., TERT. A person having ordinary skill in the art is capable of identifying reverse transcription domains based upon homology to other known reverse transcription domains using routine tools as Basic Local Alignment Search Tool (BLAST). In some embodiments, the reverse transcriptase contains the InterPro domain IPR000477. In some embodiments, the reverse transcriptase contains the pfam domain PF00078. In some embodiments, the RT contains the InterPro domain IPR013103. In some embodiments, the RT contains the pfam domain PF07727. In some embodiments, the reverse transcriptase contains a conserved protein domain of the cd00304 RT_like family, e.g., cd01644 (RT_pepA17), cd01645 (RT_Rtv), cd01646 (RT_Bac_retron_I), cd01647 (RT_LTR), cd01648 (TERT), cd01650 (RT_nLTR_like), cd01651 (RT_G2_intron), cd01699 (RNA_dep_RNAP), cd01709 (RT_like_1), cd03487 (RT_Bac_retron_II), cd03714 (RT_DIRS1), cd03715 (RT_ZFREV_like). Proteins containing these domains can additionally be found by searching the domains on protein databases, such as InterPro (Mitchell et al. Nucleic Acids Res 47, D351-360 (2019)), UniProt (The UniProt Consortium Nucleic Acids Res 47, D506-515 (2019)), or the conserved domain database (Lu et al. Nucleic Acids Res 48, D265-268 (2020)), or by scanning open reading frames for reverse transcriptase domains using prediction tools, for example InterProScan. The diversity of reverse transcriptases has been described in, but not limited to, those used by prokaryotes (Zimmerly et al. Microbiol Spectr 3(2):MDNA3-0058-2014 (2015); Lampson B.C. (2007) Prokaryotic Reverse Transcriptases. In: Polaina J., MacCabe A. P. (eds) Industrial Enzymes. Springer, Dordrecht), viruses (Herschhorn et al. Cell Mol Life Sci 67(16):2717-2747 (2010); Menendez-Arias et al. Virus Res 234:153-176 (2017)), and mobile elements (Eickbush et al. Virus Res 134(1-2):221-234 (2008); Craig et al. Mobile DNA III 3rd Ed. DOI:10.1128/9781555819217 (2015)), each of which is incorporated herein by reference.


In some embodiments, the reverse transcriptase (RT) domain exhibits enhanced stringency of target-primed reverse transcription (TPRT) initiation, e.g., relative to an endogenous RT domain. In some embodiments, the RT domain initiates TPRT when the 3 nt in the target site immediately upstream of the first strand nick, e.g., the genomic DNA priming the RNA template, have at least 66% or 100% complementarity to the 3 nt of homology in the RNA template. In some embodiments, the RT domain initiates TPRT when there are less than 5 nt mismatched (e.g., less than 1, 2, 3, 4, or 5 nt mismatched) between the template RNA homology and the target DNA priming reverse transcription. In some embodiments, the RT domain is modified such that the stringency for mismatches in priming the TPRT reaction is increased, e.g., wherein the RT domain does not tolerate any mismatches or tolerates fewer mismatches in the priming region relative to a wild-type (e.g., unmodified) RT domain. In some embodiments, the RT domain comprises a HIV-1 RT domain. In embodiments, the HIV-1 RT domain initiates lower levels of synthesis even with three nucleotide mismatches relative to an alternative RT domain (e.g., as described by Jamburuthugoda and Eickbush J Mol Biol 407(5):661-672 (2011); incorporated herein by reference in its entirety).


In some embodiments, the RT domain forms a dimer (e.g., a heterodimer or homodimer). In some embodiments, the RT domain is monomeric. In some embodiments, an RT domain, e.g., a retroviral RT domain, naturally functions as a monomer or as a dimer (e.g., heterodimer or homodimer). In some embodiments, an RT domain naturally functions as a monomer, e.g., is derived from a virus wherein it functions as a monomer. Exemplary monomeric RT domains, their viral sources, and the RT signatures associated with them can be found in Table 30 with descriptions of domain signatures in Table 32. In some embodiments, the RT domain of a system described herein comprises an amino acid sequence of Table 30, or a functional fragment or variant thereof, or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity thereto. In embodiments, the RT domain is selected from an RT domain from murine leukemia virus (MLV; sometimes referred to as MoMLV) (e.g., P03355), porcine endogenous retrovirus (PERV) (e.g., UniProt Q4VFZ2), mouse mammary tumor virus (MMTV) (e.g., UniProt P03365), Mason-Pfizer monkey virus (MPMV) (e.g., UniProt P07572), bovine leukemia virus (BLV) (e.g., UniProt P03361), human T-cell leukemia virus-1 (HTLV-1) (e.g., UniProt P03362), human foamy virus (HFV) (e.g., UniProt P14350), simian foamy virus (SFV) (e.g., UniProt P23074), or bovine foamy/syncytial virus (BFV/BSV) (e.g., UniProt 041894), or a functional fragment or variant thereof (e.g., an amino acid sequence having at least 70%, 80%, 90%, 95%, or 99% identity thereto). In some embodiments, an RT domain is dimeric in its natural functioning. Exemplary dimeric RT domains, their viral sources, and the RT signatures associated with them can be found in Table 31 with descriptions of domain signatures in Table 32. In some embodiments, the RT domain of a system described herein comprises an amino acid sequence of Table 31, or a functional fragment or variant thereof, or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity thereto. In some embodiments, the RT domain is derived from a virus wherein it functions as a dimer. In embodiments, the RT domain is selected from an RT domain from avian sarcoma/leukemia virus (ASLV) (e.g., UniProt A0A142BKH1), Rous sarcoma virus (RSV) (e.g., UniProt P03354), avian myeloblastosis virus (AMV) (e.g., UniProt Q83133), human immunodeficiency virus type I (HIV-1) (e.g., UniProt P03369), human immunodeficiency virus type II (HIV-2) (e.g., UniProt P15833), simian immunodeficiency virus (SIV) (e.g., UniProt P05896), bovine immunodeficiency virus (BIV) (e.g., UniProt P19560), equine infectious anemia virus (EIAV) (e.g., UniProt P03371), or feline immunodeficiency virus (FIV) (e.g., UniProt P16088) (Herschhorn and Hizi Cell Mol Life Sci 67(16):2717-2747 (2010)), or a functional fragment or variant thereof (e.g., an amino acid sequence having at least 70%, 80%, 90%, 95%, or 99% identity thereto). Naturally heterodimeric RT domains may, in some embodiments, also be functional as homodimers. In some embodiments, dimeric RT domains are expressed as fusion proteins, e.g., as homodimeric fusion proteins or heterodimeric fusion proteins. In some embodiments, the RT function of the system is fulfilled by multiple RT domains (e.g., as described herein). In further embodiments, the multiple RT domains are fused or separate, e.g., may be on the same polypeptide or on different polypeptides.


In some embodiment, a GeneWriter described herein comprises an integrase domain, e.g., wherein the integrase domain may be part of the RT domain. In some embodiments, an RT domain (e.g., as described herein) comprises an integrase domain. In some embodiments, an RT domain (e.g., as described herein) lacks an integrase domain, or comprises an integrase domain that has been inactivated by mutation or deleted. In some embodiment, a GeneWriter described herein comprises an RNase H domain, e.g., wherein the RNase H domain may be part of the RT domain. In some embodiments, an RT domain (e.g., as described herein) comprises an RNase H domain, e.g., an endogenous RNAse H domain or a heterologous RNase H domain. In some embodiments, an RT domain (e.g., as described herein) lacks an RNase H domain. In some embodiments, an RT domain (e.g., as described herein) comprises an RNase H domain that has been added, deleted, mutated, or swapped for a heterologous RNase H domain. In some embodiments, mutation of an RNase H domain yields a polypeptide exhibiting lower RNase activity, e.g., as determined by the methods described in Kotewicz et al. Nucleic Acids Res 16(1):265-277 (1988) (incorporated herein by reference in its entirety), e.g., lower by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to an otherwise similar domain without the mutation. In some embodiments, RNase H activity is abolished.


In some embodiments, an RT domain is mutated to increase fidelity compared to to an otherwise similar domain without the mutation. For instance, in some embodiments, a YADD (SEQ ID NO: 1539) or YMDD (SEQ ID NO: 1540) motif in an RT domain (e.g., in a reverse transcriptase) is replaced with YVDD (SEQ ID NO: 1541). In embodiments, replacement of the YADD (SEQ ID NO: 1539) or YMDD (SEQ ID NO: 1540) or YVDD (SEQ ID NO: 1541) results in higher fidelity in retroviral reverse transcriptase activity (e.g., as described in Jamburuthugoda and Eickbush J Mol Biol 2011; incorporated herein by reference in its entirety).


In some embodiments, the reverse transcriptase domain is one selected from an element of Table 1 or Table 3.









TABLE 2







InterPro descriptions of signatures present in reverse transcriptases in

















RT


Protein
Type
Accession
UniProt
Sequence
signatures





MarathonRT
Group
CBK92290.1
D4JMT
MDTSNLMEQILSSDNLNRAYLQVVR
IPR000477



II

6
NKGAEGVDGMKYTELKEHLAKNGET
PF00078,



intron


IKGQLRTRKYKPQPARRVEIPKPDG
cd01651






GVRNLGVPTVTDRFIQQAIAQVLTP







IYEEQFHDHSYGFRPNRCAQQAILT







ALNIMNDGNDWIVDIDLEKFFDTVN







HDKLMTLIGRTIKDGDVISIVRKYL







VSGIMIDDEYEDSIVGTPQGGNLSP







LLANIMLNELDKEMEKRGLNFVRYA







DDCIIMVGSEMSANRVMRNISRFIE







EKLGLKVNMTKSKVDRPSGLKYLGF







GFYFDPRAHQFKAKPHAKSVAKFKK







RMKELTCRSWGVSNSYKVEKLNQLI







RGWINYFKIGSMKTLCKELDSRIRY







RLRMCIWKQWKTPQNQEKNLVKLGI







DRNTARRVAYTGKRIAYVCNKGAVN







VAISNKRLASFGLISMLDYYIEKCV







TC







(SEQ ID NO: 1542)






TGIRT,
Group
AAT72329.1
Q6DKY2
MALLERILADRNLITALKRVEANQG
IPR000477


trt
II


APGIGDVSTDQLRDIYRAHWSTIRA
PF00078,



intron


QLLAGTYRPAPVRRVGIPKGPGGTR
cd01651






QLGITPVVDRLIQQIALQELTPIFD







PDFSPSSFGFRPGRNAHDAVRQAQG







YIQEYGRYVVDMDLKEFFDRVNHDL







IMSRVARKVDKKRVLKLIRYALQAG







VMIEGVKVQTEEGTQPGGPLSPLLA







NILLDDLDKELEKRGLKFCYRADDC







NIYVSKLRAGQRVKQSIQRFLEKTL







KLKVNEEKSVADRPWKRAFGLFSFT







PERKARIRLAPRSIQRLKQRIRQLT







NPNWSISMPREIHRVNQYVGMWIGY







FRLVTEPSVLQTIEGWIRRRLRLCW







QLQWKRVRTRIRELRALGLKETAVM







EIANRTKGAWRTTKPQTLHQALGKY







TWTAQGLKTSLQRYFELRQG







(SEQ ID NO: 1543)






LtrA
Group
AAB06503.1
P0A3U0
MKPTMAILERISKNSQENIDEVFTR
IPR000477,



II


LYRYLLRPDIYYVAYQNLYSNKGAS
PF00078,



intron


TKGILDDTADGFSEEKIKKIIQSLK
cd01651






DGTYYPQPVRRMYIAKKNSKKMRPL







GIPTFTDKLIQEAVRIILESIYEPV







FEDVSHGFRPQRSCHTALKTIKREF







GGARWFVEGDIKGCFDNIDHVTLIG







LINLKIKDMKMSQLIYKFLKAGYLE







NWQYHKTYSGTPQGGILSPLLANIY







LHELDKFVLQLKMKFDRESPERITP







EYRELHNEIKRISHRLKKLEGEEKA







KVLLEYQEKRKRLPTLPCTSQTNKV







LKYVRYADDFIISVKGSKEDCQWIK







EQLKLFIHNKLKMELSEEKTLITHS







SQPARFLGYDIRVRRSGTIKRSGKV







KKRTLNGSVELLIPLQDKIRQFIFD







KKIAIQKKDSSWFPVHRKYLIRSTD







LEIITIYNSELRGICNYYGLASNFN







QLNYFAYLMEYSCLKTIASKHKGTL







SKTISMFKDGSGSWGIPYEIKQGKQ







RRYFANFSECKSPYQFTDEISQAPV







LYGYARNTLENRLKAKCCELCGTSD







ENTSYEIHHVNKVKNLKGKEKWEMA







MIAKQRKTLVVCFHCHRHVIHKHK







(SEQ ID NO: 1544)






R2Bm
non-
AAB59214.1
V9H052
MMASTALSLMGRCNPDGCTRGKHVT
IPR000477



LTR


AAPMDGPRGPSSLAGTFGWGLAIPA
PF00078,



retro-


GEPCGRVCSPATVGFFPVAKKSNKE
cd01650



transposon


NRPEASGLPLESERTGDNPTVRGSA







GADPVGQDAPGWTCQFCERTFSTNR







GLGVHKRRAHPVETNTDAAPMMVKR







RWHGEEIDLLARTEARLLAERGQCS







GGDLFGALPGFGRTLEAIKGQRRRE







PYRALVQAHLARFGSQPGPSSGGCS







AEPDFRRASGAEEAGEERCAEDAAA







YDPSAVGQMSPDAARVLSELLEGAG







RRRACRAMRPKTAGRRNDLHDDRTA







SAHKTSRQKRRAEYARVQELYKKCR







SRAAAEVIDGACGGVGHSLEEMETY







WRPILERVSDAPGPTPEALHALGRA







EWHGGNRDYTQLWKPISVEEIKASR







FDWRTSPGPDGIRSGQWRAVPVHLK







AEMFNAWMARGEIPEILRQCRTVFV







PKVERPGGPGEYRPISIASIPLRHF







HSILARRLLACCPPDARQRGFICAD







GTLENSAVLDAVLGDSRKKLRECHV







AVLDFAKAFDTVSHEALVELLRLRG







MPEQFCGYIAHLYDTASTTLAVNNE







MSSPVKVGRGVRQGDPLSPILFNVV







MDLILASLPERVGYRLEMELVSALA







YADDLVLLAGSKVGMQESISAVDCV







GRQMGLRLNCRKSAVLSMIPDGHRK







KHHYLTERTFNIGGKPLRQVSCVER







WRYLGVDFEASGCVTLEHSISSALN







NISRAPLKPQQRLEILRAHLIPRFQ







HGFVLGNISDDRLRMLDVQIRKAVG







QWLRLPADVPKAYYHAAVQDGGLAI







PSVRATIPDLIVRRFGGLDSSPWSV







ARAAAKSDKIRKKLRWAWKQLRRFS







RVDSTTORPSVRLFWREHLHASVDG







RELRESTRTPTSTKWIRERCAQITG







RDFVQFVHTHINALPSRIRGSRGRR







GGGESSLTCRAGCKVRETTAHILQQ







CHRTHGGRILRHNKIVSFVAKAMEE







NKWTVELEPRLRTSVGLRKPDIIAS







RDGVGVIVDVQVVSGQRSLDELHRE







KRNKYGNHGELVELVAGRLGLPKAE







CVRATSCTISWRGVWSLTSYKELRS







IIGLREPTLQIVPILALRGSHMNWT







RFNQMTSVMGGGVG







(SEQ ID NO: 1545)






LINE-1
non-
AAC51271.1
O00370
MTGSNSHITILTLNVNGLNSPIKRH
IPR000477



LTR


RLASWIKSQDPSVCCIQETHLTCRD
PF00078,



retro-


THRLKIKGWRKIYQANGKQKKAGVA
cd01650



transposon


ILVSDKTDFKPTKIKRDKEGHYIMV







KGSIQQEELTILNIYAPNTGAPRFI







KQVLSDLQRDLDSHTLIMGDFNTPL







SILDRSTRQKVNKDTQELNSALHQT







DLIDIYRTLHPKSTEYTFFSAPHHT







YSKIDHIVGSKALLSKCKRTEIITN







YLSDHSAIKLELRIKNLTQSRSTTW







KLNNLLLNDYWVHNEMKAEIKMFFE







TNENKDTTYQNLWDAFKAVCRGKFI







ALNAYKRKQERSKIDTLTSQLKELE







KQEQTHSKASRRQEITKIRAELKEI







ETQKTLQKINESRSWFFERINKIDR







PLARLIKKKREKNQIDTIKNDKGDI







TTDPTEIQTTIREYYKHLYANKLEN







LEEMDTFLDTYTLPRLNQEEVESLN







RPITGSEIVAIINSLPTKKSPGPDG







FTAEFYQRYKEELVPFLLKLFQSIE







KEGILPNSFYEASIILIPKPGRDTT







KKENFRPISLMNIDAKILNKILANR







IQQHIKKLIHHDQVGFIPGMQGWFN







IRKSINVIQHINRAKDKNHVIISID







AEKAFDKIQQPFMLKTLNKLGIDGM







YLKIIRAIYDKPTANIILNGQKLEA







FPLKTGTRQGCPLSPLLFNIVLEVL







ARAIRQEKEIKGIQLGKEEVKLSLF







ADDMIVYLENPIVSAQNLLKLISNF







SKVSGYKINVQKSQAFLYNNNRQTE







SQIMGELPFTIASKRIKYLGIQLTR







DVKDLFKENYKPLLKEIKEDTNKWK







NIPCSWVGRINIVKMAILPKVIYRF







NAIPIKLPMTFFTELEKTTLKFIWN







QKRARIAKSILSQKNKAGGITLPDF







KLYYKATVTKTAWYWYQNRDIDQWN







RTEPSEIMPHIYNYLIFDKPEKNKQ







WGKDSLLNKWCWENWLAICRKLKLD







PFLTPYTKINSRWIKDLNVKPKTIK







TLEENLGITIQDIGVGKDFMSKTPK







AMATKDKIDKWDLIKLKSFCTAKET







TIRVNRQPTTWEKIFATYSSDKGLI







SRIYNELKQIYKKKTNNPIKKWAKD







MNRHFSKEDIYAAKKHMKKCSSSLA







IREMQIKTTMRYHLTPVRMAIIKKS







GNNRCWRGCGEIGTLVHCWWDCKLV







QPLWKSVWRFLRDLELEIPFDPAIP







LLGIYPKDYKSCCYKDTCTRMFIAA







LFTIAKTWNQPNCPTMIDWIKKMWH







IYTMEYYAAIKNDEFISFVGTWMKL







ETIILSKLSQEQKTKHRIFSLIGGN







(SEQ ID NO: 1546)






Penelope
non-
AAL14979.1
Q95VB5
MERSPEPSININGRHAVCTATNMSY
IPR000477,



LTR


AKIKTKYKDSKRTINKFQLTLVKLT
PF00078,



Retro-


KLKSSLKFLLKCRKSNLIPNFIKNL
cd00304



transp


TQHLTILTTDNKTHPDITRTLTRHT




oson


HFYHTKILNLLIKHKHNLLQEQTKH







MQKAKTNIEQLMTTDDAKAFFESER







NIENKITTTLKKRQETKHDKLRDQR







NLALADNNTQREWFVNKTKIEFPPN







VVALLAKGPKFALPISKRDFPLLKY







IADGEELVQTIKEKETQESARTKFS







LLVKEHKTKNNQNSRDRAILDTVEQ







TRKLLKENINIKILSSDKGNKTVAM







DEDEYKNKMTNILDDLCAYRTLRLD







PTSRLQTKNNTFVAQLFKMGLISKD







ERNKMTTTTAVPPRIYGLPKIHKEG







TPLRPICSSIGSPSYGLCKYIIQIL







KNLTMDSRYNIKNAVDFKDRVNNSQ







IREEETLVSFDVVSLFPSIPIELAL







DTIRQKWTKLEEHTNIPKQLFMDIV







RFCIEENRYFKYEDKIYTQLKGMPM







GSPASPVIADILMEELLDKITDKLK







IKPRLLTKYVDDLFAITNKIDVENI







LKELNSFHKQIKFTMELEKDGKLPF







LDSIVSRMDNTLKIKWYRKPIASGR







ILNFNSNHPKSMIINTALGCMNRMM







KISDTIYHKEIEHEIKELLTKNDFP







PNIIKTLLKRRQIERKKPTEPAKIY







KSLIYVPRLSERLTNSDCYNKQDIK







VAHKPTNTLQKFFNKIKSKIPMIEK







SNVVYQIPCGGDNNNKCNSVYIGTT







KSKLKTRISQHKSDFKLRHQNNIQK







TALMTHCIRSNHTPNFDETTILQQE







QHYNKRHTLEMLHIINTPTYKRLNY







KTDTENCAHLYRHLLNSQTTSVTIS







TSKSADV







(SEQ ID NO: 1547)






M-
Retro
ADS42990.1
P03355
TLNIEDEHRLHETSKEPDVSLGSTW
IPR000477


MLV
virus

[660-
LSDFPQAWAETGGMGLAVRQAPLII
PF00078,


RT


1330]
PLKATSTPVSIKQYPMSQEARLGIK
cd03715






PHIQRLLDQGILVPCQSPWNTPLLP







VKKPGTNDYRPVQDLREVNKRVEDI







HPTVPNPYNLLSGLPPSHQWYTVLD







LKDAFFCLRLHPTSQPLFAFEWRDP







EMGISGQLTWTRLPQGFKNSPTLFD







EALHRDLADFRIQHPDLILLQYVDD







LLLAATSELDCQQGTRALLQTLGNL







GYRASAKKAQICQKQVKYLGYLLKE







GQRWLTEARKETVMGQPTPKTPRQL







REFLGTAGFCRLWIPGFAEMAAPLY







PLTKTGTLFNWGPDQQKAYQEIKQA







LLTAPALGLPDLTKPFELFVDEKQG







YAKGVLTQKLGPWRRPVAYLSKKLD







PVAAGWPPCLRMVAAIAVLTKDAGK







LTMGQPLVILAPHAVEALVKQPPDR







WLSNARMTHYQALLLDTDRVQFGPV







VALNPATLLPLPEEGLQHNCLDILA







EAHGTRPDLTDQPLPDADHTWYTDG







SSLLQEGQRKAGAAVTTETEVIWAK







ALPAGTSAQRAELIALTQALKMAEG







KKLNVYTDSRYAFATAHIHGEIYRR







RGLLTSEGKEIKNKDEILALLKALF







LPKRLSIIHCPGHQKGHSAEARGNR







MADQAARKAAITETPDTSTLL







(SEQ ID NO: 1548)






RSV
Retro
AAC82561.1
P03354
TVALHLAIPLKWKPDHTPVWIDQWP
IPR000477


RT
virus

[709-
LPEGKLVALTQLVEKELQLGHIEPS
PF00078,





1567]
LSCWNTPVFVIRKASGSYRLLHDLR
cd01645






AVNAKLVPFGAVQQGAPVLSALPRG







WPLMVLDLKDCFFSIPLAEQDREAF







AFTLPSVNNQAPARRFQWKVLPQGM







TCSPTICQLVVGQVLEPLRLKHPSL







CMLHYMDDLLLAASSHDGLEAAGEE







VISTLERAGFTISPDKVQREPGVQY







LGYKLGSTYVAPVGLVAEPRIATLW







DVQKLVGSLQWLRPALGIPPRLMGP







FYEQLRGSDPNEAREWNLDMKMAWR







EIVRLSTTAALERWDPALPLEGAVA







RCEQGAIGVLGQGLSTHPRPCLWLF







STQPTKAFTAWLEVLTLLITKLRAS







AVRTFGKEVDILLLPACFREDLPLP







EGILLALKGFAGKIRSSDTPSIFDI







ARPLHVSLKVRVTDHPVPGPTVFTD







ASSSTHKGVVVWREGPRWEIKEIAD







LGASVQQLEARAVAMALLLWPTTPT







NVVTDSAFVAKMLLKMGQEGVPSTA







AAFILEDALSQRSAMAAVLHVRSHS







EVPGFFTEGNDVADSQATFQAYPLR







EAKDLHTALHIGPRALSKACNISMQ







QAREVVQTCPHCNSAPALEAGVNPR







GLGPLQIWQTDFTLEPRMAPRSWLA







VTVDTASSAIVVTQHGRVTSVAVQH







HWATAIAVLGRPKAIKTDNGSCFTS







KSTREWLARWGIAHTTGIPGNSQGQ







AMVERANRLLKDRIRVLAEGDGFMK







RIPTSKQGELLAKAMYALNHFERGE







NTKTPIQKHWRPTVLTEGPPVKIRI







ETGEWEKGWNVLVWGRGYAAVKNRD







TDKVIWVPSRKVKPDITQKDEVTKK







DEASPLFAG(SEQ ID NO: 1549)






AMV
Retro
HW606680.1
—
TVALHLAIPLKWKPNHTPVWIDQWP
IPR000477,


RT
virus


LPEGKLVALTQLVEKELQLGHIEPS
PF00078,






LSCWNTPVFVIRKASGSYRLLHDLR
cd01645






AVNAKLVPFGAVQQGAPVLSALPRG







WPLMVLDLKDCFFSIPLAEQDREAF







AFTLPSVNNQAPARRFQWKVLPQGM







TCSPTICQLIVGQILEPLRLKHPSL







RMLHYMDDLLLAASSHDGLEAAGEE







VISTLERAGFTISPDKVQREPGVQY







LGYKLGSTYVAPVGLVAEPRIATLW







DVQKLVGSLQWLRPALGIPPRLMGP







FYEQLRGSDPNEAREWNLDMKMAWR







EIVQLSTTAALERWDPALPLEGAVA







RCEQGAIGVLGQGLSTHPRPCLWLF







STQPTKAFTAWLEVLTLLITKLRAS







AVRTFGKEVDILLLPACFREDLPLP







EGILLALRGFAGKIRSSDTPSIFDI







ARPLHVSLKVRVTDHPVPGPTVFTD







ASSSTHKGVVVWREGPRWEIKEIAD







LGASVQQLEARAVAMALLLWPTTPT







NVVTDSAFVAKMLLKMGQEGVPSTA







AAFILEDALSQRSAMAAVLHVRSHS







EVPGFFTEGNDVADSQATFQAY







(SEQ ID NO: 1550)






HIV
Retro
AAB50259.1
P04585
PISPIETVPVKLKPGMDGPKVKQWP
IPR000477,


RT
virus

[588-
LTEEKIKALVEICTEMEKEGKISKI
PF00078,





1147]
GPENPYNTPVFAIKKKDSTKWRKLV
cd01645






DFRELNKRTQDFWEVQLGIPHPAGL







KKKKSVTVLDVGDAYFSVPLDEDFR







KYTAFTIPSINNETPGIRYQYNVLP







QGWKGSPAIFQSSMTKILEPFRKQN







PDIVIYQYMDDLYVGSDLEIGQHRT







KIEELRQHLLRWGLTTPDKKHQKEP







PFLWMGYELHPDKWTVQPIVLPEKD







SWTVNDIQKLVGKLNWASQIYPGIK







VRQLCKLLRGTKALTEVIPLTEEAE







LELAENREILKEPVHGVYYDPSKDL







IAEIQKQGQGQWTYQIYQEPFKNLK







TGKYARMRGAHTNDVKQLTEAVQKI







TTESIVIWGKTPKFKLPIQKETWET







WWTEYWQATWIPEWEFVNTPPLVKL







WYQLEKEPIVGAETFYVDGAANRET







KLGKAGYVTNRGRQKVVTLTDTTNQ







KTELQAIYLALQDSGLEVNIVTDSQ







YALGIIQAQPDQSESELVNQHIEQL







IKKEKVYLAWVPAHKGIGGNEQVDK







LVSAGIRKVL







(SEQ ID NO: 1551)






Tyl
LTR
AAA66938.1
Q07163-
AVKAVKSIKPIRTTLRYDEAITYNK
IPR013103



Retro-

1
DIKEKEKYIEAYHKEVNQLLKMKTW
PF07727



transposon

[1218-
DTDEYYDRKEIDPKRVINSMFIFNK






1755]
KRDGTHKARFVARGDIQHPDTYDSG







MQSNTVHHYALMTSLSLALDNNYYI







TQLDISSAYLYADIKEELYIRPPPH







LGMNDKLIRLKKSLYGLKQSGANWY







ETIKSYLIQQCGMEEVRGWSCVFKN







SQVTICLFVDDMVLFSKNLNSNKRI







IEKLKMQYDTKIINLGESDEEIQYD







ILGLEIKYQRGKYMKLGMENSLTEK







IPKLNVPLNPKGRKLSAPGQPGLYI







DQDELEIDEDEYKEKVHEMQKLIGL







ASYVGYKFRFDLLYYINTLAQHILF







PSRQVLDMTYELIQFMWDTRDKQLI







WHKNKPTEPDNKLVAISDASYGNQP







YYKSQIGNIYLLNGKVIGGKSTKAS







LTCTSTTEAEIHAISESVPLLNNLS







YLIQELNKKPIIKGLLTDSRSTISI







IKSTNEEKFRNRFFGTKAMRLRDEV







SGNNLYVYYIETKKNIADVMTKPLP







IKTFKLLTNKWIH







(SEQ ID NO: 1552)






Brt
Diversity-
NP_
Q775D8
MGKRHRNLIDQITTWENLLDAYRKT
IPR000477,



Generating
958675.1

SHGKRRTWGYLEFKEYDLANLLALQ
PF00078,



retro-


AELKAGNYERGPYREFLVYEPKPRL
cd01646



element


ISALEFKDRLVQHALCNIVAPIFEA







GLLPYTYACRPDKGTHAGVCHVQAE







LRRTRATHFLKSDFSKFFPSIDRAA







LYAMIDKKIHCAATRRLLRVVLPDE







GVGIPIGSLTSQLFANVYGGAVDRL







LHDELKQRHWARYMDDIVVLGDDPE







ELRAVFYRLRDFASERLGLKISHWQ







VAPVSRGINFLGYRIWPTHKLLRKS







SVKRAKRKVANFIKHGEDESLQRFL







ASWSGHAQWADTHNLFTWMEEQYGI







ACH







(SEQ ID NO: 1553)






Tyl
LTR
AAA66938.1
Q07163-
AVKAVKSIKPIRTTLRYDEAITYNK
IPR013103



Retro-

1[1218-
DIKEKEKYIEAYHKEVNQLLKMKTW
PF07727



transposon

1755]
DTDEYYDRKEIDPKRVINSMFIFNK







KRDGTHKARFVARGDIQHPDTYDSG







MQSNTVHHYALMTSLSLALDNNYYI







TQLDISSAYLYADIKEELYIRPPPH







LGMNDKLIRLKKSLYGLKQSGANWY







ETIKSYLIQQCGMEEVRGWSCVFKN







SQVTICLFVDDMVLFSKNLNSNKRI







IEKLKMQYDTKIINLGESDEEIQYD







ILGLEIKYQRGKYMKLGMENSLTEK







IPKLNVPLNPKGRKLSAPGQPGLYI







DQDELEIDEDEYKEKVHEMQKLIGL







ASYVGYKFRFDLLYYINTLAQHILF







PSRQVLDMTYELIQFMWDTRDKQLI







WHKNKPTEPDNKLVAISDASYGNQP







YYKSQIGNIYLLNGKVIGGKSTKAS







LTCTSTTEAEIHAISESVPLLNNLS







YLIQELNKKPIIKGLLTDSRSTISI







IKSTNEEKFRNRFFGTKAMRLRDEV







SGNNLYVYYIETKKNIADVMTKPLP







IKTFKLLTNKWIH







(SEQ ID NO: 1552)






Brt
Diversity-
NP_
Q775D8
MGKRHRNLIDQITTWENLLDAYRKT
IPR000477,



Generating
958675.1

SHGKRRTWGYLEFKEYDLANLLALQ
PF00078,



retro-


AELKAGNYERGPYREFLVYEPKPRL
cd01646



element


ISALEFKDRLVQHALCNIVAPIFEA







GLLPYTYACRPDKGTHAGVCHVQAE







LRRTRATHFLKSDFSKFFPSIDRAA







LYAMIDKKIHCAATRRLLRVVLPDE







GVGIPIGSLTSQLFANVYGGAVDRL







LHDELKQRHWARYMDDIVVLGDDPE







ELRAVFYRLRDFASERLGLKISHWQ







VAPVSRGINFLGYRIWPTHKLLRKS







SVKRAKRKVANFIKHGEDESLQRFL







ASWSGHAQWADTHNLFTWMEEQYGI







ACH







(SEQ ID NO: 1553)






RT86
Retron
AAA61471.1
P23070
MKSAEYLNTFRLRNLGLPVMNNLHD
IPR000477,






MSKATRISVETLRLLIYTADFRYRI
PF00078,






YTVEKKGPEKRMRTIYQPSRELKAL
cd03487






QGWVLRNILDKLSSSPFSIGFEKHQ







SILNNATPHIGANFILNIDLEDFFP







SLTANKVFGVFHSLGYNRLISSVLT







KICCYKNLLPQGAPSSPKLANLICS







KLDYRIQGYAGSRGLIYTRYADDLT







LSAQSMKKVVKARDFLFSIIPSEGL







VINSKKTCISGPRSQRKVTGLVISQ







EKVGIGREKYKEIRAKIHHIFCGKS







SEIEHVRGWLSFILSVDSKSHRRLI







TYISKLEKKYGKNPLNKAKT







(SEQ ID NO: 1554)






TERT
Telomerase
AAG23289.1
014746
MPRAPRCRAVRSLLRSHYREVLPLA
IPR000477






TFVRRLGPQGWRLVQRGDPAAFRAL
PF00078,






VAQCLVCVPWDARPPPAAPSFRQVS
cd01648






CLKELVARVLQRLCERGAKNVLAFG







FALLDGARGGPPEAFTTSVRSYLPN







TVTDALRGSGAWGLLLRRVGDDVLV







HLLARCALFVLVAPSCAYQVCGPPL







YQLGAATQARPPPHASGPRRRLGCE







RAWNHSVREAGVPLGLPAPGARRRG







GSASRSLPLPKRPRRGAAPEPERTP







VGQGSWAHPGRTRGPSDRGFCVVSP







ARPAEEATSLEGALSGTRHSHPSVG







RQHHAGPPSTSRPPRPWDTPCPPVY







AETKHFLYSSGDKEQLRPSFLLSSL







RPSLTGARRLVETIFLGSRPWMPGT







PRRLPRLPQRYWQMRPLFLELLGNH







AQCPYGVLLKTHCPLRAAVTPAAGV







CAREKPQGSVAAPEEEDTDPRRLVQ







LLRQHSSPWQVYGFVRACLRRLVPP







GLWGSRHNERRFLRNTKKFISLGKH







AKLSLQELTWKMSVRDCAWLRRSPG







VGCVPAAEHRLREEILAKFLHWLMS







VYVVELLRSFFYVTETTFQKNRLFF







YRKSVWSKLQSIGIRQHLKRVQLRE







LSEAEVRQHREARPALLTSRLRFIP







KPDGLRPIVNMDYVVGARTFRREKR







AERLTSRVKALFSVLNYERARRPGL







LGASVLGLDDIHRAWRTFVLRVRAQ







DPPPELYFVKVDVTGAYDTIPQDRL







TEVIASIIKPQNTYCVRRYAVVQKA







AHGHVRKAFKSHVSTLTDLQPYMRQ







FVAHLQETSPLRDAVVIEQSSSLNE







ASSGLFDVFLRFMCHHAVRIRGKSY







VQCQGIPQGSILSTLLCSLCYGDME







NKLFAGIRRDGLLLRLVDDFLLVTP







HLTHAKTFLRTLVRGVPEYGCVVNL







RKTVVNFPVEDEALGGTAFVQMPAH







GLFPWCGLLLDTRTLEVQSDYSSYA







RTSIRASLTFNRGFKAGRNMRRKLF







GVLRLKCHSLFLDLQVNSLQTVCTN







IYKILLLQAYRFHACVLQLPFHQQV







WKNPTFFLRVISDTASLCYSILKAK







NAGMSLGAKGAAGPLPSEAVQWLCH







QAFLLKLTRHRVTYVPLLGSLRTAQ







TQLSRKLPGTTLTALEAAANPALPS







DFKTILD







(SEQ ID NO: 1555)






Mauriceville
Retro
NC_0015
Q36578
MPNHRLPNCVSYLGENHELSWLHGM
cd00304


RT
plasmid
70.1

FGLLKRSNPQTGGILGWLNTGPNGF







VKYMMNLMGHARDKGDAKEYWRLGR







SLMKNEAFQVQAFNHVCKHWYLDYK







PHKIAKLLKEVREMVEIQPVCIDYK







RVYIPKANGKORPLGVPTVPWRVYL







HMWNVLLVWYRIPEQDNQHAYFPKR







GVFTAWRALWPKLDSQNIYEFDLKN







FFPSVDLAYLKDKLMESGIPQDISE







YLTVLNRSLVVLTSEDKIPEPHRDV







IFNSDGTPNPNLPKDVQGRILKDPD







FVEILRRRGFTDIATNGVPQGASTS







CGLATYNVKELFKRYDELIMYADDG







ILCRQDPSTPDFSVEEAGVVQEPAK







SGWIKQNGEFKKSVKFLGLEFIPAN







IPPLGEGEVKDYPRLRGATRNGSKM







ELSTELQFLCYLSYKLRIKVLRDLY







IQVLGYLPSVPLLRYRSLAEAINEL







SPKRITIGQFITSSFEEFTAWSPLK







RMGFFFSSPAGPTILSSIFNNSTNL







QEPSDSRLLYRKGSWVNIRFAAYLY







SKLSEEKHGLVPKFLEKLREINFAL







DKVDVTEIDSKLSRLMKFSVSAAYD







EVGTLALKSLFKFRNSERESIKASF







KQLRENGKIAEFSEARRLWFEILKL







IRLDLFNASSLACDDLLSHLQDRRS







IKKWGSSDVLYLKSQRLMRTNKKQL







QLDFEKKKNSLKKKLIKRRAKELRD







TFKGKENKEA







(SEQ ID NO: 1556)






RTX
Engineered
QFN49000.1
—
MILDTDYITEDGKPVIRIFKKENGE
IPR006134



polymerase


FKIEYDRTFEPYLYALLKDDSAIEE
PF00136,






VKKITAERHGTVVTVKRVEKVQKKF
cd05536






LGRPVEVWKLYFTHPQDVPAIMDKI







REHPAVIDIYEYDIPFAIRYLIDKG







LVPMEGDEELKLLAFDIETLYHEGE







EFAEGPILMISYADEEGARVITWKN







VDLPYVDVVSTEREMIKRFLRVVKE







KDPDVLITYNGDNFDFAYLKKRCEK







LGINFALGRDGSEPKIQRMGDRFAV







EVKGRIHFDLYPVIRRTINLPTYTL







EAVYEAVFGQPKEKVYAEEITTAWE







TGENLERVARYSMEDAKVTYELGKE







FLPMEAQLSRLIGQSLWDVSRSSTG







NLVEWFLLRKAYERNELAPNKPDEK







ELARRHQSHEGGYIKEPERGLWENI







VYLDFRSLYPSIIITHNVSPDTLNR







EGCKEYDVAPQVGHRFCKDFPGFIP







SLLGDLLEERQKIKKRMKATIDPIE







RKLLDYRQRAIKILANSLYGYYGYA







RARWYCKECAESVIAWGREYLTMTI







KEIEEKYGFKVIYSDTDGFFATIPG







ADAETVKKKAMEFLKYINAKLPGAL







ELEYEGFYKRGLFVTKKKYAVIDEE







GKITTRGLEIVRRDWSEIAKETQAR







VLEALLKDGDVEKAVRIVKEVTEKL







SKYEVPPEKLVIHKQITRDLKDYKA







TGPHVAVAKRLAARGVKIRPGTVIS







YIVLKGSGRIVDRAIPFDEFDPTKH







KYDAEYYIEKQVLPAVERILRAFGY







RKEDLRYQKTRQVGLSARLKPKGTL







EGSSHHHHHH







(SEQ ID NO: 1557)
















TABLE 2







InterPro descriptions of signatures present in reverse transcriptases in


Table 1.










Signature
Database
Short Name
Description





cd00304
CDD
RT like
RT_like: Reverse transcriptase





(RT, RNA-dependent DNA





polymerase)_like family. An RT





gene is usually indicative of a





mobile element such as a





retrotransposon or retrovirus. RTs





occur in a variety of mobile





elements, including





retrotransposons, retroviruses,





group II introns, bacterial





msDNAs, hepadnaviruses, and





caulimoviruses. These elements





can be divided into two major





groups. One group contains





retroviruses and DNA viruses





whose propagation involves an





RNA intermediate. They are





grouped together with





transposable elements containing





long terminal repeats (LTRs). The





other group, also called poly(A)-





type retrotransposons, contain





fungal mitochondrial introns and





transposable elements that lack





LTRs. [PMID: 1698615, PMID:





8828137, PMID: 10669612,





PMID: 9878607, PMID:





7540934, PMID: 7523679,





PMID: 8648598]


cd01645
CDD
RT Rtv
RT_Rtv: Reverse transcriptases





(RTs) from retroviruses (Rtvs).





RTs catalyze the conversion of





single-stranded RNA into double-





stranded viral DNA for





integration into host





chromosomes. Proteins in this





subfamily contain long terminal





repeats (LTRs) and are





multifunctional enzymes with





RNA-directed DNA polymerase,





DNA directed DNA polymerase,





and ribonuclease hybrid (RNase





H) activities. The viral RNA





genome enters the cytoplasm as





part of a nucleoprotein complex,





and the process of reverse





transcription generates in the





cytoplasm forming a linear DNA





duplex via an intricate series of





steps. This duplex DNA is





colinear with its RNA template,





but contains terminal duplications





known as LTRs that are not





present in viral RNA. It has been





proposed that two specialized





template switches, known as





strand-transfer reactions or





“jumps”, are required to generate





the LTRs. [PMID: 9831551,





PMID: 15107837, PMID:





11080630, PMID: 10799511,





PMID: 7523679, PMID:





7540934, PMID: 8648598,





PMID: 1698615]


cd01646
CDD
RT_Bac_
RT Bac retron I: Reverse




retron I
transcriptases (RTs) in bacterial





retrotransposons or retrons. The





polymerase reaction of this





enzyme leads to the production of





a unique RNA-DNA complex





called msDNA (multicopy single-





stranded (ss)DNA) in which a





small ssDNA branches out from a





small ssRNA molecule via a 2′-





5′ phosphodiester linkage.





Bacterial retron RTs produce





cDNA corresponding to only a





small portion of the retron





genome. [PMID: 1698615,





PMID: 16093702, PMID:





8828137]


cd01648
CDD
TERT
TERT: Telomerase reverse





transcriptase (TERT). Telomerase





is a ribonucleoprotein (RNP) that





synthesizes telomeric DNA





repeats. The telomerase RNA





subunit provides the template for





synthesis of these repeats. The





catalytic subunit of RNP is





known as telomerase reverse





transcriptase (TERT). The reverse





transcriptase (RT) domain is





located in the C-terminal region





of the TERT polypeptide. Single





amino acid substitutions in this





region lead to telomere





shortening and senescence.





Telomerase is an enzyme that, in





certain cells, maintains the physical





ends of chromosomes (telomeres)





during replication. In somatic cells,





replication of the lagging strand





requires the continual presence of





an RNA primer approximately





200 nucleotides upstream, which





is complementary to the template





strand. Since there is a region of





DNA less than 200 base pairs





from the end of the chromosome





where this is not possible, the





chromosome is continually





shortened. However, a surplus of





repetitive DNA at the





chromosome ends protects against





the erosion of gene-encoding





DNA. Telomerase is not normally





expressed in somatic cells. It





has been suggested that





exogenous TERT may extend the





lifespan of, or even immortalize,





the cell. However, recent studies





have shown that telomerase





activity can be induced by a





number of oncogenes.





Conversely, the oncogene c-myc





can be activated in human TERT





immortalized cells. Sequence





comparisons place the telomerase





proteins in the RT family but





reveal hallmarks that distinguish





them from retroviral and





retrotransposon relatives. [PMID:





9110970, PMID: 9288757,





PMID: 9389643, PMID:





9671703, PMID: 9671704,





PMID: 10333526, PMID:





11250070, PMID: 15363846,





PMID: 16416120, PMID:





16649103, PMID: 16793225,





PMID: 10860859, PMID:





9252327, PMID: 11602347,





PMID: 1698615, PMID:





8828137, PMID: 10866187]


cd01650
CDD
RT_nL
RT_nLTR: Non-LTR (long




TR_like
terminal repeat) retrotransposon





and non-LTR retrovirus reverse





transcriptase (RT). This subfamily





contains both non-LTR





retrotransposons and non-LTR





retrovirus RTs. RTs catalyze the





conversion of single-stranded





RNA into double-stranded DNA





for integration into host





chromosomes. RT is a





multifunctional enzyme with





RNA-directed DNA polymerase,





DNA directed DNA polymerase





and ribonuclease hybrid (RNase





H) activities. [PMID: 1698615,





PMID: 10605110, PMID:





10628860, PMID: 11734649,





PMID: 12117499, PMID:





12777502, PMID: 14871946,





PMID: 15939396, PMID:





16271150, PMID: 16356661,





PMID: 2463954, PMID:





3040362, PMID: 3656436,





PMID: 7512193, PMID:





7534829, PMID: 7659515,





PMID: 8524653, PMID:





9190061, PMID: 9218812,





PMID: 9332379, PMID:





9364772, PMID: 8828137]


cd01651
CDD
RT_G2
RT_G2_intron: Reverse




intron
transcriptases (RTs) with group II





intron origin. RT transcribes





DNA using RNA as template.





Proteins in this subfamily are





found in bacterial and





mitochondrial group II introns.





Their most probable ancestor was





a retrotransposable element with





both gag-like and pol-like genes.





This subfamily of proteins





appears to have captured the RT





sequences from transposable





elements, which lack long





terminal repeats (LTRs). [PMID:





1698615, PMID: 8828137,





PMID: 12403467, PMID:





11058141, PMID: 11054545,





PMID: 10760141, PMID:





10488235, PMID: 9680217,





PMID: 9491607, PMID:





7994604, PMID: 7823908,





PMID: 3129199, PMID:





2531370, PMID: 2476655]


cd03487
CDD
RT_Bac_
RT Bac retron IL Reverse




retron_II
transcriptases (RTs) in bacterial





retrotransposons or retrons. The





polymerase reaction of this





enzyme leads to the production of





a unique RNA-DNA complex





called msDNA (multicopy single-





stranded (ss)DNA) in which a





small ssDNA branches out from a





small ssRNA molecule via a 2′-





5′ phosphodiester linkage.





Bacterial retron RTs produce





cDNA corresponding to only a





small portion of the retron





genome. [PMID: 1698615,





PMID: 8828137, PMID:





11292805, PMID: 9281493,





PMID: 2465092, PMID:





1722556, PMID: 1701261,





PMID: 1689062]


cd03715
CDD
RT_ZFREV_
RT_ZFREV_like: A subfamily of




like
reverse transcriptases (RTs) found





in sequences similar to the intact





endogenous retrovirus ZFERV





from zebrafish and to Moloney





murine leukemia virus RT. An RT





gene is usually indicative of a





mobile element such as a





retrotransposon or retrovirus.





RTs occur in a variety of mobile





elements, including





retrotransposons, retroviruses,





group II introns, bacterial





msDNAs, hepadnaviruses, and





caulimoviruses. These elements





can be divided into two major





groups. One group contains





retroviruses and DNA viruses





whose propagation involves an





RNA intermediate. They are





grouped together with





transposable elements containing





long terminal repeats (LTRs). The





other group, also called poly(A)-





type retrotransposons, contain





fungal mitochondrial introns and





transposable elements that lack





LTRs. Phylogenetic analysis





suggests that ZFERV belongs to a





distinct group of retroviruses.





[PMID: 14694121, PMID:





2410413, PMID: 9684890,





PMID: 10669612, PMID:





1698615, PMID: 8828137]


cd05536
CDD
POLBc_B3
DNA polymerase type-B B3





subfamily catalytic domain.





Archaeal proteins that are





involved in DNA replication are





similar to those from eukaryotes.





Some members of the archaea





also possess multiple family B





DNA polymerases (B1, B2 and





B3). So far there is no specific





function(s) has been assigned for





different members of the archaea





type B DNA polymerases.





Phylogenetic analyses of





eubacterial, archaeal, and





eukaryotic family B DNA





polymerases are support





independent gene duplications





during the evolution of archaeal





and eukaryotic family B DNA





polymerases. Structural





comparison of the thermostable





DNA polymerase type B to its





mesostable homolog suggests





several adaptations to high





temperature such as shorter loops,





disulfide bridges, and increasing





electrostatic interaction at





subdomain interfaces. [PMID:





10997874, PMID: 11178906,





PMID: 10860752, PMID:





10097083, PMID: 10545321]


cd05780
CDD
DNA_polB_
The 3′-5′ exonuclease domain of




Kod1_like_
archaeal family-B DNA




exo
polymerases with similarity to






Pyrococcus kodakaraensis






Kod1, including polymerases





from Desulfurococcus (D.





Tok Pol) and Thermococcus






gorgonarius (Tgo Pol). Kod1, D.





Tok Pol, and Tgo Pol are





thermostable enzymes that exhibit





both polymerase and 3′-5′





exonuclease activities. They are





family-B DNA polymerases.





Their amino termini harbor a





DEDDy-type DnaQ-like 3′-5′





exonuclease domain that contains





three sequence motifs termed





ExoI, ExoII and ExoIII, with a





specific YX(3)D pattern at





ExoIII. These motifs are clustered





around the active site and are





involved in metal binding and





catalysis. The exonuclease





domain of family B polymerases





contains a beta hairpin structure





that plays an important role in





active site switching in the event





of nucleotide misincorporation.





Members of this subfamily show





similarity to eukaryotic DNA





polymerases involved in DNA





replication. Some archaea possess





multiple family-B DNA





polymerases. Phylogenetic





analyses of eubacterial, archaeal,





and eukaryotic family-B DNA





polymerases support independent





gene duplications during the





evolution of archaeal and





eukaryotic family-B DNA





polymerases. [PMID: 18355915,





PMID: 16019029, PMID:





11178906, PMID: 10860752,





PMID: 10097083, PMID:





10545321, PMID: 9098062,





PMID: 12459442, PMID:





16230118, PMID: 11988770,





PMID: 11222749, PMID:





17098747, PMID: 8594362,





PMID: 9729885]


PF00078
Pfam
RVT 1
A reverse transcriptase gene is





usually indicative of a mobile





element such as a retrotransposon





or retrovirus. Reverse





transcriptases occur in a variety





of mobile elements, including





retrotransposons, retroviruses,





group II introns, bacterial





msDNAs, hepadnaviruses, and





caulimoviruses. [PMID: 1698615]


PF00136
Pfam
DNA_pol B
This region of DNA polymerase





B appears to consist of more than





one structural domain, possibly





including elongation, DNA-





binding and dNTP binding





activities. [PMID: 9757117,





PMID: 8679562]


PF07727
Pfam
RVT 2
A reverse transcriptase gene is





usually indicative of a mobile





element such as a retrotransposon





or retrovirus. Reverse





transcriptases occur in a variety





of mobile elements, including





retrotransposons, retroviruses,





group II introns, bacterial





msDNAs, hepadnaviruses, and





caulimoviruses. This Pfam entry





includes reverse transcriptases not





recognised by the Pfam: PF00078





model. [PMID: 1698615]


IPR000477
InterPro
RT_dom
The use of an RNA template to





produce DNA, for integration





into the host genome and





exploitation of a host cell, is a





strategy employed in the





replication of retroid elements,





such as the retroviruses and





bacterial retrons. The enzyme





catalysing polymerisation is an





RNA-directed DNA-polymerase,





or reverse trancriptase (RT)





(2.7.7.49). Reverse transcriptase





occurs in a variety of mobile





elements, including





retrotransposons, retroviruses,





group II introns [PMID:





12758069], bacterial msDNAs,





hepadnaviruses, and





caulimoviruses. Retroviral reverse





transcriptase is synthesised as





part of the POL polyprotein that





contains; an aspartyl protease, a





reverse transcriptase, RNase H





and integrase. POL polyprotein





undergoes specific enzymatic





cleavage to yield the mature





proteins. The discovery of





retroelements in the prokaryotes





raises intriguing questions





concerning their roles in bacteria





and the origin and evolution of





reverse transcriptases and





whether the bacterial reverse





transcriptases are older than





eukaryotic reverse transcriptases





[PMID: 8828137], Several crystal





structures of the reverse





transcriptase (RT) domain have





been determined [PMID:





1377403].


IPR006134
InterPro
DNA-dir_
DNA is the biological




DNA_pol_B_
information that instructs cells




multi_dom
how to exist in an ordered





fashion: accurate replication is





thus one of the most important





events in the life cycle of a cell.





This function is performed by





DNA-directed DNA-polymerases





2.7.7.7) by adding nucleotide





triphosphate(dNTP) residues to





the 5′ end of the growing chain





of DNA, using a complementary





DNA chain as a template. Small





RNA molecules are generally





used as primers for chain





elongation, although terminal





proteins may also be used for the





de novo synthesis of a DNA chain.





Even though there are 2 different





methods of priming, these are





mediated by 2 very similar





polymerases classes, A and B,





with similar methods of chain





elongation. A number of DNA





polymerases have been grouped





under the designation of DNA





polymerase family B. Six regions





of similarity (numbered from I to





VI) are found in all or a subset of





the B family polymerases. The





most conserved region (I) includes





a conserved tetrapeptide with two





aspartate residues. It has been





suggested that it may be involved





in binding a magnesium ion. All





sequencesin the B family contain





a characteristic DTDS motif (SEQ





ID NO: 1558), and possess many





functional domains, including a





5′-3′ elongation domain, a 3′-5′





exonuclease domain [PMID:





8679562], a DNA binding





domain, and binding domains for





both dNTP's and pyrophosphate





[PMID: 9757117], This domain





of DNA polymerase B appears to





consist of more than one





activities, possibly including





elongation, DNA-binding and





dNTP binding [PMID: 9757117].


IPR013103
InterPro
RVT_2
A reverse transcriptase gene is





usually indicative of a mobile





element such as a retrotransposon





or retrovirus. Reverse





transcriptases occur in a variety





of mobile elements, including





retrotransposons, retroviruses,





group II introns, bacterial





msDNAs, hepadnaviruses, and





caulimoviruses. This entry





includes reverse transcriptases





not recognised by IPR000477





[PMID: 1698615].









Table 3 (below) shows exemplary Gene Writer™ proteins and associated sequences from a variety of retrotransposases, identified using data mining. Column 1 indicates the family to which the retrotransposon belongs. Column 2 lists the element name. Column 3 indicates an accession number, if any. Column 4 lists an organism in which the retrotransposase is found. Column 5 lists the predicted 5′ untranslated region, and column 6 lists the predicted 3′ untranslated region; both are sequences that are predicted to allow the template RNA to bind the retrotransposase of column 7. (It is understood that columns 5-6 show the DNA sequence, and that an RNA sequence according to any of columns 5-6 would typically include uracil rather than thymidine.) Column 7 lists the predicted retrotransposase amino acid sequence.









TABLE 3







Exemplary Retrotransposon Sequences















3.

5.
6.
7.


1.
2.
Acces-
4.
Predicted
Predicted 
Predicted Amino


Family
Element
sion
Organism
5′UTR
3′UTR
Acid Sequence





R2
R2-
.

Taeniopygia

GTCTAGTTACAACTGGGCAT
TTCAGGTTATTTAGATGCTT
MASCPKPGPPVSAGAMSLES



1_TG


guttata

CGCTGCAGAGATCGCACCTC
AGTTTTTGTACCTTTCTTGT
GLTTHSVLAIERGPNSLANS






CTCGTGGTCCCGCTGGTAGC
TTTGTTTAGGATTTTGATAG
GSDFGGGGLGLPLRLLRVSV






CCTTCGAAGGGTGACTAAGT
TGTTAGTATTTTTATATTTT
GTQTSRSDWVDLVSWSHPGP






CGATCTCTGCCCCAGGTACG
TGTACGATTGCATAATGTTC
TSKSQQVDLVSLFPKHRVDL






GAGCCGTTGGGACTCACCAG
TTTTTTATACAGTTCTGTTT
LSKNDQVDLVAQFLPSKFPP






TCCAACGTAACTCCTGCCTA
TAATAAAATAGACGATAGCT
NLAENDLALLVNLEFYRSDL






AATTCGGTGAAACAAATTCC
AGAGACGTTAGGGCAGCCAC
HVYECVHFAAHWEGLSGLPE






TCGGTAAAAAGCCCC
AAGCCAGTTAGGTAGCGGAT
VYEQLAPQPCVGETLHSSLP






(SEQ ID NO: 1140)
AGTAGGTAGGAACAGACTTT
RDSELFVPEEGSSEKESEDA







TACTATTTCATAACGCGTCA
PKTSPPTPGKHGLEQTGEEK







ATTACCACCTGATTTGGACC
VMVTVPDKNPPCPCCGTRVN







AATTCACGGGATTTGTCCAA
SVLNLIEHLKVSHGKRGVCF







GGTGGACGGGCCACCTTTAC
RCAKCGKENSNYHSVVCHFP







TTAACCCGGAAAAGGAACAT
KCRGPETEKAPAGEWICEVC







ATATAATTTATGTGTGTTCG
NRDFTTKIGLGQHKRLAHPA







ATAAA
VRNQERIVASQPKETSNRGA







(SEQ ID NO: 1263)
HKRCWTKEEEELLIRLEAQF








EGNKNINKLIAEHITTKTAK








QISDKRRLLSRKPAEEPREE








PGTCHHTRRAAASLRTEPEM








SHHAQAEDRDNGPGRRPLPG








RAAAGGRTMDEIRRHPDKGN








GQQRPTKQKSEEQLQAYYKK








TLEERLSAGALNTFPRAFKQ








VMEGRDIKLVINQTAQDCFG








CLESISQIRTATRDKKDTVT








REKHPKKPFQKWMKDRAIKK








GNYLRFQRLFYLDRGKLAKI








ILDDIECLSCDIPLSEIYSV








FKTRWETTGSFKSLGDFKTY








GKADNTAFRELITAKEIEKN








VQEMSKGSAPGPDGITLGDV








VKMDPEFSRTMEIFNLWLTT








GKIPDMVRGCRTVLIPKSSK








PDRLKDINNWRPITIGSILL








RLFSRIVTARLSKACPLNPR








QRGFIRAAGCSENLKLLQTI








IWSAKREHRPLGVVFVDIAK








AFDTVSHQHIIHALQQREVD








PHIVGLVSNMYENISTYITT








KRNTHTDKIQIRVGVKQGDP








MSPLLFNLAMDPLLCKLEES








GKGYHRGQSSITAMAFADDL








VLLSDSWENMNTNISILETF








CNLTGLKTQGQKCHGFYIKP








TKDSYTINDCAAWTINGTPL








NMIDPGESEKYLGLQFDPWI








GIARSGLSTKLDFWLQRIDQ








APLKPLQKTDILKTYTIPRL








IYIADHSEVKTALLETLDQK








IRTAVKEWLHLPPCTCDAIL








YSSTRDGGLGITKLAGLIPS








VQARRLHRIAQSSDDTMKCF








MEKEKMEQLHKKLWIQAGGD








RENIPSIWEAPPSSEPPNNV








STNSEWEAPTQKDKFPKPCN








WRKNEFKKWTKLASQGRGIV








NFERDKISNHWIQYYRRIPH








RKLLTALQLRANVYPTREFL








ARGRQDQYIKACRHCDADIE








SCAHIIGNCPVTQDARIKRH








NYICELLLEEAKKKDWVVFK








EPHIRDSNKELYKPDLIFVK








DARALVVDVTVRYEAAKSSL








EEAAAEKVRKYKHLETEVRH








LTNAKDVTFVGFPLGARGKW








HQDNFKLLTELGLSKSRQVK








MAETFSTVALFSSVDIVHMF








ASRARKSMVM








(SEQ ID NO: 1016)





R2
R2-
.

Geospiza

AGACTTAAGTGAGTTTGGTT
GGTAGATAATCTTTGTATAG
VGLCPSPGVDGTHQPNDSFQ



1_


fortis

ACAACTGGGCATAGCTGCAG
TGGGGGGGGATCTCATGTAC
NFGETNFSVQVARLVTRNLA



Gfo


AGACCGCGCCTCCTCGCGGC
CGGGTTTCTTTTATTTGATT
PRSVRGNGFGSGMATHPVPA






CCCGCTGGTAAGCCCTTAAC
TTCAATAAAACAGACGGTAG
DESGHESDPFLVGRSCGQPA






AGGGTGACTAA
CTAGGTTCGCAAGGCAGCCA
RLTRQSVGTQTSRDDILPSK






(SEQ ID NO: 1141)
CAAGCCAAAGATAGGTAGGG
TTKLTENELDLLVNFSLELY







TGCTCATAGTGAGTAGGGAC
RSDLQGFVQEGIHFSVNREV







AGTGCCTTTTGATTCACAAC
LEGFPEVYEQPAPQPAVGDD







GCGTCAATACCATCTGACAC
LNTSLPPDNNICVLEKGSSE







GGATACCCTTACCGGACTTG
AVEDGTPEVAHPVPETQGKE







TCATGATCTCCCAGACTTGT
SPNNIVMVTLPNKNPPCPCC







CCAAGGTGGACGGGCCACCT
RVRLHSVLALIEHLKGSHGK







TTACTTAACCCGGAAAAGGA
KRACFRCVKCGRENFNYHST







ACATATATTAATTATATGTG
VCHIAKCKGPKVEKAPVGEW







TTCGGAAAA
ICEVCGRDFTTKIGLGQHKR







(SEQ ID NO: 1264)
LAHPLVRNQERIDASQPKET








SNRGAHKRCWTKEEEEMLIK








LEVQFEGHRNINKLIAEHLT








TKTSKQISDKRRLLPRKQLT








DLSKGVAGQKVLDPGLSHQP








QLGVVDNGLGGGHLPGGPAA








EGRTIEPLGHHLDKDNGHRE








IADQHKAGRLQAHYRKKIRK








RLSEGMISNFPEVFEQLLDC








QEAQPLINQAAQDCFGCLDS








ASQIRKALRKQNTQKDQGDQ








PKRPAQKWMKKRAVKRGHFL








RFQKLFHLDRGKLAKIILDD








VECLSCDIPPSEIYSVFKAR








WETPGQFAGLGDFEINRKAN








NKAFRDLITAKEILKNVREM








TKGSAPGPDGIALGDIRKMD








PEYTRTAELFNLWLTSGEIP








DMVRGCRTVLIPKSSKPERL








KDINNWRPITIGSILLRLFS








RIITARLTKACPLNPRQRSF








ISAAGCSENLKLLQTIIRTA








KNEHRPLGVVFVDIAKAFDT








VSHQHIIHVLQRRRVDPHII








GLVKNMYKDISTVITTKKNT








YTDKIQIQVGVKQGDPLSPL








LFNLAMDPLLCKLEEHGKGF








HRGQSKITAMAFADDLVLLS








DSWEDMNANIKILETFCDLT








GLKTQGQKCHGFYIKPTKDS








YTVNNCAAWTINGTPLNMIN








PGESEKYLGLQFDPWVGIAK








TSLPEKLDFWLERIDRAPLK








PFQKLDILKTYTIPRLTYVA








DHSEMKAGALEALDRTIRSA








VKDWLHLPSSTCDAILYTSM








KDGGLGVTKLVGLIPSVQAR








RLHRIAQSPEETMKDFLEKA








QMEKMYEKLWVQAGGKRKRM








PSIWEALPEVVPSIDTATTS








EWEAPNPKSKYPRPCNWRRK








EFKKWTKLIAQGWGIRCFKG








DKISNNWIRHYRYIPHRKLL








TAIQLRASVYPTREFLARGR








EDNCVKSCRHCEAAEESCAH








IIGMCPVVRDARIKRHNRIC








ERLMEEAGKRDWTVFQEPHI








RDVTKELYKPDLIFVKEGLA








LVVDVTIRFESTKTTLEEAA








AEKVNKYKHLETEVRNLTNA








KDVIFMGFPLGARGQWYNKN








FELLDTLGLPRSRQDIIAKT








LSTDALISSVDIIHMFASRG








RRQHA








(SEQ ID NO: 1386)





R2
R2-
.

Zonotrichia

CGACTTGAGAAGGTCTGGTT
GTAGTCACATTGCACTTTCT
NKFLGKSRVAYCLKPGPPVS



1_ZA


albicollis

ACAACTGGGCATAGCTGCAG
GTAACTTGCACTGGGTGTGG
DRGKEFGSGLTTHPEPESES






AGATCGCGCCTCCTCGTGGC
GATGTGGGCCTGGGGTGTGG
GHDPTVPNPGPSLGAGEGAQ






CCCGCTGGTAAGCCCTTAAC
GTTATGGGGTATATATGTGG
PLPLLRVSVGTQTCEEDFIT






AGGGTGACTAAGTCGATCTC
GATATTCTGGTGGGAATGTC
SRPTKLPGIESELGPLVKFS






TGCCCCAGTCCAGGAGCCGC
CATTCACTGTATGCCTATCT
LEVYRSDLKGDVQFEGIHFP






TGGGTTTCACCAGCCCAGCG
TTTTAATAAAAAGACGGTAG
DNWGVLEGFPEVYEQLAPQP






ATTCCTTCCAAATTCGGTGA
CTAGGTTCGCGAAGCAGCCA
NGGDELNHSLPGDREGDVLE






(SEQ IDNO: 1142)
CAAGCCAATAGCCAGTTAGG
KDSSEKEKEAAPEALPSVQR







TAGCTCATAGTGGGTAGGTG
ARSEQLPDNIVKVTVPDKNP







ACAGGAACCTTTGACTCAGA
PCPCCGVRLNSVLALIEHLK







ACGCGTCCATTAACATCTAG
GSHGRRRVCFRCAKCGRENF







AACGGACCAAACTTCGGACA
NHHSTVCHYAKCKGPQIERP







TGCACCGATTAACCGGATTT
PVGEWICEVCGRDFTTKIGL







GTCCAAGGTGGACGGGCCAC
GQHKRHMHAMVRNQERIDAS







CTTTACTTAACCCGGAAAGG
QPKETSNRGAHKRCWTKEEE







GAACATATATAGTTATATGT
ELLMKLEVQFENHKNINKLI







GTTCGTAATA
AEQLTTKTAKQISDKRRMLL







(SEQID NO: 1265)
KKGRGTTGNLETEPGMSHQS








QAKVKDNGLGGDHLPGGPVV








DKGTIGKPGQHLDTDNSHQI








TAGKKKGGGLQARYRRRIMK








RLAAGTINIFPKVFKELIND








QEARPLINQTTEDCFGLLDS








ACQIRTALREKGKSQEERPR








KQYQKWMKKRAIKRGDYLRF








QRLFHLDRGKLARIILDNTE








SLSCDISPSEIYSVFKARWE








TPGHFNGLGDFEIKGKANNK








AFRDFITAKEIEKNVREMSK








GSAPGPDGIALGDIKKMDPG








YSRTAELFNLWLTAGDIPDM








VRGCRTVLIPKSTTPERLKD








INNWRPITIGSILLRLFSRI








ITARMTKACPLNPRQRGFIS








APGCSENLKLLQSIIRTAKN








EHKPLGVIFVDIAKAFDTVS








HQHIIHVLQQRRVDPHIVGL








VNNMYKDISTYVTTKKNTHT








DKIQIRVGVKQGDPLSPLLF








NLAMDPLLCKLEESGKGFHR








GQSSITAMAFADDLVLLSDS








WENMKENIKILETFCNLTGL








KTQGQKCHGFYIKPTKDSYT








INNCPAWTINGTPLNMINPG








ESEKYLGLQIDPWTGVAKYD








LSTKLKIWLESIDRAPLKPL








QKLDILKTYTIPRLTYLADH








SEMKAGALEALDQQIRTAVK








DWLHLPSCTCDAILYVSTRD








GGLGVTKLAGLIPSVQARRL








HRIAQSPDETMKDFLEKAQM








EKMYEKLWVQAGGKKKGMPS








IWEALPMTVPPTNTGNLSEW








EAPNPKSKYPKPCDWRRKEL








KKWTKLESQGRGVKNFRNDT








ISNDWIQYYRRIPHRKLLTA








IQLRANVYPTREFLARGRGD








NYVKFCRHCEADLETCGHII








GFCPVTKDARIKRHNRICDR








LCEEAAKREWVVFKEPHLRD








ATTELFKPDVIFVKEDRALV








VDVTVRYESAKTTLEAAAME








KVDKYKHLEAEVKELTNAKD








VVFMGFPLGARGKFYKGNFN








LLETLGLPKTRQLSVAKTLS








TYALMSSVDIVHMFASRSRK








PNV








(SEQ ID NO: 1387)





R2
R2Dr
AB097

Danio

AATCCCCCCTACCCAATCCC
AAATCCCAGCGGGATACAGC
MESTAKGKSYWMARRPVEGA




126

rerio

CCCGTCGTGACCTCCAGGCC
AAGAAGGTATCGGATCTAAT
TEGSLGRVPFVTRDPKRKPE






AGGAATCACGAGCGTACGAC
AAGGTTGAGCGAGGAGAGGG
AKRTLTHGLGLRECSVVLTR






AGTGGCCATCCGGCAATGAC
TGGAGATCCTTTGGGGGGGG
LIEGRRGRDHTPSGWNAQRG






AATAGCGTGACTAACGACAA
TCGGGCTAAGTTCCCCTCTC
MPNDESSVEEPNGPIPSNPI






TGAGTCAGATCCATGACCCT
GGGTCCTCCCACGGTGACGC
PTGTQALPEPMADGEQGEHP






TGGAGTGGGTTAACCTCCGC
TCTACCCCTCCCTCCTCGCT
GVVVTLPLRDLNCPLCGGSA






CTCTTTAAAAAC
CGTAGAACCCAACGGTGAAC
STAVKVQRHLAFRHGTVPVR






(SEQ ID NO: 1143)
ACGGTTGGCAGGATGAAGTG
FSCESCGKTSPGCHSVLCHI







ACGTGAGGGGTAAGACATGC
PKCRGPTGEPPEKVVKCEGC







GTACGTGAGCGCGCATTTTT
SRTFGTRRACSIHEMHVHSE







GCTGTTCTCTGGACTGGGTT
IRNRKRIAQDRQEKGTSTDG







TCGTCCCCCTCACAACCATC
EGRAGVERADAGEGPSGEGI







ACTTACACTATAGGGGCACA
PPKRPRRARTPREPSEPPAN







GCGGCTCCTACCTCCCTCCC
PPILSPQPDLPPGGLRDLLR







TATGACCCCCCCTTCCCATA
EVASGWVRAARDGGTVIDSV







CCGATCCATGGCTGTTCTAG
LAAWLDGNDRLPELVDAATQ







TCTGGACCGAGGGTCGGACG
RTLQGLPAGRLARRPATFVA







GGGCATTTGAAGGTAGCTGG
PNRRRGRWGRRLKLLAKRRA







AATCCTCCGCTGCTGCGAGC
YHDCQIRFRKDPARLAANIL







CTGAGGTCGATGGTTAGAGG
DGKSETSCPINEQAIHEHFR







TGAAATACTTGGGAGGAGAC
NKWANPSPFGGLGRFGTENR







ACAGCCTCCGGAGAGCCCCT
ANNAHLLGPISKSEVQTSLR







CCCGGGTGGTCATCATGGCA
NASNASTPGPDGVGKRDISN







ACCGGGTGAAACCTTACGGT
WDPECETLTQLFNMWWFTGV







TTCACTTACGAAACAGCACC
IPSRLKKSRTVLLPKSSDPG







ATAACAGCGCCGTAATAGCG
AEMEIGNWRPITIGSMVLRL







CACCGGTGTGACTACTGTCC
FTRVINTRLTEACPLHPRQR







AGTGCTGATATTCTCATCTG
GFRRSPGCSENLEVLECLLR







GAGAATACAACACGGGTAAT
HSKEKRSQLAVVFVDFAQAF







GGCAGAGTATTCAAAACCCA
DTVSHEHMLSVLEQMNVDPH







AATGTTTACGATCGACCAAC
MVNLIREIYTNSCTSVELGR







GGAGTCGTTCCCTTGCATCT
KEGPDIPVRVGVKQGDPLSP







AGGCCGGACCCGAAACTGCC
LLFNLALDPLIQSLERTGKG







GTAATTGCCCGTCCCCAAGG
CEAEGHKVTALAFADDLALV







TAGCCTCTTAGAAAACCGAA
AGSWEGMAHNLALVDEFCLT







GCCCGGTCGGGGCGGTGGTT
TGLTVQPKKCHSFMVRPCRG







GCGGCGGCGCTGCGGGGGCC
AFTVNDCPPWVLGGKALQLT







TGCTGCTCGGGCGGCGTCGG
NIENSIKYLGVKVNPWAGIE







TGTGCCGCGGTGGTTGCGGT
KPDLTVALDRWCKRIGKSLL







GGTGCGGCGGGGATCTCGGT
KPSQKVYILNQFAIPRLFYL







CCTTGCGGTGCCGCTGTGCC
ADHGGAGDVMLQNLDGTIRK







GCCGCGGTCGCGTCGGTGGC
AVKKWLHLPPSTCNGLLYAR







GCTGGGGTGGTGGCCCGAGT
NCNGGLGICKLTRHIPSMQA







GGCGTCGGCGTGCCACTGCC
RRMFRLANSSDPLMKAMMRG







CATAGTCGCCCGCGGGGGCG
SRVEQKFKKAWMRAGGEESA







ACCGATCTGGAGGGGCGAGG
LPRVFGANQYQEGEEVANDL







GGGCTCGCGGGACTTTAACG
VPRCPMPSDWRLEEFQHWMG







AGAAACGGAACGCAACTTCT
LPIQGVGIAGFFRNRVANGW







CGCATCGCTCCCGGGACTTT
LRKPAGFKERHYIAALQLRA







CCCCCCTCGTTCAGCCGAGG
CVYPTLEFQQRGRSKAGAAC







GATGCCAAAAGGCATGAAAG
RRCSSRLESSSHILGKCPAV







GTAAGTACCATACCGGTCCG
QGARIRRHNKICDLLKAEAE







CAAAACTCTCTTCTGACTCG
TRGWEVRREWAFRTPAGELR







GTTCTCTGTTGGTTTTCTAG
RLDLVLILGDEALVIDVTVR







AGTAACAACGAGGTGGAGGA
YEFAPDTLQNAGKDKVSYYG







GAGGGACATGGCAGGGACTC
PHKEAIARELGVRRVDIHGF







CCATTCGTGCCAGCGGGTGG
PLGARGLWLASNSKVLELMG







GGACAGATCGAAGGAACGGT
LSRERVKVFSRLLSRRVLLY







TCGAGGGCGTAACAGACGAG
SIDIMRTFYATLQ







AGGGAATCCGGTCACACATT
(SEQ ID NO: 1388)







GATGCCATGCCTAAATAGGC








GAGGTTTGTATTTCTACTTT








GTGGGTTCAGTATAGTCGGA








GCATATGGTCGGTTGTCCCG








TTGTTTTCACGGCGGGCAAG








CGACTATCATGATAAAGTAG








AATGGGAGACGGGCTCCCTG








ACAAACCCGGAAAGGCGCCC








CCCCGTGGTTCGTAGCAGCT








GACGGATCACGCTCGAAGAA








AAATGAGTGAGAGGGGACGC








CGCAACCAC








(SEQ ID NO: 1266)






R2
R2-
.

Gasterosteus

CATATTGGGGTCTCAGGAGG
GGAGGGGAGTAGGTCTCTAC
MLRGGVGTPPAGGAGAVGPG



1_GA


aculeatus

AGACACAGGGTCTGTTGCGG
TCTGACCCGAAGGGCCCCCC
MASPGGCSVRFSPGGRRLLG






CTCCGGTAAACGGTACCGGA
CGTTTCAGACCTGATTCTAG
HRTGGLSPSVSWRLKRLSVS






GTCGGTTAAGCATCGTTTGG
GCTACCTGTGCCTAATTGGG
LRRWSGPGLLGADGAGGGAA






GCCCGCCTCCACGTGGTGGT
GGGGTCCCAAAGAGATGTTG
VASPRGTQVLGSGAGRRWLG






CCGCGGTAACACCAATAGGG
TCTGTTGTAGAAGGGTTTGC
HGSRGSSPSAARGLRRLTVR






TGGCTAAGAGGCCCAGTAAT
GCCACTGACTGCACGGAAGG
LKRLSGGLLSPKACRDAEEG






TTCCCCGAATTGTCTTCCCC
GTGGGCCTCGACAGGTAGGG
SSSSPGFRNPKGLGGRGLTP






CCCGCGCGGGGGGGACCCCC
GTTACATGACTCCGTGCTGC
LGSRRFCRLTVSLNRWRGSL






CTTTAGTGTCGGAGCGGTCG
TCAGCAGACCCGCGCCTCTG
VKLNASSRASGRRTPVKPAC






CGCCTCCGCGTTTGGGGTGT
AGACCGGGTAGGGCTACTTG
DSRAGRGSEHAEGGGVSAAP






CGCAGGCGTGAGCCTTCGTC
AACAAGCGACGCCCTGGTGT
MVLRSRRKLTFSVDGDSNSG






CCCTTAAGTTCAGACGGTCC
ATGTCCGTATCCTAACCTGG
DRARSGSVSAARPGHLLVDG






CGGCTTCTTGCCGGGCCAAC
TTTGGGAAAGCCGATACCGG
ESASSRSGPAGDARLAGPST






CCCCGGTGCAGCGTTCTCCC
CAATGCCCGCCACAGGTGTC
RSRRKGCLPPVDFENPKKRT






ATGTTGGATCGGCACCCAGC
GCGCACCCCACGGGATGACG
RLMAKMTNGNPTSHVPCPAP






CCCGGGTGCCATGCGAGTTC
TATGGGCCCCGGGGGACCTC
CSNGHEGGGRVAVIEGRLPE






AGACATTTTGTTTATGTATC
ATGGATACTCCACTGGACTT
LSGSRISGIQPALPVETSFV






GTCTGCGTGGTTGACTTGCT
GCACAATCCTGGTGTACTGG
GQSTGRGADGDANANSSPPS






AAGCTCATTTCCTCCTCTCA
ATGCAGCGACGTTGGTGACA
PNLGGSVGMVPAVRDGTPPL






CTGCGTCCCCCCAGGTGCTG
TAAGCAATCGCTAAGTCGGG
GRPGEDHSRECAGGNTPLWM






ATCGGTTGAAGAGGATTCGT
GTAGGGGAGGTGGGGACCTC
LEDSFRCDYCPREFGTRAGR






CGTTGACCTCGGCGGTGAAT
GGCACGGCTGTAGGAACGGG
SLHMRRAHLAEYDGAGFCWG






TTGGGATTGTATTATACAGG
TGTATGGGCTCCGGCAGCCG
ERLSEFAATRLWSTEETKKL






TAGGTATAGAGGGCGTGCGG
TCGTCACTCCCATACAACAC
AVFCERGVPSPSECRAIAAS






(SEQ ID NO: 1144)
AGGGGCTGCATCCTGGTGGC
LGAGKTHHQVRSKCRLVFEA







CGGTGCTAGTTGGTTCTGGA
IRRRELLEVAAATERLEKSA







AGCCCGCCCGGGCTGGTTCG
RRKQPAVPPAPVHGVRGVLR







CAGAAGCAGGGTGCGCCCAG
GLLGKRVPREGGTTGSTSAR







GGTAGGTTTGGTATATCTGG
IVRRDDCRQGAVASASLNLI







GTCCGGTGCGATACCTATCG
RRLGRKATGRSGRRRVLGRP







ATGGGCAGCGAGGGCCGCCT
PRMDVRRSVRMRRMRRFLYR







CGTGACGCGCTGTGTGGAGC
LARLGWAKLAMFVLDGQMGA







TGGAGCCGGCCTGGGTATGA
SCPVPLVEVSAVFRERWSIV







ACAGTTCTTGCGGATGTGGC
RAFLGLGQFGGFGTADNAGF







GTAGCTAGATAGTACCCGTG
GKLIDPAEVRAHLQSIKNRS







GTTGTGGGCGTGGTGTCGAC
SPGPDGITKVALSKWDPEGI







CAAATGTTGTCCTGTGTGCA
KLAHMYSTWLVSAGIPKVFK







CATAGGCCAAGGGTTACGTG
KCRTTLIPKTGDVSLHGDVG







GGTGGCAGTCAGAAGCACCC
QWRPITIASLVLRLYSRILT







GCACCTGGAAGTGATTGCCC
ERMTVACPSHPRQRGFIASP







CGGGATCCCGGCTCTCTGTG
GCSENLMLLEGCMSLSKAGN







AAGAGCTACCTTGAGGAAAG
GSLAVVFVDFAKAFDTVSHE







GTGTTCCGCTGGAACTCAAG
HLLSVLVQKGLDQHMVELIK







ACCCTACAGTAGGGGATATC
DSYENSVTKVHCQEGCSTDI







AACTGGCTTTGAGGTGCTGT
AMKVGVKQGDSMSPLLFNLA







GATTCCGGAACCAGGGCGAG
LDPLIQQLEREGRGFPVNGK







GGCGAGTACTTAGAGCATGT
SITAMAFADDLAIVSDSWEG







CCAAAAGCCCGGGGAACGTT
MRANLDILVDFCELTGMRTQ







CCGGGGGCCTGCTTGGGTCG
PSKCHGFLIEKSGSRSYKVN







TTGGACCCACATCCGTAAAA
RCEPWLLNDTALHMVGPKES







CGATGGATCTCGCGTCGGCG
IKYLGVQVNPWTGIFAEDTV







CTCGGGAGAACTTCCCGCAT
AKLRQWVVAISKTPLRPLDK







GAACGCTGATTGCATGTGAG
VSLLCQFAVPRVIFVADHCM







AACGCCCCCACGGCGGCGGG
LSAKALTEMDRSIRQAVKRW







GCAGGCGCTCCCCCTGGGTG
LHLARCTTNGLLYSRKSSGG







TAAGGCTCGGGGGGGTCACG
LGIPKLSMIVPAMQARRLLG







GCTCCGCTCTAAAAG
LSRSKDETVRWMFLETTDHV







(SEQ ID NO: 1267)
AFERAWLRAGGSPDEVPELG








PDLVEGSPAEGNADPVSTVR








PRKRIVPCDWRQVEFDRWAG








QLVQGKGIRTFEADKISNCW








LYDYPPNKLKPGDFTAAVQL








RANVYPTRELAGRGRTDTID








VCCRHCGEAPETCWHILALC








PKVKRCRIQRHHKVCQVLVA








EAERHGWEVEREKRWMLPSG








ECVAPDLICWLDELALIVDV








TVRYEFDEESLERARIEKEC








KYRPLIPVIRASRVQTKKVT








VYGFPLGARGKWPAKNELLL








ADLGLSKARTRSFAKLLSRR








VLLHSLDVMRTFMR








(SEQ ID NO: 1389)





R2
R2_B
AB076

Bomby

GGGCGATACGCATAATTTTA
GCCTTGCACAGTAGTCCAGC
MMASTALSLMGRCNPDGCTR



M
841

x mori

ATTTCCCGATTGAAATCCAG
GGTAAGGGTGTAGATCAGGC
GKHVTAAPMDGPRGPSSLAG






TCGTCTTAATCTGGTGACCA
CCGTCTGTTTCTTCCCCGGA
TFGWGLAIPAGEPCGRVCSP






GTGGCGCGGTCACCAGTATA
GCTCGCTCCCTTGGCTTCCC
ATVGFFPVAKKSNKENRPEA






GTGCACAGGACGTGAATGGC
TTATATTTAACATCAGAAAC
SGLPLESERTGDNPTVRGSA






TCCGAGGCTGGCGGAGTCAC
AGACATTAAACATCTACTGA
GADPVGQDAPGWTCQFCERT






TCACTATAAGTGTGAGAGAC
TCCAATTTCGCCGGCGTACG
FSTNRGLGVHKRRAHPVETN






GATGTCCTGTGCCAAGTATA
GCCACGATCGGGAGGGTGGG
TDAAPMMVKRRWHGEEIDLL






CGTCCAACCCTAACGGGTTA
AATCTCGGGGATCTTCCGAT
ARTEARLLAERGQCSGGDLF






AGTGAAATTAGTTGCTCATA
CCTAATCCATGATGATTACG
GALPGFGRTLEAIKGQRRRE






ACAGGGACGGTGTACCTGTT
ACCTGAGTCACTAAAGACGA
PYRALVQAHLARFGSQPGPS






TGCTCGTGGCTGGCTATCGA
TGGCATGATGATCCGGCGAT
SGGCSAEPDFRRASGAEEAV






ATGGACGGGACCAATACACC
GAAAA 
EERCAEDAAAYDPSAVGQMS






CCCCTGTTAGTAATGGGGTA
(SEQ ID NO: 1268)
PDAARVLSELLEGAGRRRAC






AGAGAGAGCGGTCTGAAACT

RAMRPKTAGRRNDLHDDRTA






ATGGCCGAAATCACGACGCC

SAHKTSRQKRRAEYARVQEL






CCACTCCTACCCATAACCTG

YKKCRSRAAAEVIDGACGGV






CACGTGGTACCGCCGCACAT

GHSLEEMETYWRPILERVSD






TGACCGATACGGGAGGAGGG

APGPTPEALHALGRAEWHGG






GCAGCACTTGAATCACGTAG

NRDYTQLWKPISVEEIKASR






TCTTGGTGTAGCCATTGCGG

FDWRTSPGPDGIRSGQWRAV






GACTACAGCCCTCGTAAGTG

PVHLKAEMFNAWMARGEIPE






CCGCCTTAGAACGCAACGGG

ILRQCRTVFVPKVERPGGPG






GCAATAGGTGGGCCGGGGCG

EYRPILIASIPLRHFHSILA






CTAGCGGGGGGGAGTAATCT

RRLLACCPPDARQRGFICAD






CCCCTGTTGGCGTGCACCGC

GTLENSAVLDAVLGDSRKKL






ACTGCTCCCACTGGGGGCAG

RECHVAVLDFAKAFDTVSHE






TGTCATCCGGAAACAGGTGG

ALVELLRLRGMPEQFCGYIA






GCCGGGGCGCCACCAGGGGG

HLYDTASTTLAVNNEMSSPV






GAGCAATCCCTCCTG

KVGRGVRQGDPLSPILFNVV






(SEQ ID NO: 1145)

MDLILASLPERVGYRLEMEL








VSALAYADDLVLLAGSKVGM








QESISAVDCVGKQMGLRLNC








RKSAVLSMIPDGHRKKHHYL








TERTFNIGGKPLRQVSCVER








WRYLGVDFEASGCVTLEHSI








SSALNNISRAPLKPQQRLEI








LRAHLIPRFQHGFVLGNISD








DRLRMLDVQIRKAVGQWLRL








PADVPKAYYHAAVQDGGLAI








PSVRATIPDLIVRRFGGLDS








SPWSVARAAAKSDKIRKKLR








WAWKQLRRFSRVDSTTQRPS








VRLFWREHLHASVDGRELRE








STRTPTSTKWIRERCAQITG








RDFVQFVHTHINALPSRIRG








SRGRRGGGESSLTCRAGCKV








RETTAHILQQCHRTHGGRIL








RHNKIVSFVAKAMEENKWTV








ELEPRLRTSVGLRKPDIIAS








RDGVGVIVDVQVVSGQRSLD








ELHREKRNKYGNHGELVELV








AGRLGLPKAECVRATSCTIS








WRGVWSLTSYKELRSIIGLR








EPTLQIVPILALRGSHMNWT








RFNQMTSVMGGGVG








(SEQ ID NO:1390)





R2
R8Hm-
.

Hydra

TTCAAGTGGATGAAGCTGGG
TAAATGCCAAAAGTTGCTTG
MNLLIVTSSIKESDVPSSGK



A


vulgaris

AAGGTAATCTGTAGTTGGTT
GGCTAAATGATACGTACGCT
GGVAVNNITAGASGKDTCVI






GAGTTGGTTGCAGATTACTG
AGAAAAAGCGACTTGCTGCA
IHPGTDGIWCCTECVEIHNS






CTGTCGATTTTGCTTTCTAT
CGGATGACGGTTCATCAGAG
GKDLKRHLAKRHPSVTISGY






TGAAAGCCTGTCTCTACGGG
CCCGATATGTGCATGTCAAG
KCNLCPFVSERQLSVGTHLR






TCCTGAAGCTTGAATTTTGG
GCGGCAGGGAGAATCACTAG
YCRGVKEVVKREFACASCSF






TAGCTATAGTTTTGTGGGAG
TGTAGCTGTTCTTTCCATTA
SSDTFSGLQVHMQRKHIAEW






GAAAGTGGAATTTTGTACCA
CGACTTACGCGGTTAACGTG
NDQLKEKTEFAWTDRELREL






TCTTTTGTCTCTCGTATCTA
GCACGATAGATTTACACCAG
AEKELTTPSFRYNKIFYAAL






CTATAGTAAATCCGGTCATG
GAAATAATACGTGAAGGGTT
GTSRTYDAVRKIRYNDRYKS






CAGCCTCTACGCGGCGCAAC
CCACCATATACTGGAGTTTA
AIAEMRSQIADAAAAAQERD






TAGAAACTTGGATCAGTGAT
GATCTATGAGGGAAACATTT
VERGLVSAHSDRGKEMLPVV






CAAGGCTAATGCATGCCGGG
GTAATAAGTCAGTCTGGTAA
ETKSDIQVNNDIKKDIELTP






TCTCCTCAGATTAGGAGTAT
CCTGGCGCCGCTGTTGAGTC
NSRQKQTNLALARPAVIEVE






AATACAAATCTGACTTCATC
AAATTAACTATGTCAATACT
EDLGRQDVKQYLASLRQDDY






ACTAAGAGGCTATGGGGCTA
CATTAAGTTATCGACTTTGA
TSPAERSIFAYCREETNWSA






ACGATCCTATAGTCTCG
TATGGCATGGGGTGATTCCG
TKRQVLKISRTTRGLRQPKK






(SEQ ID NO: 1146)
CGTTATATCAAAGTCAAACA
VRPFEFPEGFKPNRNMRKWR







TGATGATTGCAATGAGAAAC
KYRFLQECYREKRAETVSKI







TACCACGCTTGGTCACGTTT
LDGTFIDEPEEEIRPELEEV







GTGAGGAGAACATCTCATTC
QRMYIDRLEKRTQLDTTKIV







AAGCCTCCCGGATGTCGGCA
QTDEVFCLQSYGRITIGEVR







CCCGCTGACATCTTCTGGCT
DALGASKKDSASGPDGLLLQ







TATGAAAATTTTCATTAATT
DVRRLGPLLLCNIFNMWYLH







TTTGTAAGTCATGGGCGGCT
GIPVEENRCRTILLYKSGDR







TGAAAGC
HLASNYRPVTIGNMLNRLYA







(SEQ ID NO: 1269)
KIWDKRIRKNVRLHVRQKAF








IPVDGCFENVKTIQCVLQSY








RKRKLEHNVVFIDLAKAFDT








VLHDSIRKALWRKGVPSGVV








KVVDSLYAGAVTSISVGKTK








TRSICINSGVKQGCPLSPLL








FNLILDELAERIEATGCGLD








LDGHVLSSMAFADDYVLLAK








DSVEMNELIRVCSTFFKEKG








LSVNPGKCQSLRVLPVKEKK








RSMKVLVRPHRWWRIKDQDV








DIPSMTYDSLGKYLGVSIDP








TGKIALPIEEWKNWMTKLKE








CKLKPEQKVKILKEVVCSRV








NYVLRMSECGISELRSWTRF








VRNWAKNIIHLPTWCSSDWI








HSIKGLGIPDVSKGIVIQRM








RASEKMSTSEDGIVRVVGAR








LVQKNRVLWEKAGFEGIELK








AARRHCEVERLNNIGNITNG








VALKTIAAVSSVNRYWMIED








NLKSGNKILVWKAMAGAIPT








KINLSRGVADQTLKKCRRCG








LTAETDGHILAGCHTSSDAY








SKRHNMLCDKLAKELKLNGG








PNRRVWRERTCFTSTGRRYR








PDIIVKDDSKITVIDMTCPY








EKSEGHLIQCESAKVTKYEP








LKLDKYWTRELEGANGIVAE








KVELMGLAIGAIGTIMRSTL








RKLCELKSGRIVRRLQMIAC








NNSAQIIKGHLSRATRRNLR








(SEQ ID NO: 1391)





R2
R8Hm-
.

Hydra

CTTGGGGTCACTGACACATT
ATGCCCGAGGTAGTTGGGAT
MSNRITIGDVPSVGKGGLTV



B


vulgaris

TTTCGGTAGCCATAGTTTTT
AATGATGCACAAGCTCGTAA
NKQTAGADGAEACVVIHPGA






TGAGAGGAAGAGTGGAAGTT
GGCGACTTGCTGCACGTATG
KGIWSSPACLRKFTIGKELR






TTTCCATGAGTCGTCTCTCG
CCGCTAAACGCTTAGCTCGA
AHLAQIHKLAPSAVRYRCNK






TATAAACTGTGGTAAATCCG
TGAGTGCATGTCAAGACGGT
CPYEGDVQLSVGTHLRYCKG






GCCATCCAGCCTCTACGCGG
CGGGAGTATGATCAGTGGAG
IAGVVEEKKQFACAICNFSS






CGCAACTAGAAACTTGGATC
CTGACTTTCCAGACAACTCA
DTFSGLQVHKQRKHVVEWNE






AGTGATCAAGGCTAATGGAT
CGCGGATTCGCGTGCGGTGG
QLKEKTEFAWTDRELRELAV






GACGGGACTCCATGGATAAG
ATACAACACCTGGTATAACA
KEVTIPFSVVNTETFAVLDI






GAGATATAAAGATCTTATTT
TATGAAGGGTTCCATCTAGT
TTRTKDAVRKIRYTDRYKSI






GAACGCATCTTAAGGGGTTA
ACAGGGATAACGATCCATGG
LAEVRAQVNAVAEEAPQASD






TGGGGCTAACACCCCCTTAA
GAGCAAACTAATTAGTTGGA
ESQITLLVNTGRGAELQPAV






TTCTGGTGCACATTTATTGA
GGTAATCCAACGCCGCTGTT
INITDSIELVTDVNEVEMVT






CCGTT
GAGTCAGTTTTTAACCGCCA
SNSTNEEQPINAPVEPAVIE






(SEQ ID NO: 1147)
GTCAACTCTTGTAGGTTATC
ADLGRQDAKLYLASLRQSDC







GGTCTTCGGCAGACCTTGGA
TNASDRWTLAYCRGEVDWCK







CCGCCTAGCGCCGGCCAACA
TKSRLFKVSRHARGLRQPQR







GTTTGTCGTCGACTAACATG
VENWEFPEGFRPNRNLRKWR







ATGATTTGCGAGAGAAACCC
KYSFLQSCYRTKKKETVSKI







ACGCTTTGTCACTTATGTGA
LDGTFKDTPEEEIRPELEEV







GGATAAAATCTCTTGTCCAT
QRVYVDRLEVRTQLDTTRTV







ATGATCCTTTGAAGGGAACA
HIDERFDLVSYGRITIREVQ







GCGCTTTGAGCTTGCTCGGC
DAISASKKDASGGPDGLLLQ







GTTGGCACCTTTAGTCTGTA
DVKKASPRQLCIIFNMWYLH







ATATTTTCTTGATATTATGG
GIPVVENRCRTILLHKGGEK







ACGAAAAAGGTAGTATGGTT
HLTSNYRPVTIGNMLNRVYA







GCA (SEQ ID NO:1270)
KIWDRRIRKNLQLHVRQKAF








VPLDGCFENVKTIQCILQSY








RRSRREHNVVFVDLAKAFDT








ILHDSIEKALLRKGIPRSVI








KVVDSLYAGAVTSITVGKTK








TRPICINSGVKQGCPLSPLL








FNLVIDELAERLEATGCGLD








LEGHVISSMAFADDYVLLAK








DSVEMNVLMNVCNTFFEEKG








LAVNPAKCQSLRVLPVKGKR








SMKVLTRTHRWWKINNQDVE








IPSMTYESVGKYLGVMIDPA








GKIALPIEEWKLWLTRLREC








KLKPDQKVKVLKEVVCARAN








YVLRMSGCGICELRKWSRFV








RGWVKSIIHFPAWCNSEWMH








SSKGLGIPDVVSGIVIQRMR








AAEKMAKSTDGVVRVVGARI








VQTNRVLWKRAGLAGIELDA








ARKFCEVKRVNKIGNQTNGG








ALKTIAESSVSRHWLLEKNI








RPGNKILVWKAMAGVIPTKI








NLSRGVADQTLKKCRCCGLT








AETDCHILAGCPTSRDAYSK








RHNLLCDKLAKELRLNGGPS








RRVWRERMCLSGNGRRYKPD








IVVKDDGVITVIDMACPYEK








SERHLSQCEDAKVAKYEPLR








LDRSWTQELEGNNGRSANEI








SVVGIAVGAIGTITRKTQRI








LSKLKLAKVGRPLQIIACNE








SAQIIRRHLSGSRLRNLR








(SEQ ID NO: 1392)





R2
R9Av
GQ39

Adineta

GAAATAGTTTGCAATGGTAG
ACTAGTCTCCTTCTTCTATT
MNLPIREHAVSVHNINKFNY




8057

vaga

GTGTATGGCGCCTCTGTGTC
AGTCAGTCTAATTAATTTTT
LCQLCSKSYDTINSVKAHYV






TCTCTTTCGCTGGATATAGT
CTTACATTCTACATCTAGTT
ACRRQKNASSTTAVPTNVIN






TTGACGATTTTGTACCAGGT
CCATTATTAAATTGGTATGA
NNQLAINTNQVISRNPLQCV






ATCTGTTTCTTGTGAGTTCA
TCAGTGCTATCTCTGCTACA
ECLMKQVDFYAKDTKALVTH






GCACCAGTTTGAACAGGCTT
CTCAATGCTTAATCGTATGT
MRTKHAAAYEESKKVATRRV






AGCGATAGACCTTCGAACTT
TATTGACAGTCTGACACTTG
AWSPDEDQILAELEVKLKKI






GAAACACTGTTGTGAAGCTG
ATTACTCTTACGACATATGC
QKGQLLSRLVVEYNKCADKS






GCTGGGCCCCTGCAGATTTT
ACTGTTTGCTTCAGAGAAAC
KAPSRSKDAIRTRRQQHDYK






CTCGATTAGAACGTGAGTGT
CACTGTTCATATAGTGAAGT
LLLRSLQSQQPPVGSEDSDS






TACGTCCAGAATGACCCACC
TCCTCAGTTTTCTGTTGATA
DISSSNNNPLTTTHNVTPTP






AGTGGTTAGTTCTACGTTGC
TATTCTTCTTTCATTCTCGC
DSSNVVLLIQKIRESVDSIV






CCTGGAAAGGAGAAAAGTTG
TTCTCCTTTTCTACTGTGTT
KITNLKLNTNMLNAASAFIN






AGCTAAAATCGCACGGCCTA
CTTTTTATCAGTTTTTTGTG
QNNNMDPLELSMRGIEEDVK






GTTGTTTATCAAATAGGCAC
GAAAAATTGAGAATAAATAA
AIRDKELQKPTRNVPSSTTS






GGTGAGGAACTCTTCTATGT
AGT
RKPTRNAKRLEKSKKYGYYQ






ACCCTGACTAAAGTACTCAC
(SEQ ID NO: 1271)
HLYYNNKKKLVAEILDGETS






TTGTGCGCTGGGTTTGCTCC

GAKPPPMNLVEDYYRNIWSR






CCCTCGCATTGACTTATCTG

STIDDSPVNNIKTVNSDSIF






ATCGCACTACCCACCAAACG

APISRDEIKLALSNTKKDSA






AAACATAAACTTAGCTCGTG

AGPDAVTIKEAKAIIDNLYV






GTATCAGTCCACAGCGTGTG

AYNIWLGVQGIPEQLKLNKT






CAGTCGGATTCAGGGGAGCG

ILIPKGNSDLSLLKNWRPIT






TGTTAGTGACAAGCAGGATA

ISSIILRVYNRLLAYRMNKI






ATATTAACATAGTTAATGTT

FKTNDKQVGFKPVNGCGINI






AAGGCGTTCAACATTCCTTA

SWLHSLLKHARLNKNSIYAC






TCCAATTGGAAGAGTTGACT

LVDVSKAFDSVSHQSIVRAL






GTGAAGTTTGTCATGAAGAC

TMNGAPSLLVKLIMDQYTNV






ATTGGACAA

NTVITCSGSISNKINISSGV






(SEQ ID NO: 1148)

KQGDPLSSLLFNLVIDELFD








VIKDQYGYTIDNIGTTNARC








FADDLTLISSSRMGMNKLLE








LTTKFFKERGLNVNPSKCMS








IGMSKGYKGKKSKIESEPLF








SITDAQIPMLGYIDKTTRYL








GVNFTSIGAIDAKRIKKDLQ








DTLDKLEHLKLKAQCKMDLL








RTYMIPRFMFQLIHTELYPK








LLIKMDILIRKLAKRILHLP








ISTSSEFFYLPFKEGGLQLT








SLKEAVGLAKIKLHKKIMSS








NDPMLCYLIESQRSRIVEHF








MKDLKLGDSLTLNEMNNIKE








CFMKEKRISFAQKIHGVGFE








VFSSSPLTNQWINGEIKTMT








TKTYINSIKLRTNTLETRVT








TSRGLNIIKTCRRCHVADES








LMHVLQCCSSTKGLRYSRHH








KICAKVANKLVMNGYGVFRE








KSYPDPNNSGSYLRPDIIAV








KNGHVIVLDVTVVYEVTGAT








FINAYQTKINKYNAIMVQIE








QMFNCVNGELHGLVIGSRGS








IHHSQLHIWHQMGFSSIELK








YVAIGCMEDSLRIMSTFSKA








IT (SEQ ID NO: 1393)





R2
R2Ol
LC349

Oryzias

CGCACAGGGGACACAGAGCC
GGGGGACAGCTGGGAGTCTC
MGTDTVYVGQDYPSGLSKRV




444

latipes

TGCCCAAGTACCGCTCCCGA
GGCATGATTACAAATCTTGC
PARLVAGPMLRERSCHAHVF






GGGAGCGGGAAACGGGGGGG
GCTGCACTCGGATGTCGTCC
RAGHMWNWRTSLPSGRWDQP






TGACTATCCCCTGGGGTCCG
CCGTGACGGACACATTAATC
ALEKSRVLTRSVATATDPEI






GCGAGAGCGCTGGTCTACGG
CGGAAAGCGAGTGGTGACTC
TSYPGKSVSTSTQVQEEDWC






ACCAGGGGTGGCTGTGGGCA
GCCTCAAG
SRESGWISPGLAPEEPSVVS






GGCTGCTCCTCAGGCCAGTT
(SEQ ID NO: 1272)
EITASMVATMRVATEEVVLE






GATTAGTTACGCATGGGCTG

PQPEQVVTILPEHGRNVPPG






TACCTCCACGTGGTCCCGCT

LAEQDTASPIEVSVLLPDLA






GGTAACGACTTGTCGGCTAA

ENCPLCGVPSGGLRLLGKHF






ATCAGCCCGCCCACCATCTG

AVRHAGVPVTYECRKCAWRS






GGATATGGTTGACCGTCTAA

PNSHSISCHVPKCRGRARMP






CCCCAGTACTCAGGTCACAA

SGDPGIACDLCEARFATEVG






ACAAA

VAQHKRHVHPVEWNKVRLER






(SEQ ID NO: 1149)

RGARGGGIKATKLWSVAEVE








TLIRLIREHGDSGATYQLIA








DELGRGKTAEQVRSKKRLLR








IDTASNSPDDAEVEEERLES








LAVRSSSRSPPSLVATRVRE








AVARGESEGGEEIRAIAALI








RDVDQNPCLIETSASDIISK








LGRRVDGPKRPRPVVREQTQ








EKGWVRRLARRKREYREAQY








LYSRDQARLAAQILDGAASQ








ECALPVDQVYGAFREKWETV








GQFHGLGEFRTGARADNWEF








YSPILAAEVKENLMRMANGT








APGPDRISKKALLDWDPRGE








QLARLYTTWLIGGVIPRVFK








ECRTKLLPKSSDPVELQDIG








GWRPVTIGSMVTRLFSRILT








MRLTRACPINPRQRGFLASS








SGCAENLLIFDEIVRRSRRD








GGPLAVVFVDFARAFDSISH








EHILCVLEEGGLDRHVIGLI








RNSYVDCVTRVGCVEGMTPP








IQMKVGVKQGDPMSPLLFNL








AMDPLIHKLETAGTGLKWGD








LSIATLAFADDLVLVSDSEE








GMGRSLGILEKFCQLTGLRV








QPRKCHGFFMDKGVVNGCGT








WEICGSPIHMIPPGESVRYL








GVQVGPGRGVMEPDLIPTVH








TWIERISEAPLKPSQRMRVL








NSFALPRIIYQADLGKVTVT








KLAQIDGIVRKAVKKWLHLS








PSTCNGLLYSRNRDGGLGLL








KLERLIPSVRTKRIYRMSRS








PDIWTRRMTSHSVSKSDWEM








LWVQAGGERGSAPVMGAVEA








APTDVERSPDYPDWRREENL








AWSALRVQGVGADQFRGDRT








SSSWIAEPASVGFAQRHWLA








ALALRAGVYPTREFLARGKE








KSGAACRRCPARLESCSHIL








GQCPFVQANRIARHNKVCVL








LATEAERFGWTVIREFRLED








AAGGLKIPDLVCKKADTVLI








VDVTVRYEMDGETLKRAASE








KVKHYLPVGQQITDKVGGRC








FKVMGFPVGARGKWPASNNT








VLAELGVPAGRMRTFARLVS








RRTLLYSLDILRDFMREPAG








RGTRVALIPAATGAAN








(SEQ ID NO: 1394)





R2
R2_LP
AF015

Limulus

TGGGAGGAGACCCAAACTAT
ATTTTGTCTCTTTCCCCAAT
GIDGYMFGYARASGSTSVSI




814

polyphemus

CCTAGGATGGGGCGGAACCG
GATGTCTACTAGCACGCTGC
QSSSMTEGETNERATPRASD






ACCATATGAGCCATATTAAC
CGAAGCTAGATAGATTGAGG
SSSVSIQSSCVTEGECLPPT






ATTGCCCACACTATCCTCTG
AATCTGCGTAATCTGTAATG
DNCNPSVENQLPCVTEGRFE






GAGGTACCTCCTCGTGGTAC
ATTACGCCTCATGGGCATCT
RVGSLVTVRLPFRKVACDLC






GGCTGGATATAGGTAAATCC
ATCGGTAGCGTCGACCCTGA
SKEFLTYSKFAVHQANFHNS






TGTAACCAAATCCTCCAACC
CGTTAAATTGGGTAATAAGA
ETQACCTYCGKSDGNHHSIA






CGTGAAGGAGAACACTAAAA
AATATCGA
CHVPKCPWRRTVTFAANLSN






CCCATATAGTGGCCTCGCCA
(SEQ ID NO: 1273)
FLCDLCNDSFKTKSGLSQHK






ACCACTATATGTCCAACGGC

RHKHPCSRNAERILSLGVRT






AGGAGAAGCTATCTCCCGGA

PSARPRQVVWSEEETRTLRE






TGGGAAGGAAAACCCTAAAC

VEVVYSGQKNINVLCAGHLP






CGTGATGGGAACTTACCGGC

GKTSKQVSDKRRDLHRIRSS






CCCATCAGCTATTGGGTACC

NVHGTPTTQSRGDPVEQVEE






CGGTAGGGACTTGCAACCCT

YEELDWEGMHPFPDPDSKFC






ACCCTGTATTTGCATTTTAT

SYLDQLRDQKGLTEPVWQEI






AGGGAACCGGTCGGCCCTAT

EIVAQEWVENLAHVQSSWNH






ATCAGAGTAGACCGTTTATT

ERTTKQVPENNTPARRPFKR






AAATATGGGTGAAAATATTA

RLHRVERYKRFQRMYDLQRK






ACAGTAAAAGCTATGGTTTG

RLAEEILDGREAVTCNLKKE






GCGTCCGTGTGGTGCCAGGG

EIKDHYDQVYGVSNDRVSLD






CGGCGGCCAAACCCGAGCTA

DCPRPPGANNTDLLKPFTPT






CTTGGCACCAACTGGGGATG

EVMDSLQGMKNGAPGPDKIT






GTAGCTTCCGAGCGATTCCC

LPFLQKRLKNGIHVSLANVF






TGGCGACGTGGGACCGATCG

NLWQFSGRIPECMKSNRSVL






ACGATGGAGTCCAAACATCC

IPKGKSNLRDVRNWRPITIS






GGAATAGAGGAATTGAGAAA

SIVLRLYTRILARRLERAVQ






TACCTATTCCACCACCGGCT

INPRQRGFVPQAGCRDNIFL






CACATACCCAAGGTGAACCC

LQSAMRRAKRKGTLALGLLD






GGTGCAACTAGAGTACAACC

LSKAFDTVGHKHLLTSLERF






TATCTGTGGCGGTAGGTGCC

AVHPHFVRIVEDMYSGCSTS






GAACCACTCAGGTGACGGGC

FRVGSQSTRPIVLMRGVKQG






TTGTTTATTGATGTCTCCCT

DPMSPILFNIALDPLLRQLE






ACGAGACACGAATTGTGACA

EESRGFMFREGQAPVSSLAY






AATCCACTCCGGTGGACAAT

ADDMALLAKDHASLQSMLGT






TACCCGATCTATGAACCTGT

VDKFCSGNGLGLNIAKSAGL






TACCGATATTAGACAAGAAA

LIRGANKTFTVNDCPSWLVN






ATAAAGAACTGACAACGCCT

GETLPMIGPEQTYRYLGASI






AGAGCTTCAGGCAGCATGTC

CPWTGINSGPVKPTLEKWIA






TGTAAGTATCCAGTCATCGA

NITESPLKPHQRVDILCKYA






GCGTGACTGAGGGCGAAATT

LPRLFYQLELGTLNFKELKE






GATAATAACTCTGAAACTGA

LDSMVKQAVKRWCHLPACTA






(SEQ ID NO: 1150)

DGLLYSRHRDGGLAVVKLES








LVPCLKIKTNLRLVHSTDPV








ISSLAESDGLVGAIEGIAQK








AGLPIPTPDQRSGTYHSNWR








DMERRSWERLALHGQGVELF








KGSRSANHWLPRPVGMKPHH








WVKCLAMRANVYPTKRGLSR








GNLSKNKDSAKCRGCTSMRE








TLCHLSGQCPKLKSMRIRRH








NKICEHLIAEASFKGWKVLQ








EPTLVTDNGERRRPDLIFHR








DDKAVVVDVTVRYEISKDTL








REAYASKVRRYGCLTEQIKD








LTGATSVVFHGFPMGARGAW








FPESSDVMADLNIRSKYFEE








FLCRRTILYTLDLLWKSNNE








QYLERLAP








(SEQ ID NO: 1395)








NeSL
NeSL-
Z82058

Caenor

GCTCACTTTCTATCGTGTTA
CCTCCAGGGCACGCCGCACG
MLRRKGRHRMVMVNSVKWQP



1


habditis

ACCGTACGTTTACACTCCCA
CCAAAAGTCCTGGCATAACT
SAHAEAIGTGKSWAPQRSQA






elegans

GTGAGTGTAATAAAGGTTAT
CTGCAAATAACATCAAACGT
SEHGWQSNAMFDPPNRILFA






TCGATAGAGGGTGTCTCCCT
CAATCAACTCCACAAACTCT
RDSWSLNQSTHLQNQRSGSG






CTTTCTTGGGTAATTCTTCG
CCACTCTCTTCAAGTCTTCT
LGIRPGQVRNNMVGGGPHRA






GCGGTCCGGGGTCTCTCCCT
CGGTGCTTCCAACACCACAA
GDPKRRVELVSIQGSEVTVR






CGTCTTTTTTTTAAACTTTT
TGGTGAAAGCTCCTTCACCT
TIYPSDEIFSCYSKSCDIKT






CTTTCTCATCCACTCTTTTG
TTTCCCTCCAAAATTCTTCC
KAGYGPEDLKHLTRHIKNEH






CTCCTTTTTACTAACTCTTG
CATGTGGGGAAGTCCTGTTC
GLKARWAYQCGLCNEKSDPS






TACTCTATAGTCTTTTCTCA
TTGTAAGCTCTCCGGAGGCT
VSEGHKWMEAHMVAVHQSSA






TCCCCCATCCGCCGTTGGGC
GCAAGAGCAGAAGAAATTCT
EKRIKSYQKCTGARVAEQLQ






AAAGTTTATTTACTTTGTTA
TCTTTCTGACAAGGTCAGAA
AAAPSLTVPGKHKSGSRDAA






AATCCATATTTTATCTCTCT
GGAAGTCCTGTTCTTGAGGC
KDSMTPTKDDDPKTRIYQTR






CACCCGTACAGAAAGCGTCT
GTCCATCCCGGGCGTCATAG
SVVKKSTQKTAEPTDEGSRG






CCTTCTCAAACGCTTTTCTG
GAGAGATCAGATGCACCTTC
PKYASIFQKSVKARKSLALL






TACTTTTTCTTATATTTTCA
TAGCAGGAGCTAGAAGGGCT
CELSSPKPMNPLPTNELTLK






TTAACATATTTTTCCTGTTT
GCCCTGTCTTGAGATCCCCA
EGNSRELAKEEAPSEGIDDI






ATACTAACCTAACCTCCATT
CGGGGGTCAATAGACGGGAG
VIIDLDESEESPPRRKRFNT






GTCAATTACTAACTAACTTG
GGGCTGCTGGCTTTCTCTTT
WCLDHESSREAWLDDTAIFW






TACAACGGATTTCG
TTAAGAGGAAGCACCAATCC
YISYLCRGSTKYSALDPCLW






(SEQ ID NO: 1151)
GGAGATCCTTAGGGGTCAAA
SMYKVKGSRYILDRLESSIT







GGATTAAAAGGCAGCAGGTC
YFFPICEEDHWTLLVLKDNS







CAATTCTCCTCACTGACTTC
YYYANSLHQEPRGPVRDFIN







GGTCAGAGAGGAGTCCCGCC
DSKRARKEFKVQVPLQRDSF







TTGGAGACCTCCCCGGGGAG
NCGVHICLMTNSIMAGGKWH







GTTGCTGAAGAGGCGGAAGC
SEEDVRNFRKRLKKTLQEEG







TCCTTCTAGCAAGAGCTAGA
YELYSVNSLGIPFQAPTTEQ







GGGAGTTCCCAGTCCTGAAA
MDYKETRCKRSYASVLTQIS







CCCTTGCGGTTGATGATGGA
PPAKRPDCKPDNNIFVPTKD







ATGGAAGAGTACTTCGGTAC
CAAEGNPQEKGRNESPEEIN







TGCTCGTTGCTCTCTCTGCG
TEHIVVAGKPANNISPRCRS







TTTTACTGCCGAGGGCCGGA
TSEMLFEMVKATTSSGRSSL







TTTGCTCGAATCGCGAAAGG
GTMTQDEFIRTSTIAEAVPL







TCTCAATCGACCATTCAAGA
MSIKLPPMELPRKILPPIPP







TGACGGCTTATCTAAGGTCC
RKPTQTNGGQKGKQQRVPTG







GAAAGCAGTTGGGAGAGTAA
KPDTLNAKVRNWENNQLESY







CGTGTTCTCCTACCTTTCAA
AMEGRSFQRLEWLTEVLTAS







GTTGAATGGTCGTTTTACTG
IQKAAAGDEGIVDIICKRNP







TTTGGGATAGCTGACTTGAT
PLEVAKGEMCTQTENKRKTT







GCTAGTACGCTTCATCTGTG
NNAARIADPIQSSKGAGDVK







GATGACGCTCCCCAAGCAGT
ASYWKERARTYNRIIGSKEE







CAAGTAGACTTGAAAGGTGC
LCKIPIDQLEDFFKKSTSRT







CCTCGCCCTAGTTAGCTCTT
NVQESIMKEKSSKIPALKIG







AGACCTTATGGGTCGCCATG
NWMEKKFIGKEVAFALRKTK







GTTGTGGACGGGTATGCTTG
DTAQGADGLRYHHLQWFDPS







CCGGAGCCGAGTCGTGTTTC
GELLAKVYNECQRHRKIPKH







TTAGAACCAACCTCGACGAG
WKEAETILLFKNGDQSKPEN







GCGAAAGCTTGCACAAGTTA
WRPISLMPVIYKLYSSLWNR







GCACAATTGTGGTAGGGCCG
RIRAVPNVLSKCQRGFQERE







ACTAGAAAATGAGTCCCTTA
GCNESLAILRTAIDVAKGKR







GGGGGTTACGCCTTGGCGAA
RNLAVAWLDLTNAFGSIPHE







AGTGAGGACAATTGGCATTG
LIEYALTAYGFPQMVVDVVK







ACGGGTGCTTCGGCACTAGG
DMYQGASMRVKNATEKSDRI







CAAAGGCGCCACCACACTGT
PIMSGVKQGDPISPTLFNIC







CCAATCTCTAAAAAGTTCAC
LETVIRRHLESANGHQCLKT







ATTCATCGAAGAACTACCGG
RIKVLAFADDMAILTDSPDQ







AACCAACCACACATGTGTTG
LQRELSKLDNDCTPLNLIFK







AAACCTACACGGTGGAAGGG
PAKCASLVIQKGVVRSASIK







AAAGGAAAGCTTCGCTGGAA
LKGNAIRCLDENTTYKYLGV







CGAAAAGAACGGATAGGTTC
QTGSAARISAMDLLEKVTKE







CCCTTCTTGATGGCTGTGAG
LECVVKSDLTPPQKLDCLKT







GCTTAGGATGGACGGGAAGG
FTLSKLTYMYGNSIPLITEI







CCGTGAGGCCTCAGGCGGGT
KMFANIVIRGVKVMHRIPVR







AACTCGGCCAGACGCTAGTT
GSPLEYIHLPVKDGGLGVAC







GATCTTCGGATCACGACAGC
PKTTCMITFLVSTLKKLWSD







CCTGGCTAAGAGGAACCCTG
DEYIKTLFTSLAEEVVKKES







GATGGAGTGTGAAGGATGGG
KKSTVTMDDIADYLNVEERI







CGGGTAGGGGGTTAAGCCTG
NRSEFGYNSITRLRDVMRNL







TTGACAGACCACCGACTGCA
AITGDSPLYRLKMVVKNGKI







GTCACAAAATCAGTGATTAT
ALLVQATSESMERIYTEEDA







GCGGGTGGACCAATCTGTTG
KKLQRSLKDQVNKALKHRFN







GCGGGTGTTTCCCTCTACCT
TTKVVKSKVVRVVQQHPASN







GACCCCGCAATATGGTATGT
RFVTKGGNLSLACHRFVHKA







ACGATCCTCGGATCTAAAAT
RLNLLACNYNNYDKSKSKVC







TCATAATGGCCCACCACAAC
RRCGKDLETQWHILQNCPFG







CATAAACCTCCCTAGCAGCT
FSKKITERHDAVLHKVKTLI







GGTGGTCCCGATAATTCGGG
ESGGKKNWTMKIDEELPGFS







TTCTTGCCACTACTGCGACC
RLRPDICLKSPDEKQIILAD







CAGGCTCGCC
VACPYEHGVEAMERSWQAKI







(SEQ ID NO: 1274)
DKYETGFAHLRKSGTKLTVL








PIIIGSLGSWWKPTGDSLKE








LGIKGSVINSAIPELCATVL








EHSKNTYWNHIFGEAYIPNP








MRNGHAKPAGNGWKKERLQK








APVRPTN








(SEQ ID NO: 1396)





CRE
Cnl1
.

Cryptococcus

CCCTCTTAATACCCCATAAC
TGAGGAAGAGGAGGTTGGAT
MSLQRAKNARGDPGRCNLCS






neoformans

ACATAACAACCCCCTAATCA
TATTTTTTCTTTTCTTTAAT
ADYRDLKDHLNKQHSTHFFV






ACGTTCTCTGCACCTTAAAC
AAGTTGTTTATTTAAGTAGT
PSDLRGSSLVACPRCGTPCS






ACCACCAAC
TTCTTTCATTCGGGCAACCC
AGTGLSRHQSRYCGLTAPRI






(SEQ ID NO: 1152)
ACACGACAACCCAATAAATT
RRNRVGNSTNTSRCPPSNTA







AAACAACGAAAAATGCAACC
ASPIVPSPSPERPSPPQPAE







TCTATAACCC
VVASLEPLSEAEEVLEVAQV







(SEQ ID NO: 1275)
DAETVDTLEGTRRAPESVPR








SAEEGSTRVRELNMTAPEEE








HRGEEESSHTNPTAPAGLEN








AVSSTLGPSPGTLPSLLPSQ








ECANERFLYLAHLPVRSKPL








PNNLVTDFMDAAERCALAYI








AQPSDSTLLAFLALPKVGLT








QALAPEQPLRPSTFLKQFPH








IPWPEQPPARRPPSNIRPDT








TKQVIKLVENGRLGAAERVL








EEDASVAELDQGVIDQLITK








HPKGPSCPFGNAVGPTPGKA








PDIDTIQKALDSFKPDTAPG








VSGWSVPLLKTAAKREPVKQ








FLQLLCAAIANNTAPGRSML








RTSRLIPLKKDDGSIRPIAV








GELIYRLCAKALIISHFQPD








FLLPFQLGVKSIGGVEPIVR








LTERVLEGSAGAEFSFLASL








DASNAFNRVDRAEMAAAVKT








HAPTLWRTCKWAYGDSSDLV








CGDKILQSSQGVRQGDPFGP








LFFSITLRPTLNALSQSLGP








STQALAYLDDIYLFSNDSQV








LSKTTQFLADKQHIIKLNEK








KCKLISFDEIRQEGFKMLGT








MVGGKEKRAEFLEGRIRKEM








AKVGKLKDLPHQHALLLLRF








CIQQNLRHLQRSLRSDDLVD








LWERLDTMLWEEVKRMRMRQ








REDTAEEEALGRSLTKLPAR








LGGLGLLSFKDVAPLAYRSA








AEASDTLLDNLGLLSSPEEP








PTPIPQRTRCAELWESQQEA








ILHNLGDTERKRLTENASRL








GRSWLSVIPYLQPLRLSNVE








IASGLHDRTLVGSSIPVCRF








CGSDSPLGHDELCRARNPWT








QRRHNAINRVIYQHLKQIQG








ATVEIEPHTLSGQRRNDLRV








RGSSALAFTDYDLKVYSLGD








RDARSTVTPCAPNGKLADFC








LDRCVNWLDKVGQVVSKNAP








KVTGGVFKPIILSTGGLMSR








STADEWKDWRDAMPVGGFEK








MEKRIGVELVKARARTLVL








(SEQ ID NO: 1397)





CRE
CRE-
.

Chondrus

ACGCCCCCTATCCATTTCTG
TAAGTCCTTGACGCCTGCCC
MSQPNISSAETPLSQLPTPV



12_CC


crispus

CCAGCCTCCCATCGGCTCGC
CGTGATACAGCATCGGTACC
PTPPSPSNPSLSLPTVRDLL



ri


CGTCTCCGCAACCCCTCTTC
CCTAGCATTTGAATAAAAAA
LCPIRSSHVYSSIPSSCLHS






CTCGGCTGTACCAGTTCCGC
(SEQ ID NO: 1276)
FTMLLIKTVRAASATMTPTE






TCCCACAACCTCCCTCGCCA

SHRAFIHLHILPIAVLRRSF






CA

RGETGWRSRTGQHHALRQRI






(SEQ ID NO: 1153)

RRASSGRHWAALWHEALAAH








QVDLDYRTRHSRRYQASATS








RHRIGRAMRLAADAQYGRAM








SALKAKPLPDLHAAATRDTL








TALHPPPASPVQPLSPTDLP








PVPEITEGQVLRAARALNPT








SAAGPDHLSPRILQLLARTT








ISPEAGVTGLSALTNLVRRL








ARGDIPDRTAPLLAAATLIP








LQPRPHKIRPIAVGQALRRL








VTKVLLPPAIQDTRDHLLPE








QLANSVASGMDAIVHDTRML








MHRHGRNPDYIMVSVDARNA








FNTFSRQSLLDRLPLQTPSL








ARFLNLIYGRTVPDLVLPSS








PRFLMKSQEGTQQGDPASML








LFSLAIQPLLRRLTRECRLD








LNRWYADDGTLVGPISEVIK








ALRILRDDGPQSGFHVNINK








CRAYWPTVMPEKLSELLRIF








PLHVECGEGGVALLGAPLGT








DAFVRRHLMNKVQSCHASLS








LLDEIPDARTRFHLHRVTGS








VCKVEHVFRLTPPHLSLPAA








TKFDEQQIAAYSRLNDVAVS








TSMATQIGLPFRLGGHGFTP








LSPFIHASYAASLIEAAPVR








VKGPHNPSESFYRRMARRHI








VHVLGALNPEVRTRGILGTH








SPLGPFEPEALLSRPERVHH








TLIQAMQGATSRLYWEHTAW








DLDPLPRNHSAASVRRRARY








NSLRAPGAASFLCSHPSLTS








RVPSAVWSCMLRRHLDTPVY








CDSIRPLICSHCCKPMDARG








DHAAICRHGFGVVHRHNTVR








NLLARHAFRAAGLCCDLEVP








SLLPNTANRPADILVQPAPP








PSGALPDRPTAYDVTVRSPY








CRSTMSLAAKGLAGAAEAAD








LDKLRVHSRTVRDAFHLQPD








SPLPLLDWHFVPLAFDTLGA








TSSRTMAVLEYLAHRIANRT








YSSYGTAKIRLLORISFAVW








SSLASATLSRMPYHGAALSS








PAQV








(SEQ ID NO: 1398)





CRE
CRE-
.

Chondrus

CNCCAGCCAMCGATCCCGCC
TAATTCACCTTCATATCTGC
MAXXPXISPPGAPPAPLRYR



13_CC


crispus

GCCACTCGCMGCCCGGCCGT
TAGTGTCTCTGTAAGCGCAC
MLQCPPPLPKXXXXPVPHPM



ri


CTCGACCGCCACCTCCCCGA
CCCTCATGCATTGATAAAAT
SSPIRXRLPHRXMRGPPSXT






SGCCCCAGCCCATC
TACCCCCCA
PPRDMHRPHGTPGPHSHRXC






(SEQ ID NO: 1154)
(SEQ ID NO: 1277)
GRPPXHCTHASXQPRXAXHX








LQXPKLRSPPPHPHVSPLIL








CXGPLPTPMTQPRMKRALSX








SAKAPPTKRPSASQGPAASS








HDXPRTPPPXPPRPPPYRFP








PPTLDQHXFALSXAYPHPXP








RRPPSPXRXLRHSFPPRFGX








QTFSSIPGPRLHSTVLLLIR








LVRAATAANTPETTTLXSCT








FTCSRLPFFERPSXAXLAGG








PRAVNFMLSACXYGERVXDE








SGXSYXXKXHCITSHPPRPR








RYSRQHSRNHHTXFLHLHLL








PTAXLREAFRGEXGWRSSRG








QLHALRLXIRRACTGREWGL








LXXEALDAHSXRTEWQHTHA








RRPSPPVSPSARAARAMRLA








SQAQYGRAMRTFTNPPLADL








NDPATMERLQALHPTPTVPV








VPLPPSAQPRPPEVTXEAVX








RAVRRLNPNSAAGPDRMSPK








LLHLLAHTPISPEAGVTGLS








ALTNLVSRLARGSLPPCTIP








LASAATLLPLQPRPGKIRPI








AIGQALRRLVTKXLLPAAID








DCRDHLAPEQXANGIPNGID








AIVHDARMLVRRHGNDPHYX








MVSIDASNAFNNFSRQQVLD








QLPTRAPSLSRYLDMVYARA








PSPLVLPSXPPTILHSRXGS








QQGDPASMLLFSLALQPLTR








LISRECXLXMNRWYADDGTI








IGRIDEVXKALDIITKEGPR








FQFFLNPSKTRVFWPSRQXD








LLSPLMTVGPLRVIDEGGVX








LLGAPIGSPSXMAQYIREXL








NTCKTALAHLDHIPEARMRF








HLHRVSASACRLQHLFRLVP








PDFAXPFAQQFDRDQLXAYX








RFNSVTMSPRIVPKYGCXFX








TXATASPHWHLPYTPXTLLA








SSIPLQHGYKVPTFPPSLSI








SVLHEARCGSFFEIYLHSXN








PHTSRCDYVAKNRAQIRLXF








SHGGHGLTSLASTIHASYAA








SLIDTAPARLQGPHFPAVSQ








YQRFARGPLRVVLRNLPSFV








QPAHFSMTEXDLGCLEPXAL








LARPERIHTFLLQAQYSAAA








SSYWQXPLWESFPNPGDHSA








ASLRKRVRYNSLLAPGATSF








LTAHPAATSRVHNATWSTML








RRHLDAPVTNDSISPLRCXH








CSKPMDARGDHAXIXSHGFG








TLHRHNTVRNVLARQLFRVA








GLAYSLEVPFLIPNTAARPA








DILVQPPPPAPGLPPDXPTA








YDVTICSPFRRGMLYHAARH








RGGAADAASVRKXKALERTI








RXALLIEDDNXPPPLDWHFQ








PLSFDALGAPSQSTVHVIED








HAKLMALRNSCTIATAKSRI








QQRLSFAIWSSAAAAILSRL








PTHAADISYPIEV








(SEQ ID NO: 1399)





CRE
CRE-
.

Acanthamoeba

TAACCCTAACCCTCTCCCTC
TAAGCCGCGCGACGAGGACG
MATTTISRSPSSSSSSSSAR



1_ACas


castellanii

GGCCCCTCTACCCTAAAGCG
GCCAGGACGACCAGGACGAC
SRASASTSASVASIPRLFRD






CCCTAATCGACCGGCGACGC
GGCGACGGCGACCACCTAGC
GRFHCPLAHCQTRTSTWQDL






CCTAATCGCTACCCTCTACG
ACCGCACGCGCCACGACATA
SAHLTRMHDGDVPRDVAAAC






CCCTAATCGACTTTGGCGCC
TTGTCGCGCGCTGTACAGGC
GIVQCLHEGCRKWFRGAAGL






AAAGCGACTTTCCCCGGCCG
GGCTAGGTCGAGCCCAGCCG
ASHRGKARHAPPPAPRAALA






ATTTTCTTCCTGCCTTTTTC
ACCGTTCTGAGCCTCAGTCG
VAAVPRADSRGRTPAPTPSV






TTTTCTCTCCAAGCGACGCG
GCTTGAGCCCCCGGCTTCCC
APPXAGPPPRAAPRAAPSPL






CCTTTTACTTTGCCGCCGTT
AAGGCCTACCGGGGCGGCTC
PCPPALPHPPPSASPPTSSV






CTGTTTTTTCTTTTCTCTTT
TTTTTCGCCCTGGTTTTTGC
TSPCSPPTTPPSQPSPDLFS






GCACTTCGCTTCTACTTCAC
CGGCCTGTTTTTTCTCTCCC
GFANAPTTPSPPSTPXSSPA






ACCTCCTCCTCCTCCTTCTC
CCTTTTCCCCCCTTTTCCAT
GSPIPAARRFVLPVATPYPA






GACCCGCGCGGCCTCGAGCG
TTGTACTTAGTTTTTCCTTC
PAPRANRPKLSPVARPFVPK






ACTTGCTGCAGCGGCTCCCG
GGCCGCGGCAGCTTGTTGCC
ARAGAIPEASSPVTPQDRAV






GCCTCCCCCACGCGGCCTGC
CGGCATAGTGTTAATATGTT
SRREDAAAAPSSAPGLGLAD






TACTCCCGCTTTCTAGACGC
TAAAAAACGTGTAAATAAAT
EHEDDDTYGGDTIALTAPHA






CCCCGGTCTTGCTCTCAGTC
AACTGTTTAACCCTAACCCT
PRETRAPFEFEACFLEEEAP






TCCCGCATCGAAGCGGTAGT
AACCCTAA
ATAGDLPPYARAFLACPSAR






CGGGGTACGTGCTCAAGTGA
(SEQ ID NO: 1278)
LQEIPRRLKSAWQAAAKTIA






CTCAAGCCTCTTTTCAGCCT

EAALDCHTAGDTQGYNAHLR






CGGCGCTCTCTCAATCCGCC

LFIELPARGLAVPTNCRGAA






TCAGTCTTAGCCTTTCAAGT

RTKLQRERLLDIAAGRIPAI






TGCTCGATTACGCTCTCGAA

PDPPCDAPGADDALRGFPVS






TCGCTCTCTCTCTCAGTCTC

GTTAGDVSNDDDSGGVHDRP






AGTCTCAGTCTCAATCTCGA

AATASARQAKRLVEQGLSSR






TCTTGCCTTCGCCTTCGTCT

ALRALERGEPAVASADTLGR






CGACGCCTTGCTCTCGWAAT

LEALHPPNPTDRGLWPGAPK






CGCTGCCACTACGTGCCAGC

AAIPRVTAKHLAQVAKELPR






TTTTTCGTGCCTTGTCTTCG

GSAPGPSGWTFELVQAAIDR






TGTCGACCCGGACCGTTTGC

QPTGTVAAFLIDMAQRALRG






AAGCCCTCGCCTTCGTACCC

TLHWRGLLTASRLVALKKPD






CGCTCTCGTAGCCGTTCTCA

GGVRPIAVGEALYRVIGRLV






TCGCTGAAGCGTTCTACGCG

LKADRVMSSADATQYVGRHQ






CTGGCAGCAAGCCTCGGCCC

YGVAYPGGVEAPVHAVRELH






TAGCTTGTAGCGCCGCCGGT

DSGQLRAVVSLDWRNAFNSL






GGCCGCTCGCCAAC

DRVHTALLIADRAPALARLY






(SEQ ID NO: 1155)

EWSYREDSVLVLPRAFEKAG








LPASLLSQAGVRQGDVLGPL








FFAIGAAPVLDEIDAIPYVT








PRAYLDDIFVTIPHGVTDAA








TKAAVAATFATAEREGAAAG








LRLNRCKSAVWAADAEALLP








PHAAGAREDVESCAPVREGL








KILGAPVGSPAFVAKSLDGI








IKRAIGTLDLVADAELPLQH








KLVLLRQCVAQIPTFWARAV








PDAGPALAVWDTALLRRTGA








LVGLDVRDGSLQADIARLPV








RLGGLGLRSMKDTAPRAFVA








SILFAAALANTRRSELTCSA








STARRLRAALPELARTDACN








DEAAWRRSIARGVFPDVDKL








GTTQLQRVLQGMADSKSAHR








TRRQVPFLFAAVFEDAATPG








SGAWLAAIPSDPTLVLPDAE








LAEAVRIKLLTTTANAAGVC








PACHKTGIDPSHAYTCVSLS








HLRTARHDVVVRRVELACKT








EKPVREHVLAIPPVAPTDNN








NNGDEDGSPVTTADDNADGH








AVATKRRPETRASARAAAAA








ATAAAAAAIINDNSLLSDDD








DDDDHDDNCHGEERGEGERN








VTCPGHYTATPFAADDTLDN








SDEDNEDNAHEDDDEDGKDD








NDDDVYNNCNSSSSDGDEGG








DDLDYEYSDQSVTRSVDAAT








GESPNPERPTTPTRALLRAD








LWLPATSTAVDVMVAAACRR








SRAKAFDRAVSRKAAKYGPA








VADGSIAKVVPFVVSPFGVL








SRPAKAFLKRAMGDTTAAKQ








AKARLRLAVAAVRGTARLSY








AWGACAALIVGGN








(SEQ ID NO: 1400)





CRE
Cre-
.

Fragilariopsis

ATCAATCTAATACTGAAGGC
TAGCACCACCATCTATTCAT
MAPLPWNAATSSPPSPVPLT



1_FCy


cylindrus

AATACCAAACTCAACCCGAA
ATCCACACACTGACCACCTC
NDKKKDSTLPTATSKNLSKN






ATCAAAATCGTTAGAATCAA
CACCTTCACAACTCCACTCT
NNNKNNNTNRINNIKNNDNT






TATACGACCCCCGCTGCTGT
CAATTCCCCTGACTACTAAG
NDGSNKINLKLPPAAVKITN






ACATGTCCAGCCGGATCTCG
AAATATTTCATGGTGGTTAC
PYKNKKKNKKKNNAGKSNPK






TTGTAAAGAAGATTGCAGCT
ATTGGAGGTATCCCAACACC
TNQNPNSSPLSDNDDDDTDS






GTAAAAAAGTTGGACTTCTT
AAGCACAAAATGAACCCACT
SNITINRRLKFGTDDLAPPN






TGTTCTTCCTGTGAAGAAGT
AACCCTCTCATCCTATCCAC
PPSNTNTIGTATAATAATAT






TGATTGTGGCTGTTCAAATT
GGGGACCACCTTTGAGCAGA
TTATAATATTATNTTTTTTT






CTTTTCATAATAAAGAATTA
ACACCATTCATATTACAACC
TNNTTGDNLASNINNNNNNN






(SEQ IDNO: 1156)
TTTAGCTAGATTAAGATAAT
NSGSNNSNTNNINNTDGNGS







TATTTAGTACATATTTTATA
NNRPPPRVYTVDPRSDLPGA







CTATTAAAAAAAAAAAAAAA
EISAANKMLDEVYGDHVHDN







A (SEQ ID NO:1279)
PGSHLSGLISSSQDQLWQGY








FRRLIPHNQSLYDCPKGKLG








KDITNEYSNLFEAIMNGKCN








MEKLLVFPVVVLQRRHGVTK








NADVKRRLLSRLTAWKEGKF








KYLVEDTHRDLIAKQSKARG








DTTPAHRAKVYSSKLMRGHL








QSAVNYITDREGGGILYPYD








VDEKSGHTVSRVLQDKHPSM








RDPGPTAMPAYESVPELPTL








EITADTVEIVAGKLSGGAGL








SGVDSIQLKHLLLHHGQASQ








RLRNVCAKFGRWLANEHPPW








ASYRAMLANRLIALDKMPGI








RPVGIGDTWRRFFAKLVLAV








SMSYATDCCGSDQLCAGLRA








GVDGAIHGLSAMWREMESEE








NTGFVLIDADNAFNEVSRIN








MLWTIRHEWPAGARFAFNCY








RHHSLLVVRNPGGKPFTFFS








KEGVTQGDPFAMIAYGVALL








PLIRKLKELNVLLVQSWYAD








DASAAGKFDEILRLFQDLLR








MGPDFGYFPNASKSILITHP








DNVVAAHHFFNETHGLGFKI








STGSRFLGGFIGDTTSRDEY








VSTKIADWIHGTKELAAVAR








LKYPHAAYTGITKCLQHKWS








FTQRVIPGIDDLFQPLEDEL








TNNLLPALFGDPPSTMDDKL








RLLTALPVKHAGLALPNPVT








SSATNYKNSTLMSSHLLLAV








QGKINFSLQDHRDTCQSSLS








ASRELRQTENDSSLTNLLAA








LPPAAAGQPSTTRAIKRAGE








TGLWLTTIPNHINGNILGCD








EFIDAIRLRYQKVPHNLPAK








CDGCGSAFDVGHALQCKSGG








LIIRRHDELNLELASLAKMA








LRESAIRAEPEINPSASIMD








SPTTITAIDTNGDRGDLLIK








GFWDNGMDAIIDVRITDTDA








KSYRTRDPKKVLQSQEKEKK








KKYLDQCLLQRRAFTPFVVS








VDGLIGYEASNVLKQLSKRL








ADKWNKPYSVTCGIVRSRIS








IACARASNQCLRGSRIPFKT








MSRQIQWEDGAGAGLYRIVR








(SEQ ID NO: 1401)





CRE
Cre-
.

Hydra

TTTCTAATGTTACGTGATAT
TAACTTGTATTTTTAAATTG
MNMVSICKRCDRSFTTLKGL



1_HM


vulgaris

GATATGGTTAGTTCATGGTT
TTTTATTAGTTT
NIHKGQCKIFVSNTNKQINN






AGTTTATGTTTATGCTTAGT
(SEQ ID NO: 1280)
VVNNELTTPNKNKVEINTIL






TTATGGAAAATCGTTTATTT

NCDEISVEHYSTNTPYLPKI






ATGGCACAATATTGTTTGCT

NICESIIDPNDYLWGHMPFS






GTTTTTAAATTTATGTAACG

FLLNHVNTIYDEIVFYHKNL






TGTGCATTTGATGTATATTC

FKVPSGKGGKMFIEELTFWL






TTGAACTTTTTAATCTGAAT

KQFNNRTKLNGIAMKCFMIV






TTTTACTTGGTTTAATACGT

PSLMLQKPSIRSKAKEHAEC






TTATTATATTCTTCGATTGA

LVRRITLWRNGNFSELMREI






GCAATTTATCCTATCAAAGC

RYIQSKINTSKKKRTFEDIS






AATTTATCCTTCGATTCGAG

RIFAKLMMEGKVAAALKVLD






CAATTTATCCTTCGATTCGA

RESSGILQCSESVLKELKSK






GCAATTTATCCTTCGATTGA

HPDETPVQDNCLLYGPLQNT






GCAATTTATCCTATCAAAAT

PECLFDSIDEISIFNSALQT






TAGCATATATACTGCAATTT

KGSAGPSGMDADLYRRVLCS






TCAAATAATCTACGAAATAA

KCFGPSCKTLREEIATFTKN






GTTCACTTACTGAAAATCAT

IATKSYQPDIVQPYIACRLI






TAAGTAAAAGAAGAAAGGAA

PLDKNPGIRPIGIGEVLRRI






GAAAAAATAAAAATAAAAAG

VGKTISHHCQKEIKEAAGPL






TAGTAAATCCTTTCATAACA

QTCAGHGAGAEAAIHAMQKI






ATAATCATTCTATTATTAAA

FHQEDTDGVLLIDARNAFNC






TTTAAAGGAATATTTTGGTT

LNRSVALHNIQITCPILAMY






TTGTACTAAATCATGCGTTC

LVNTYRKPAKLFIYGGETIF






ATATTTCACCGAAGAAGGGG

SKEGTTQGDPLAMPWYSLST






GCTGCTATATTTTTGTTTGA

VTIINTLKLVIPDVKQVWLA






AGTTGTTTATCTTAAAACTT

DDATAAGKLQSLKKWYKCLE






TAAACTTGTGTTCAACCAAC

DVGGLYGYYVNQSKCWLIVK






CGTAAACATTAGTTCGCTGT

SDNQAEEAKLIFGNSINITT






TCGCTCAAATTATCTACAAT

QGKRHLGAALGSEAYKKVYC






ATAAAATTTATCAATCTTTI

EDLVSKWSKELNNLCEIATT






TTCGTTACGGTAAACAATAA

QPQAAYSAFIKGYRSKFTYF






ACAATAAAATAACTATAGTT

LRTIEAFENFVTPVEKILSE






ATTTTATTGTTTACCGCATA

KLLPVLFGTDCSIIKENRDL






TTGTTTAACTATAGTTAAAC

LALNPSEGGLGICNLITEAK






AAAGTATTTGTTTATGGAAC

EQHTASKKITNLHIKSILDQ






ATTACCAGTATCTCTTGTTA

SDVMKEKDDFGKTFSEIKTK






AGGTAAACAACAAAACATAG

TNMDKSKKKKEEVKKIHAGL






ACGGCATCTCTTTTTAAGGT

PENLKLLVEQACDKGASSWL






AATTAAGTATACGGCTAATA

NTLPIKEQHLDLNKEEFKDA






ATAAAAATATACAGCTAATA

LRLRYNVPLANLPSYCACGE






ATAAAATCTTCA

KFDELHAMSCKKGGFVCNRH






(SEQ ID NO: 1157)

DNIRDLLTVCLNKVCTDVQA








EPHLIPLTNEKFNFKTANTN








DEARLDIKAKGFWRKGETAF








FDVRVTHVNSKSSKKQPTKH








IFRRHEDAKKREYLERVLEV








EHGTFTPLIFGTNGGFGDEC








KRFTALLAQKLSLKMGERYG








AVINWLRTRLSMEITRASLL








CLRGSRTPFRHYNTDDVGLE








NVQCGLI








(SEQ ID NO: 1402)





CRE
CRE-
.

Lactuca

ACATTAAATTAGAGAGGTTG
TGAACTATATTTTATATATT
MASSSTSSSDICLCPFRSFH



1_LSa


sativa

ATGTTTCAATGGAAGAAGAT
AAAAAAA
CCPNGEVGSKGIXRMISHIK






GAAATTCCAAGAAGCTATTT
(SEQ ID NO: 1281)
RHHLLTEDRKCVLREALSSD






TTGTTGCCCACCAAGTGTTT

VGLFMAVEETLKAFGQWMCG






GATAAAATGTCCAAACTAAT

KCMTLHALSRYCHHPDGRVX






TTTTCTCTTGTTGCAGCTTT

FVTGADGSSRYIVGILKPST






ATTGTTCAAGATAATGTAGT

KESVTNALGGLVFDVGLLDR






TTGCTTAGTTTGAGCGTTCC

VFKEPITTVKSIPHSCRLAF






TTGTGCACACCAACAGTGTG

SQALKTALYKVIAQPGSVDA






TTGGTGTGCCATTTCCTTTC

WICLLLLPRCTLQVFRPKNR






CTTCCTTTTTAACTATTGCT

QECRSGNRKSLQQSSILKSL






TCATAGCTTAAGCTTCATCT

DTWGKEDGIRKLVQNMLDNP






CGAGGCTTGTTCTCTTGT

EVGAMGQGGGILQKESTSSN






(SEQ ID NO: 1158)

TNIRQCLRKVADGHFTAAVK








VLCSSGVAPYNGDTIKALED








KHPFRPPPSMPSPIISEPPL








VADFDCVFGCIKSFPKGTSC








GRDGLRAQHXLDALCGEGSA








IATDLIRAITSVVNLWLAGR








CPTILAEFVASAPLTPLIKP








DNGIRPIAVGTIWRRLVSKV








AMKGVGKEMAKYLNDFQFGV








GVSGGAEVVLHSANRVLSEH








HADGSLAMLTVDFSNAFNLV








DRSALLHEVKRMCPSISLWV








NFLYGQAARLYIGDQHIWSA








TGVQQGDPLGPLLFALVLHP








LVHKIRDNCKLLLHAWYLDD








GTVIGDSEEVARVLNIIRVN








GPGLGLELNIKKTEIFWPSC








DGRKLRADLFPTDIGRPSLG








VKLLGGAVSRDAGFISGLAM








KRAVNAVDLMGLLPQLCDPQ








SELLLLRSCMGIAKLFFGLR








TCQPVHIEEAALFFDKGLRR








SIEDMVVCGGPFFGDIQWRL








ASLPIRFGGLGLYSAYEVSS








YAFVASRAQSWALQDHILRD








SGICGMDSDYLCAMTRLRDT








IPGFDCSGFTNKDTPPKSQK








ALACALFSKIVKDMEVDFDM








TVRQKAVFECLRAPHAQDFL








LTIPIDGLGQHMSPVEYRTI








LRYRLMIPLFPIDEICPVCR








KACLDTFGEHAVHCRELPGF








KYRHDVVRDVLFDACRRAGI








SAKKEAPVNFLTDPQDGRST








LRPADILVFGWVGGKHACVD








LTGVSPLVGLRSGGFTAGHA








ALKAAACKVAKHENACIENQ








HVFVPFAFDTFGFLAPEAVE








LLNRVQRVMHSNVISPRSTD








VVFKRISFAIQKGLAAQLVA








RLPSIDMY








(SEQ ID NO: 1403)





CRE
Cre-
.

Monosiga

CATCTTGGCGTGAACCACGT
TAGGTAGGCACCGTCTCGGG
MATESGGEDSWTQVRGAKRP



1_MB


brevicollis

TGTCAGACAAAATCTGCAAC
GGTCCCTCTGTGGGGATCCC
SAESPPSNTTTSPSQTHRSA






CCCGCTCTTTGCGGCCCGCG
TGTGTGCACCTGTCGCTCCC
KHTKHGSARHDRNHVFPDPM






TTTTGGCGGCGCCCTCGCTC
TAGGTGGTTCCTCGTTGTGT
TTPLRPHARHSVPTARASSH






CCACCGTGTCCGCTCGCTTG
CTTTTGATGGCTTGACTTGT
VPSTSPAAGATESSARAVVP






CTCGCTTGCTTGCCCCGCGG
ATTTTTGTTTTAATTTTGCT
AAEPVTRTSNGGGEQHPIIG






AC
TTAATTTTTGCTGTATTTGT
NTSNASPRTPRTPSSPRSFA






(SEQ ID NO: 1159)
GTGGTATTTTTGCTGAATTT
QVAAAMPAAATATSSAPMTE







TTGTAAGGTCCTTTGTATGA
DLSASVPSEPNGSGEQQPSP







TGTCTTTGTCTTCTGTGGTC
ESTGQTHHSIPNTPSDFLTM







GGTTGTTTTCCTCAATCCGA
SSDESDSPPRSTALRAPTPI







CGTTGTGTCTCGTTGGATGT
APPAHDGDGDTNGSATPEPL







GAGCGTGCCGTGGTGTTCTT
VQSPTPAQMVLPYPSGTQQT







TGTGTTTGTGCTGTGATGGC
HSDPSPPSASPPATTILPAA







TTGTAGTTGTGATGTGTGAC
ISHPVEHSEHANSAPLGEVS







TGCCTTTTTGGGTGTCTTGT
ESETHNTAGEHSESEQDVLL







GTTTGAAATGGCCGTATCTC
SDPAPPIAANVLDAQRKVLL







TGGTTATACTTGGTCGTTTT
KTSGHRQLLACPFGLCKCKG







GTACGATTTTTGTTTCTATG
PRLDRKAWVNHVLREHPYDE







TGCGTGATTCTTCGCGCTTG
QATDLVKQVMEAKLVAQCNK







TACTTCTTGGCATGATAGAA
CHLFFEAAGISQHRSRCGAN







GCCAATGAATGTGTCTTGTT
LKRATEALFHAAGHDLLEIM







CTCTTGTGTTGTTTTGCGTG
RGAWPQQCVGSRISVCELLK







CCGTCGTGATTTTGATGTCG
LARHPLMQRSRYPSNATETK







GGGTTGCACAGCTTTGCTTT
LMAATLSQLYWSAVHSDYTA







CAGCTCTGAGGTTCAAACAC
EEREMCWALILALPSMLLSA







CTAATTTA
PSTALSTIDLRNMFHDRLRW







(SEQ ID NO: 1282)
LVTGQLGRVVDAMRKAVARK








QSRRGQLNAGAGAHPNDAVD








QSLRSLVRDPDLADEAWANH








VTNRLNRGQIAKAFDADKAR








AVIGNSEVQAVRDLLVPPGL








TPYIASTPASTSTLAPATAV








SSPTVSFTKGELPKALAATK








GVTDPYGWSGELLASIYRIK








EHFSQVLGPRQGSTSDPTAP








SDGDAPQGPTTATGGPQVAL








NKIFHHIANNTVPESIRHAL








CSINYTILEKANGKFRPVGT








DSIFNKVVNRALLEQQQPHI








AHLLQASPELAVGVKDGISA








AVGMAFGELQACESTPGWTM








LSLDFKSAFNYTDRARLHEI








VADKVPGLLRAFERHYARPT








THCIVDKFFKVIDIDVGQGI








VQGNELSPFFFALYSCEVLG








LLDATTDYRCKVIKYLDDIV








LMGPAEDVAADVEIVKARAE








SAGLHLQPSKSRFYMPRHHS








ASITAIKSVLPDAVRETANT








GMTVLGTPIGRREWMKKQLN








DKAKHIAGKLNDMLTTGVSL








QALLTAMQYVPSLINHLYTL








PPSLTSGLSELLNRACKDTF








VKAFFAKVNLSAPAGAEGHD








VTLEQLLEARLFTRANTGGF








GLHDLVERGPVAYVCNMAKL








ATRYPRVYDRLLEDASRAAD








FEAHVORAGFQMATVKDAAT








QRPAEIIALRSKAALDDLMA








KCALDLQQAYLASREWGVST








VLTMRGRDKLRRLSDTTFAI








AVVSMMGFGLHELINVKPTD








KCPLCSSKTPQPRLTREHLL








TCRPIKRHNALRDEMGRLLR








YATLSHVWVEKSGYNANGQS








CRIDLHCRNPFPGGALGPAL








PDLGIDVTVRTAQPPTTSQA








CIKVGAALRRAEKEKRDYYT








GFNHGKTLIVPAAMTTTGGF








ASSFVDLLGQLARCAEARGV








YQPGLDEAFVPRWKGRFAAL








VHQMNADHIQRHFGGVCLRS








S








(SEQ ID NO: 1404)





CRE
CRE-
.

Hydra

AATTTAAAAAAAAAAAATCG
TGAGCTCTTATAAATTTATA
MSSCKVTIPHVCPYCKVELK



2_HMa


vulgaris

TTTATTTATGGCATAATACT
TTATAGCATTTTGTTTTA
TICGINRHILKCKKNPLQIP






GTTTGTAATTTTTGAAAATT
(SEQ ID NO: 1283)
SLQKTNTSLTLEPNTKVIPS






CGTGCAACAACTGCAGTTAA

ITKQNDIIIASTSSNNLAFN






ATTGAAGAGCTGAAATTTAA

QKKDYTLTPTYSRKTTPVSI






GATCTGAGCTTTTCAATCAG

LSSMKMTPISITSHIVRRKL






AGTTTTTTACCCTAAAACAT

PELPSQTTNHLFNENFINVP






TAAATTTTATCATAACAAAA

FLPEIMNHLPVPNNNVMWGV






ATCGTTCTAATATTATTAAA

YSYQQFKLFVDSTYDEIVNY






CTTAAAGAAATTCGTTCTTA

RRNIFNIPSGKAGKEFIEEL






TATCAAATCTTATTTCAGTG

TFWLRKFNSTSSLNSIALKV






TTTCACAGACGAAGGGTTTT

TMILPNLLLQKPSAKSKSKE






ACTAGATTTTTATTTTTTCA

HTLCLTRRIDLWKKGDTSLL






ACTTTTGAATTTGTTTATTA

LKEVRNIQKKFVNSKXKRSM






TAAAACTGTAAACTAGTGTG

DDISRIFAKLIMEGKITAAL






CAACCAACCGTAAAAAWTAG

KFLEKEASSGILPLSDNTLK






TTAGCTGTTCACCCAAAATA

DLKSKHPEPSRVEDYSLLFG






TTATTCAGTATGAAAATATT

PIDLIPKCFFDCIDEQLVMK






TAATCTTCTTTATTCGCAGT

AAFATKGSAGPSGMDADIYR






AAACAATAAAATATCTAGTT

RILCSKNFIKEGKELRKEIA






AAACAAAATATTTCTTAATA

KMTQNLLTETYEPTFLEAFT






ATAAAAAACAAAAACTTTTT

ACRLIPLDKNPGIRPIGVGE






CTTAACAAGTACA

VLRRIIGKVISWSFNSEIKE






(SEQ ID NO: 1160)

AAGPLQTCAGHGAGAEAAVH








AMKEIFDNVQTDAILLIDAK








NAFNCMNRQVALHNIQIICP








LISIYLINTYRNPSRLFVAG








GKEISSQEGTTQGDPLAMPW








YSCNTTIIIEHLLVNYPQVK








QVWLADDAAASGSIANLHSW








YQHLIDEGCKHGYYVNQSKC








WLIVKSPSLAENAGIVFGKS








VNITTEGQRHLGSVIGSQNF








KNKYCTEKVAKWLTELKQLC








KVAETQPQAAFIAFTKGFRS








KFTYFLRTIPKFEQYLAPVD








EILSHLLLPTLFGKDTPFED








HIRKLFTLTPRDGGLGIPIL








VEEAPHQFLSSVKLTKNLVQ








QIIDQDKILKTKNSSGNVLE








DLEKILTTDRLKHRKEKIIA








VDSMQPDSMLRNIQQTRSEC








ASTWLNALPLENQGFVLNKE








EFRDALCLRYNFDLKNIPRI








CECGEPFNVTHALSCKKGGF








ISSRHDNIRNLFTTLLKRVC








INVQSEPHLIPLDNENFYFH








TANKSNQARLDIKANGFWRN








GQTAFFDVRVTHVNSMSNKN








LDIAAIFRKHEKEKKREYGE








RVREVEHGSLTPLVFGTNGG








MGKECHRFVRRLAEKLAEKQ








NEKYSVVMTWLRTKLSFEIL








RSTILCLRGSRTPWTKKNDF








EIGVDFKMDALEARI








(SEQ ID NO: 1405)





CRE
MoTe
JQ747

Magnaporthe

CCCGAACCCGAACCCAAACC
TAATAGGTAACGTCCCTATT
MVCPTCNGVYADYNDHIRKK



R1
487

oryzae

CAAACCCAAACCCAAACCCA
TTTGTCTTTGGTTTTGTTTT
HPDERYTALQLQPLGLTPCP






AACCCAAACCCAAACCCAAA
TATCTTTGTTTTTGTTTTTG
ICKTACKNDLGVKTHLSKIH






CCCGGAGGGTTCCCAAGTCG
TTTTCGTTTTTGTTTTTGTT
KISGASKISTQPRIRTENTD






CCTAAACCCGAAGGGTTTAG
TTCGTTTTTGTTTTTTTTTT
NTNSVPTSSFNPVLPEIQTL






GATATTATTTCGTTTATTAG
TGTTTTTGTTTTTGTTTTTG
TPGLNNSRWADNPRKRRADT






AATTGGATAATTATTTACCC
CCTTTGTTTTTGTTTTTATC
PSPTRGRNTRPRRFSYTDID






CTGTTGGACAGGGGGGTTGC
TTTATTTTTGTTTTTGTTTT
LTNDEPADNPRANNPRVNNP






AGGGGTTAAATTAAGGTTTT
TACTTTGTTTTATTTGTTTT
RVNNEPPSSPNSLPSISEFH






TTATTATTTATGCGCCGTTT
ATATTTACCTTTTGATTTTT
TPGTLPLTNSNISLKDQHDK






ATTTGTTTACCCCCCCAAAT
TCTATTTTTCCCACCCTTAT
ITGPILQKPLIQKLIEYSKI






ATTATAAAAGCGCGTTCCAT
TATTATAACCCCAACCTACT
PIPEHHLHARQAKIFADAAN






CCTCTTAGGAAAAGCGAAGC
AATATTTTTTCTTTTTTCTT
RIAKNFIQSPTEKTLFNLLI






TTTTCCTTGTAAAAGTCGCT
TTTTCTTTTTACGGTTTTAT
LPRIFGIGLINGKVTKIMQN






AGACTTTTACTATAAAAGTC
TTTCCCGTTTGTTTTTTCTA
FPSQIPPIPKIDFPSEKTDS






GCTAGACTTTTATACCAATC
TTTTATTTGTACGACAAAAC
DPVLNAKKLLEKGYIGRAAK






TTTTAACAAAAAGCGTAGCT
CCTTAGCAAATAAGCTTAGA
AIIDPTPVAPETPESLNILR






TTTTGTTGCCAATCTATTAA
ATATAATAAAGCGCGAATTA
EKHPIGQNNPFNTKSQPISG






AAAAAGCGGAGCTTTTTTTA
AAA
RQITEKAILLAISSIGREKA






ACTTTTTCTTTTTTTTTTTT
(SEQ ID NO: 1284)
PGLSGWTRSLLDAAIKIPTQ






TTTTCTTTTTTTTTTTTTTT

NDVIPALRLLTDMIRQGTAP






TTTCTTTTTTTTTTTTTTTT

GRELLCASRLIGLSKPDGGV






TTTTTTATATATATTATTAT

RPIAVGDLLYKIAFKAILNT






TATTATTATTAGCGGTGGGG

LWSPNCLLPYQLGVNSIGGV






CTATTTATGCGCTTTAATTT

EPAIFTLEEAIMGPNINGIK






GTGCGGGGCTATTTATGCGC

SITSLDLKNAFNSVSRAAIA






TTTAATTTGTGCGGGGCTAT

SSVAKYAPTFYRSTCWAYNQ






TAATGCGCTTTAACTTTACA

PSILITENGSVLASAQGIRQ






AATTTTATTTATGCGCTTTA

GDPLGPLLFSLAFRPTLETI






ATTGCTGCGGGCCTGTTAAT

QKSLPYTYIAAYLDDVYILS






GCGCTTTAATTTACAAATTT

KTPVKDKIAKIIEKSPFTLN






CATTAATGCGCTTTAACTTT

SAKTTETDIDTLKTNGLKTL






TATATTTACTAATGCGTTAT

GSFIGPTELRKEFLQNKIQN






TTATATAATTGCTATTATTA

FESSINALKKLPKQYGLLIL






TCGTTGCTATTATTATTATT

RKSTQLLLRHLLRTLNSQDL






GCTATTATTATCGTTATTAT

WELWEKTDKLIADFVINLTV






TATTGCAATTTTATTATATA

TKRKKRPITDFVTPLITLPI






AACCCTCGTTTGTCCCTCGA

KDGGFGLLRHNGIAQDIYFA






TTTATCCCGTTTCTTTTCCA

AKDLTTEIRHKIQRISNDFP






TCCCATCGCGCGTTTTCGTA

QNQSPTATEILHLLHNGVLA






AGCTTTGGTTTTCGTAGGAT

DCKNGLTNAQLNALTENASY






TTGCTTTCGTAGGCTTTGCT

LGRKWLNILPIQKSNRLTDW






TTCGTAGGCTTTCGTCAGCT

EMAEAVRLRLLAPVKPLTHP






TTTACCTGCTTTTATTTTTT

CNHCGNRTNINHEDVCKGAV






CTTTTTCTTTTTATTCCCCC

RKYTARHDQINRSFVNSLKS






CCCTTTTTTTTACCTGGTTT

RPEIDVEIEPDLNNENNVNN






ATTAGCGGTTTACCTGCTTT

ANTTTENPTPSPNGQNDTGC






TATTACCTGGTTCCCCTTTA

LFTTPIRSGTRNGQNGLRAD






CCTGTTTTATTAGCGGTTTA

FAVINGVSKYYYDVQIVAIN






CCTGCTTTTATTACCTGGTT

KDSGNTNPLNTLADAANNKR






CCCCTTTACCTACTTTATAA

RKYQFLDPFFHPIIISAGGL






GCGGTTTACCTGCTTTTATT

MEKDTAQAYKQIQKLIGPVA






ACCTGGTTCCCCTTTACCTG

AHWLDTSISLILLRSRTTAA






TTTTATTAGCGGTTTACCTG

ISIAKNRPRA






CTTTTATTACCTGGTTCCCC

(SEQ ID NO: 1406)






TTTACCTGTTTTATTAGCGG








TTTACCAGCTTTTATTACCT








GGTTCCCCTTTACCTACTTT








ATTAGCGGTTTACCCGTTTC








TATTAGTGGGCATTTATTTC








CCGTTTTTATTAGCAGTTAA








ATTTACCCTTTTAAGGTTAT








TTACCTGCTTTTATTCACAG








GGCACCCCTGTTTTTACTAG








CAGTTAAATTTACCTTTTTA








AGGTTATTTACCTGCTTTTA








TTCACAGGGCACCCCTGTTT








TTACCAGCAGTTAAATTTAC








CTTTTTAAGGTTATTTACCT








GCTTTTATTAACAACCCTTT








ATTTTTTCCTATTAACGGGT








ATTTATTTACCTGTTTTATT








GGAATTCACCCGTTGGACGG








C








(SEQ ID NO: 1161)







HERO
HERO-
.

Branchiostoma

TTTTCAGTCTGGCTCAGCCA
TGATTAAAGACCCGAAACAC
MNAVCVCGKVCKNQRGLRIH



2_BF


floridae

GTGACCGCCGGGAAAGTCCG
CCAATGACCCCGGGTTCATC
QTKMACLRRVQAEHRSGAVA






GCTGACTACCACGAATAGGG
ACTGATGATGTGTCCCTGTT
TTVEPVLSASAPGQTEEDQG






TGGTGACAGCTGGATAGACA
CGCACTACCAGAGTGTATTC
PEAPHSARNLRATPAPPQGR






GACGACAGCTCGGAAAGACG
TAGAG
KSDHHRVKWPAANSKEWSQF






GCATTGGGGCAGTATGGGTT
(SEQ ID NO: 1285)
DEDVDMILESVSRGSTDQKL






GGCACCCCTAACTGCATCTC

QSMCTVIMSMGAERFGTIGQ






CCCTAGGAGAGCATCCCGCA

RKPTDTMKPNRREVKIRQLR






ACACGCTACAAAGAACCACA

QELKSLRRSFKASTSGEERA






AAGAGCAATACCCCCAGGGA

ALAELTHHLREKLRTLRRAE






TGCCCGAGAGGGGGGGAGGA

WHKKKGKERARKRSAFITNP






TGAGCATCCCATTCGGACGG

FGFTKRLLGQKRSGNLTCPV






TCCAATCGGTATTGACCCCA

EEINLHLSNTFSDASRDVDL






GCAAACGGAGAATCGACA

GPCPLLVTSPEPEVHFDISE






(SEQ ID NO: 1162)

PTLKEVRETVKAARSSSAPG








PSGVVYKVYKHCPRLVVRLW








RILKVVWRRGKVAADWRQAE








GVWIPKEEESSKVDQFRLIS








LLSVEGKIFFKIVAQRLIKY








LLDNQYIDTSVQKGGVPGVP








GCLEHTGVVTQLIREAKENR








GDLAVLWLDLANAYGSIPHK








LVETALTRHHVPESIQNLIL








DYYSNFWLRAGSSTATSAWQ








RLEKGIITGCTISVPLFALA








MNMIVKGAEAGCRGPVSRSG








TRQPPIRAFMDDLTVMTATV








PVCRWLLQGLERLITWARMS








FKPAKSRSLVLKKGKVAERF








RFTLGGTQIPTVSEKPVKSL








GKVFNSSLKDTASVQQTRSD








LTTWLEGIDKTGLPGSFKAW








MFQHGVLPRVLWPLLVYEVP








MTMVEQLERTISRFLRKWLG








LPRSLSNIALYGRSTKLQLP








LSGLTEEFKVTRAREVLMYR








DSSDSKVSSAGIHVRTGRKW








KAQEAVDQAEARLRHSVLVG








SVAVGRAGLGSCPKPRYDKV








SGKEKRLLIQDEIRAGEEED








RRCRMVGMRKQGAWTRWEHA








DSRKVTWPELCRAEPSRIKF








LISSVYDVLPSPANLHVWGL








AETPSCQLCQRRGTLEHILS








CCPKALGEGRYRWRHDQVLR








VLADTVSNAIQSSRSQQPPK








KSIVFVRAGEKTRQQPTSAG








GLLSTARDWQLLVDLGRQLK








FPEHIVATSLRPDMVLVSES








TRQVVLLELTVPWEERISEA








NERKRAKYAELVVQSQSNGW








RARCVPVEVGCRGFAGQSLA








YVLKLLGVRGFRLRKSIRDI








LEAAEKASRWLWFRRGEPWK








PHGHRSGNDQPRLGRPGEGV








W








(SEQ ID NO: 1407)





HERO
HERO-
.

Danio

TTCAAGCCTGGCGCAGCCAG
TGATCAACCCCGGCTGGGTC
MTHANEQTTNKIYVTCICGK



2_DR


rerio

TGACTCCTAGGAATAGACTA
ACCTGGGTGAGAGTGTATGA
LCKNHWGLKIHQARMKCLEQ






GGTGGCAACCAAGAATAGTT
TGTTGAGAGACCCGAAACAC
ESKVQRTGPEPGETQEEPGP






TGGTCGACTACTGGAGAGAC
TCAATGATCCCAGGATACAT
EATHRAKSLHVPEPQTPSEV






AGTTGACGGCACGGAAAGAC
CACTGATGATGTGTCCCAAA
VQQRIKWPPASKGSEWLQFD






GGCACTTGGGACAGTATGGG
TGCATCCATGAGATGTTTCT
EDVSNIIQAIAKGDADSRLK






TTAGCACCCCAGCCTGTGTC
TGCATAA
TMTTIIFSYALERFGCIEKG






TTTCGTGAGAGAGAACCCAA
(SEQ ID NO: 1286)
KTKPTTPYTMNRRATQIHHL






ACAAGCTACGGAAAGCCCCA

RQELRSLKKLYKKATDEEKQ






CAGAGATATACCCCCAGGAG

PLAELKNILRKKLMILRRAE






ATCCCGAGAGGGGGGGAGGA

WHRRRGRERARKRAAFITNP






TGAGATCTCCAATCGGACGG

FGFTKQLLGDKRSGRLECSI






ATCAAAGGTTA

EEVNRFIEETVSDPLREQEL






(SEQ ID NO: 1163)

EPNKALISPTPPAREFSLRG








PSLKEVKEIIKASRSASTPG








PSGIPYLVYKRCPGLLLHLW








KILKVIWQRGRVAEQWRCAE








GVWIPKEENSKNINQFRIIS








LLSVEGKVFFSIVSRRLTEF








LLENNYIDPSVQKGGIPGAP








GCLEHTGVVTQLIREAHENR








GDLVVLWLDLANAYGSIPHK








LVELALHRHHVPSKIKDLIL








DYYNNFKMRVTSGSETSSWH








RIGKGIITGCTISVILFALA








MNMVVKSAEVECRGPLTKSG








VRQPPIRAYMDDLTITTTTV








PGSRWILQGLERLIAWARMS








FKPSKSRSMVLKKGKVVDKF








HFSISGSVIPTITEQPVKSL








GKLFDSSLKDSAAIQKSKKE








LGAWLAKVDKSGLPGRFKAW








IYQHSILPRVLWPLLIYAVP








MSTVESLERKISGFLRKWLG








LPRSLTSAALYGTSNTLQLP








FSGLTEEFMVVRTREALQYR








DSRDGKVSSACIEVRTGRKW








NAGKAVEVAESRLQQKALVG








TVATGRAGLGYFPKTLVSQV








KGKERHHLLQGEVRASVEEE








RVSRVVGLRQQGAWTRWNTL








QRRITWANILQADFQRVRFL








VQAVYDVLPSPSNLHVWGKN








ETPSCLLCSGRGSLEHLLSS








CPKALADGRYRWRHDQVLKA








IAASLASAINTSKNHRAPRK








AVHFIKAGEKPRALPQLTTG








LLHKASDWQLEVDLGKQLRF








PHHIAATRLRPDIIAISEAS








RQLIILELTVPWEERIEEAN








ERKRAKYQELVEECRERGWR








TYYEPIEIGCRGFAGRSLCK








VLSRLGITGVAKKRAIRSAS








EAAEKATRWLWIKRADPWTA








VGTQVGT








(SEQ ID NO: 1408)





HERO
HERO-
.

Branchiostoma

CTGACCAGCAGACGGGAAGC
TAGAAACCCACAAGGCTGAG
MALPAVRSGPASTWTLLITL



3_BF


floridae

CCGCGACCAACTAGTCTCCG
AAATGTAGAGCATCTGTATG
VIVAAKGTDGFMSFKLPLLS






CAAATATTGCACACAGGGCG
GACAATATTGATGATTGAAA
TDTWSGYNNDVKTLLGPLHH






ACCCTATGGAGCTGATTCAG
TGTTGTGATTTTAGATCAAA
ELATNEMSPKLAGEGFSDIM






TCAAATTTCCTCTGAGATAT
TTTAGAAATATGAAAACCGA
CDFMASKPEFSHTTEESHSE






ACCGATAACTATCTACAGAA
ACTAAACTAAATATAATGTT
GYISHEPQSLAQVKRLKNKL






ACTGCACAGTTAGTTTGGAA
TTTTTTAAAGTAATGATAAG
RKKAFRADATPEDRKAFRDA






AGAGCTTTTCTACTGAAAGA
CAATACCCACATTGTGCAAT
IKTYSFMKRQQKRKETTKSA






CAGCAAAATCCGCCACTTTA
ACTATCTATGTTATGTCCTT
AHQEKEYHKNFWKFAGKCAK






GACGAGCGTCAAGACTGCCC
TGTCCCCCCTGCATGTTTGG
GQLDIPPVKPAFSVYYANEY






TCCCCATAACCAAT
TCAATAATGACCATCGTGTC
YKNKYSHPTRVDFNKLLWFP






(SEQ ID NO: 1164)
CTGGGCTCCGTGTACCTTTC
HLPVEEQLPANSFDMSPVRP







TTTACTATGAATAAAGAATG
KDIKAVLSKRCATSAPGPDG







ATTTTACTAC
IMYGHLKHLPACHLFLSTLF







(SEQ ID NO: 1287)
SKLLESGDPPTSWSSGNVSL








IHKDGSPEAAENFRMICLTS








CVSKIFHQILSERWAKYMTC








NDLIDPETQKAFLTGINGCV








EHVQVMREILAHAKKNRRTV








HITWFDLADAFGSVEHELIY








YQMERNGFPPIITTYIKNLY








SRLKGKVKGPGWESDPFPFG








RGVFQGDNLSPIIFLTVFQP








ILQHLKGVEQQHGYNLNDKH








YVTLPFADDFCLITTNKRQH








QKLITQISSNTKSMNLKLKP








RKCKSMSIVSGKPSDISFTI








DGDPVKTTKDAPEKFLGGYI








TFLSKTKETYDILAKTIETT








VENINKSAIRNEYKLRVYME








YAFPSWRYMLMVHDLTDTQL








QKLDSIHTKAIKTWLRMQPS








ATNAILYNTRGLNFKSISDL








YLEAHALAYSRSVLKADEKV








KHALQAKLDRESQWTRKMQK








WGIGKCHTIHQQAIHVAKDS








EWTSVRKHVKQQVTDMRHDV








WTKHQENLLQQGQMLQLLEE








EKCDLTWRSAMYNLPRGILS








FAVRASIDALPTLCNLTTWG








KRNTDKCKLCGNRETLHHVL








NHCGVALQQGRYTFRHNSVL








KHITDTIIESIDTSRINATI








YADIQGYTTNGGTIPVHTIP








TTQKPDLIIYLPEQKTLHIH








ELTVPFEKNIKTSHDRKVNK








YSTLAADLETAGISATLTCF








EVGSRGLVTPENKTRLRTLF








KIVKAKPPKTLFTDISRIAM








LSSYAIWNSRHEPYWESETL








L








(SEQ ID NO: 1409)





HERO
HERO
.

Danio

AAAGCAGTAGAG
TAGCATGCCACTTGGACACA
MTTHRAEVTTSGKTQEEPGP



Dr


rerio

(SEQ ID NO: 1165)
GGCCGGGGTCTGATCAGCCT
EATHSAQSLLVSPTPAAGRS







CGGTCGGGTCGCCTGGAGGA
PATQSCPQVTAAHNSPQSPQ







GGGTGTCTGTTGCAAGACCC
SQQVAVTRSDCVPLAQPRIQ







GAAACACCCTGTGAGCCCAG
WPQSSKKAEWLQFDKDVNQI







GAAACAACACTGATGATGTG
LEVTGKGGVDQRLSTMTTLI







TCCAAGGTTGTGCATCAGGA
VNIAAERFGTVTPKPTPSTY







GATGTTTCTGTAAC
TPSHRVKEIKRLRKELKLLK







(SEQ ID NO: 1288)
RQYKAAGEVERAGLEDLRGI








LRKQLVNLCRAEYHRKRRRE








RARKRAAFLANPFKLTKQLL








GQKRTGKLTCSKEAINNHLK








ATYSDPNREQPLGPCGALLT








PPEPTSEFNMKEPCRSEVEE








VVRRARSSSAPGPSGVPYKV








YKNCPKLLHRLWKALKVIWR








RGKIAQPWRYAEGVYIPKEE








KSENIDQFRVISLLSVESKI








FFSIVAKRLSNFLLSNKYID








TSMQKGGIPGVPGCLEHTGV








VTQLIREAREGRGDLAVLWL








DLTNAYGSIPHKLVEVALEK








HHVPQKVKDLIIDYYSKFSL








RVSSGQLTSDWHQLEVGIIT








GCTISVTLFALAMNMMVKAA








ETECRGPLSKSGVRQPPIRA








FMDDLTVTTTSVPGARWILQ








GLERLVAWARMSFKPAKSRS








LVLRKGKVRDEFRFRLGQHQ








IPSVTERPVKSLGKAFNCSL








NDRDSIRETSTAMEAWLKAV








DKSGLPGRFKAWVYQHGILP








RLLWPLLIYEVPMTVVEGFE








QKVSSYLRRWLGLPRSLSNI








ALYGNTNKLKLPFGSVREEF








IVARTREHLQYSGSRDAKVS








GAGIVIRTGRKWRAAEAVEQ








AETRLKHKAILGAVAQGRAG








LGSLAATRYDSASGRERQRL








VQEEVRASVEEERTSRAVAM








RQQGAWMKWEQAMERNVTWK








DIWTWNPLRIRFLIQGVYDV








LPSPSNLYIWGRVETPACPL








CSKPGTLEHILSSCSKALGE








GRYRWRHDQVLKSIAEAISK








GIKDSRYRQATAKVIQFIKE








GQRPERTAKNCSAGLLSTAR








DWVMTVDLERQLKIPPHITQ








STLRPDIILVSEATKQLILL








ELTVPWEERMEEAQERKRGK








YQELVEQCRANGWRTRCMPV








EVGSRGFASYTLSKAYGTLG








ITGTNRRRALSNNVEAAEKA








SRWLWLKRGEQWGQ








(SEQ ID NO: 1410)





HERO
HEROFr
.

Takifugu

AGACTAGGTGACAACCAAGA
TGATCACCCCGGCTGGGTCG
MTPAMEMTTTVTCICSKLCK






rubripes

ACAGTTWGGTCGACTACTGG
CCTGGGCGAGGGTGTATGAT
NQRGLKIHQARMKCLEREVE






AAAGACAGTTGGCAGCTCGG
GTCGTGAGACCCGAAACACC
VQRTGPGPGETQEEPGQEAT






AAAGACGGCACCCGGGACAG
CTATGAACCCAGGATACATC
HRSQSLHVPEPPNPNRVVQQ






TATGGGTTAGCACCCCAGCC
CTGACGATGTGTCCCAGTGC
QRIKWPPANRRSEWLQFDED






TGTATCTTTCGCGAGAAGGA
ATCCAGGAGATGTAKCTTTA
VSNIIQATAKGDVDSRLQAI






ACCCAAACAAGCTACGGAAA
AGT
STIIVSYGSERFGRIEKGNT






GCCCTACAGAGAAACACCCC
(SEQ ID NO: 1289)
ETTSYTMNRRSFKIHQLRKE






CAGGAGATCCCGAGAGGGGG

LRTLKKQFKRAXDGDKQALK






GGAGGATGAGATCTCCAATC

ELYNILRKKLKTLRRAEWHR






GGACGGACCTAACGTTA

RRGRERARKRAAFIANPFRF






(SEQ ID NO: 1166)

SKQLLGDKRSGRLECSREEV








NRFLQNTMSDPLRGQDLGPN








RALISPAPPSAEFKLAEPSL








KEVEEVIKAARSASSPGPSG








VPYLVYKRCPEILRHLWKAL








KVIWRRGRVADQWRCAEGLW








IPKEEDSKNINQFRTISLLS








VEGKVFFSIVSRRLTEFLLK








NNYIDTSVQKGGIPGVPGCL








EHNGVVTQLIREAHESKGEL








AVLWLDLTNAYGSIPHKLVE








LALHLHHVPSKIKDLILDYY








NNFRLRVTSGSVTSDWHRLE








KGIITGCTISVVLFVLAMNM








VVKAAEVECRGPLSRSGVRQ








PPIRAYMDDLTVTTTSVPGC








RWILQGLERLILWARMSFKP








TKSRSMVLKKGKVVDKFRFS








ISGTVIPSITEQPVKSLGKL








FDSSLKDTAAIQKSTEELGG








WLTKVDKSGLPGRFKAWIYQ








YSILPRVLWPLLVYAVPVTT








VESFERKISSFLRRWLGLPR








SLNSAALYGTSNTLQLPFSG








LTEEFKVARTREALQYRDSR








DCKVSSAGIEVKTGRKWKAE








KAVXVAESRLRQKALVGAVA








TGRTGLGYFPKTQVSHARGK








ERNHLLQEEVRAGVEEERVG








RAVGLRQQGAWTRWESALQR








KVTWSNIMQADFHRVRFLVA








AVYDALPSPANLHAWGKSET








PTCSLCSGRGSLEHLLSSCP








KSLADGRYRWRHDQVLKAVA








ESIALAISTXKHHHAPKKAI








SFIKAGERPRAGPQITTGLL








HTAXDWQLHVDLGKQLIFPQ








HIATTSLRPDMIIISEASKH








LIMLELTVPWEERIEEANER








KRAKYQELVEECRGRGWRTF








YEPIEVGCRGFAGRSLCKAF








GRLGVTGTAKKRAIKXASEA








AERATRWXWLKRADPWVATG








TQAGS








(SEQ ID NO: 1411)





HERO
HEROTn
.

Tetraodon

AGATTGGTCTGGCTAAGCCA
TGATCACTCCCAGTCGGGTC
MATTQASVKPTAVATCVCGK






nigroviridis

GTGACGTCCAGGAACAGACT
GCCTGGGTGAGGGGGTCTGA
ICKNPRGLKIHQTKMGCLAS






GGCTGACGACCACGAATAGA
TGTTGAAAGACCCGAAACCC
VQPEQRARFSLSESREVPAR






GTGGTGACAGCTTGGATAGA
CCGATGACCCCAGGTACTAT
AEPYGPQQPHSPEALGETQE






CAGCTGACAGCAGGGAAAGA
CACTGACGATGTGTCCAAGA
ERGQESPHSAQNLRAQVAQA






CGGCAACCGGGGCAGGAAGG
CATGCATCAATAGGTGTATT
PDNPQHHRRVKWPPASKVSE






GCTAGCAACCCAGCCTGCAT
TAGAAATC
WQQLDEDLEGILESTAKGGV






CTTCCGTGAGGAAGAACCCA
(SEQ ID NO: 1290)
DRKLQTMTTLVISFATERYG






AAACTTGCTACGAAGAGCCC

TMEKRAAPEKYTKNRRAEKI






GAAGCAAAGATACCCCCAGG

SQLRQELRVLKKQFKGASED






GGAGCCCGAGAGGGGGGGAG

QKPGLAELRCTLRKKLLTLR






AATGAGCTCCCCAAACGGAC

RAEWHRRRAKERAKKRAAFL






GGATAAC

ANPFGFTKQLLGQKRSAHLE






(SEQ ID NO: 1167)

CAKEEVDSYLHDTFSDAERE








NSLGECRVLISPPEPACSFN








TKAPTWKEIQTVVRAARNNS








APGPNGVPYLVYKRCPKLLA








RLWKILRVIWRRGKVAHQWR








WAEGVWVPKEEKSTLIEQFR








TISLLNVEGKIFFSILSHRL








SDFLLKNQYIDSSVQKGGIP








GVPGCLEHCGVVTQLIREAR








EGRGSLAVLWLDLANAYGSI








PHKLVEMALARHHVPGPIKT








LIMDYYDSFHLRVTSGSVTS








EWHRLEKGIITGCTISVIIF








ALAMNMLAKSAEPECRGPIT








KSGIRQPPIRAFMDDLTVTT








TSVPGCRWILQGLERLMTWA








RMRFKPGKSRSLVLKAGKVT








DRFRFYLGGTQIPSVSEKPV








KSLGKMFDGSLKDAASIRET








NDQLGHWLTLVDKSGLPGKF








KAWVYQHGILPRILWPLLVY








EFPISTVEGLERRVSSCLRR








WLGLPRSLSSNALYGNNNKL








TLPFSSLAEEFMVTRAREVL








QYRESKDPKVALAGIEVRTG








RRWRAQEAVDQAESRLHHKE








LVGAVATGRAGLGTTPTTHL








SRLKGKERRDQVQLEVRASI








EEQRASQWVGLRQQGAWTRW








EEAMARKISWPELWRAEPLR








IRFLIQSVYDVLPSPSNLFL








WGKVESPSCPLCQGRGTLEH








ILSSCPKALGEGRYRWRHDQ








VLKAIAESISSAMEYSKRLP








LPGRGVRFVRAGEQPPPQPR








AQPGLLATARDWQLRVDLGK








QLKFPENIVETNLRPDIVLH








SQSSKQVILLELTVPWEERM








EEAYERKAGKYAELVEDCRR








AGWRSRCLPIEVGGRGFAGK








SLCKAFSLLGITGMRRRKAI








CAASEAAERASRWLWIQRDK








PWTSASWTQAGN








(SEQ ID NO: 1412)





NeSL
LIN9_SM
.

Schmidtea

AAACGACATCATGAACGCTT
TAAAATGGCAAAAAGATATT
MMDSRQLNTPKIRKYQNPKM






mediterranea

GGCCGCAACAATCCAGTTAT
TCAAGATGAATTGTGGACTC
TNDIMKSYNYAVLSDVTPQE






CCCTGCGGTAACATTGTGGA
ATCTAAAAAATGACCACCTT
TTQTTTHLNVDIDNETTQPK






ACTCATAAGACAAGTACTAA
GAGTCCAAATATGCCTAGCT
QPLTKSGKPKSKPIAVSYKF






AAGAAGAATTAGAAAAATTA
ATCATGGTTGCTGATGGAAA
KDATFIWDTTPQTNPPRDCT






GAAGAAAAAATTGAAAATAA
CAGTAAGGCACCTGATAGCT
KLIDKTRPRKTIFKKSAFQS






TTTATTTATAAAATTTAAAA
AACTTTTCACTGTGAATATC
YLKKELSNETFVEVKTFLMA






ATTTAAATAAATTTAAAAAT
TTCAGATATTCACAGTGACA
THKYRFKDENSRLLAYRIIN






TTAAATTTAAATTTAAATGA
CGAAAGGACACCACTAGTAA
RYVMETANEFKETEFDMARF






AGATAAAAATTTATTTAATC
AAACCACTAGTTTTTTCTGA
AKFFTIPENWLKHLKPYSTA






CAATAAATAATCAAGAAAAT
CACCTCTTGCTACAAACTCT
TETSPADRIKVQKLVDLTCR






CAAGAAA
GTAAAAATCAAAAGGATCGA
YPFKTQEEQTSVANFLHFFT






(SEQ ID NO: 1168)
TAGGCCGCGCTTTCACGGTC
QRSIIGISRDYKFQKFIPFM







TGTATTCGTACTGAAAATCA
ARKNTRPETTSTMVTTSPTE







AGATCAAGGAAGCTTTTCCC
QNRLPMVIITPLEEPKSEHR







CTTTTAGTCAACACCAGGTT
RPEKRGASNDTIVLSDEEFP







TCTGTCCTAGTTGAGCTTCC
LLKRRTLPTRKSKNPTGAGN







CTTGGGACATCTGCGTTACC
VPTETECTDEVKFILNNEYQ







ATTTGACAGATGTACCGCCC
IECKECGKVWENVRNGLNHL







CAGTCAAACTCCCCACCTGA
RQKHDFPNRTDVMVSCVRCE







CACTGTCCTCAAAACAGTTC
VPIKGAECVNHIKNHKKDDK







AATTGCATCCGAAGATCGCA
EESEAGSLVANTQDIPNESS







ATTTTTTCACTAAAATAAAT
LSQAAIEVYLRNILKMKENQ







TAACAAAAGTTAATTATACT
ERNIQYLEPSTANFLINRNL







GCTTCATTGAGTAAGTAGAA
RAFYQNVKIEKLIGWEQVIW







AAACAATC
LIHWNKCHWIVYLANCDSKT







(SEQ ID NO: 1291)
SVILDSDNQMTLQQRCNIKA








KFDKFLEGTFEEKTVLGTLE








RKVPQQPNNFDCGIYVIQYI








SDFLKDPQRIDYHTPDSKRI








RKEIGELILEEMKNPASKIK








NPNKEIQSLLOKFRLLQINV








NDVFHWFAAEYQKSLPKIRT








KRDGKLNKLSCSYQIQRLFG








LAPKRAVKEIYFQETSTADL








ETRVLNEHFKKDESTMKECK








IKNGNHYQDWITKAQIDNKE








ILEALKNSTDSAPGEDNIPL








RQWIIWNNDGVLFDMFNYIK








RTHDIPDMWKNYTTTLLIKP








GKSQESNIPANWRPISILPT








SYRIFMKVLNKRVLEWANRG








ELISKWQKAVDKANGCDEHS








YVIQALIEKANRSYYKNEQC








HLAFLDLADAFGSIPFQVIW








HTLKNMGMDEETINLLKEIY








KDCSTKYKCGKNESEKIKIT








KGVRQGCPLSMTLFSLCIQY








LIQGIAEKKKGATIAGQEVC








ILAYADDLVIVANTAKDMQM








LLTTIENLAKQADLIFKPAK








CGYYRDPRDKKSMMKIYGKE








ISIVDEKNVYTYLGVRIGDT








KKKDLNVRFEEVKKKTTAIF








KSKLRSDQKLEAYNIFCQSK








FVYILQGEDIAKTKIETYDE








EIKKMIKEDILKLQDKSPFT








DFVIYSPREKGGLGITKIID








EQTIQTINRTAKLLNSSHRA








IRAIIYEELIQVANLRGEKE








INTIEEALKWLEGTNKYKKN








SNAKTTWITRVREAFQTLEK








KHKIKVRFVPKENCIGYKIK








CDTQEKIVELDNSKELSKSL








HWMIKEAYYKEWKALKCQGY








IISLKTSEFMEWKMPRGLPD








PDWRFLTKVKANMLDVNMKQ








ANQGGRLGSTKCRKCEDKES








ASHVINHCASGNWSRVEKHN








QVQNELAKELTKRNISFEKD








SIPKETKESLRPDLVIRLKD








KIMIVDIKCPFDEESAIESA








RNKNIDKYRELAKEIQAKTG








LQTTVSTFVVCSLGTWDKRN








NELLRQMGIRYEESKEMRIN








MIQKAIHGSRKTYDHHRNFN








NG








(SEQ ID NO: 1413)





NesL
NeSL-
.

Caenor

AAGGACGCTGGTTTAAGGCC
TAAACCCACACGAGAMCTAC
MPLXISDCVHLVSAEGDTMN



1_CBre


habditis

GAATTCGTTCGTTCTTTTTC
GACGCCATAAGATCAGGCAT
GRSTCGPLSRSSSVVSRSRS






brenneri

TGGCGGTCTTGCTTTGAGCT
GTACGGATGTGAATGAGACT
SPSPSVPPHPSPSIGPDTGL






TGGTTTCCGATCCT
GATGAACGGAATGAGCACGT
SAGIIGTSRGCSLWLPEVDN






(SEQ ID NO: 1169)
GCCCATAAGATCGGGTATKA
ALSQWLRKGLERDHEVLVCG







AAGAWCAGAGACGATCCCTA
FEAAKPLSLSKARLLRKTPR







MCATCGGGAAAACACGAGTT
NTGVVRHILEFDGRLVHTNC







ATACTGCTTCACTGAMCTCG
NETECVLSTLXSXXAVEVVR







CTAAGCTCTCATAATGACCG
ISLKCEPREPCEPKCVLSIL







AACTTGTTCGCAACTGCCTC
CSDKIVXISFECETREPFPF







CTAACCGGGCGGGTGTGAGA
FXDRKFREPIPFVFERMYDP







AGGGAGGTCGCCTTGAGGCG
RDPIPSFICWMYDLRQRMTP







GACGCAATGAGGGATGTGTG
GTLPXNPLSXENKDSWGRPA







CAGGTTCCCCCTCTTGAGAT
VIKNEIRSMRSYLEENVKEN







CCGAAAGTCTAAAAGTACTA
RLNLLRRLRGGGEGKKMIRK







GACCGAAAGATCGAGGACGG
LVAEKKSDTEAVCRILYPLD







ACGGGATGGCCGCGAGGCAC
DRYECFVDGCETTSTMGYGS







ACGGCGGGTAACACAGCCAG
SDLKYMTTHIKKEHGVKVQW







ATAACCTAGTAGATCTTCGG
TYECSLCNKQAPFMGGAASK







ATCTCGTCGGCCTGGAGATA
WVTAHMATKHTETVKLKLKP







TGTGGAACCCTGGGAAAGGA
SISTTAKVAAKLDEIAVSLP







GAAAGTTGTTTGTTGGGCTG
KPRQVRVLRDPDEVKEKVAK







GCAAGAGTGAAGTTTGAATG
PTLASTREEVKRNALRNMAP







TGAACCACCGTCATGCAACC
LVELSSQNQLTGAERPEETS







ACTAAACCAGTGGCGATGCG
EAMRLEECRTPEKIAELEGK







GGTGGAGTCATCACAGGAAA
IQTRTVTKKLSALKESMEKR







ATGTTTCTGTTGCTTGACTT
TREEKVGKPSLAPIHEEVKK







ATCAGTGTTTGATATCGCCC
TARRSLAPLVEPSTFTHLTG







TCAGGCACAAGTATGAAGGC
ASRLQAVRDAFSKANKDAAA







CCCCACCCACATAAACTCCC
KRRSSLAKPARLSEIMNTTF







TAGCAACTGGTAGTCCAGCA
TKETVNETKEPVNDTDESIA







AGCGCTGGTWCTTGCTACTA
TIQPQVRVYRFNTWCLDHET







TTGCGCCCCAGGCTCGCCC
TREAWLTGEVVDWFMGKVTE







(SEQ ID NO: 1292)
KKDQYRVFDSLVWSMYKFHG








VGYVLDLMRDPLTYFLPICE








HDHWVLLVIDEKGIWYGDSK








GAEPCREIAKFIEETKRERR








MFPVPVPLQRDGVNCGVHIC








LMVKSIVNGEPWYTEEEVKV








FRRNVKRGLKEFGFELYSER








IVYVGDDSIKVNDEHDDDVV








FLSEETNNTTFTIEQAEDPA








EEDAQHLESPVKPVKLMELK








IPKIEIKKKEIRRKPKQQIE








KKRKVPTGKPDELLVRVRLW








LEREVQSYFDSGKRFQRLEW








ILDVLTAAIHKATAGDEQAI








ERIEKRSPPLEVEEGEMSTQ








TEPKKRERKEKESGCEMKAS








HKEMYFKNRSKAFNVIIGKD








SKQCEIPIETLQKFFEGTTA








ETNVPAEVLKEMGSRLPKLE








ALDWMEANFIESEVSDAMKK








TKDTAPGVDGLRYHHLKWFD








PEYKMLTLLYNECKNHRKIP








SHWKEAETILLYKGGDETRP








DNWRPISLMPTIYKLYSSLW








NRRIRSVGGVMSKCQRGFQE








REGCNESIGILRTAIDVAKG








KRRNLSVAWLDLTNAFGSVP








HELIKSTLESYGFPEMVTEI








VMDMYRGASIRIKSKNEKSE








QIVIKSGVKQGDPISPTLFN








MCLENVIRRHLDSASGHRCI








KTKVKVLAFADDMAILAENR








DQLQTELNKLDKECESLNLI








FKPVKCASLIIERGMVNKNA








EVVLRGKPIRNLDENGSYKY








LGVHTGIATRVSTMQLLESV








TKEMDLVNQSGMAPFQKLDC








LKTFVLPKLTYMYANAIPKL








TELKVFANLTMRMVKEIHEI








PIKGSPLEYVQLPPSQGGLG








VACPKITALITFLVNVMKKL








WSSDSYIRKLYRDYLDEVAE








TETGMEEMTKEDIAKYLSGD








VPIDKKAFGYNTFTRVRDVC








NSLTXIXGAPLHKLKIVERD








GDFAILVQATKEGMEKIFTC








AQEKKLQQLLKAEVNTALAH








RFFTEKPVKSAVMSVMRQYP








QSNAFVKNGKNVSIAVHSWI








HKARLNALHCNFNTYGENKS








KVCRRCGKDVETQLHILQXC








EYGLPKLINERHDAVLHVVR








NLIRKGSKKDWKLKIDETVS








SCNQLRPDIYMCSPDGKEVI








MADVTCPYESGMQAMQESWN








RKVTKYEGGFSHFXKMGKKF








TVLPIVVGSLGTWWKPTTNS








LVQLGIEKXTIRRVIPELCS








MTMEYSKDVYWNHIFGDTFR








KPPMRFGVEKPKGNSWKKEG








SEPKGAASSD








(SEQ ID NO: 1414)





NeS
NeSL-
.

Caenor

GCGCCCCGGGTTACATTGTC
TAAAAGCCAAAAGCCACGGA
WRRPAPKQTKNSSLHHLGHE


L
1_CJap


habditis

GGGGCCACCTTTCTCTTGGA
GCATCGGGAAAGAAAAATGG
VKRIARLKPGIFEFHAKPKN






japonica

GTAGAGTACAGTCTACTAAT
AAAAGGACTGAAAACGAGAC
SSLHHLGHGVKRXARLKPGI






TTTTTGATAAGCTAGTCGGG
TGAAAAATCCCAAACAAAAC
FEFHAKXKNSSLHHLGHEVK






TCCGAACCACTAGAGTTTGC
AAATCCAAAACAAACTGAAA
RIARLKPGIFEFHAKPKNSS






TTGAAAATGCGTCAAACCAG
AAAAAAAAAAAACAAAACAA
LHHLGHEVRRNSRLKPGIFG






CATTTTAGAACTCGCCCAAA
AAACTGGACAGACACTGGAA
FYQKSKNSSLHHLGHEVRRI






AGTTCGGCCCCGACCCCCAA
ACAGTGTCAGGCAAAGTCGC
ARLKPGILEFHAKNRIKSGL






ACAAATGGGACCTTCTTGAC
CGATTATACTGTTCCACGCC
KVTFLSDLXAHAGALACSRF






GATTTTCCCTGAAAATCGGA
TTAAAAGTCCCGAAATGGCG
LASTLKTEHCRQKSFKPVGF






GGATGGAATGGTCCCCTATT
CAAAACAACCTGAATCTATC
LLHFLKNSSINEVASLRNVK






CTTGTAAATAGKACTGTGCA
TGAAAGTGCTCCAAACCACG
KXFLEFFSGKPIGGMASFSR






ATACCCCTTCGTCATCTGTG
CACAACTCGGAGAAAATCAG
TKITFFKLCLKNFVLSAENP






GGGAACAGATGACACGTGAC
GGACAAGTTGCTTCACGCAA
PIIRQKTNONKASXVQIARG






GTCATCCGTGTAGACGTCAC
CGGGCTGGGACAGGTACCCC
GHLSDCLPSQKMAGVLGRLF






GTTTTCCCGTGCCTGCGGGA
CTCCTGAAACCGCGAGGTTG
LSVQSTLSHRPFDTLLRSDD






GCCCCCAATCGAGCAATTTT
AGGATGGACGGGAAGGCCGC
DKRGRKTIKLQFFIKENLVT






TGCTCTTTTGAGTGTCTGGA
GAGGCTTATGGCGGGTAACT
PXVARDVKILXKQTKNNSGN






ACGCTTGAAACCCCAGACAA
CGGTTGGTGTGCTAGTAGAT
SDSNSETKNFSKNKVSRQNG






ATCAGGCCCAGTCGTCGGAA
GATTTATATCCGACAGCCCC
PLIGGGNHKKIGENQITRTL






AATTTCTTTTTGAAATTTTT
AACTAAGAGGAATCCTGGGA
EIESKSDDNKVLVLRILYPT






TGGCGCCTGCGAAAAAAATT
AAGGAAAACTTGAAAAAGTT
NDWYKCYSQWCQHKSLVGYG






TTTTAACCGCCACAAACCCC
TTTACAGGGCTGGTAATAGT
AHDLKYLTDHIKSTHSKKVE






CGGGAGGCGCGGWTAGGGAT
TCAGCACAATTGTAGTCTAC
WSYQCSICDAKAEGTGTKAA






ATCGATGTCATCGACTCGTC
TGTCTTGCAACCACAACAAA
RWITAHMPKVHGIEATHRIK






GGTGATCTTTGATTTTCTCT
CCAGTGGTTCTGCGGGTAGA
QNSEKTTNVKTANSLQEMAL






CTGCGTCTCCTATTTTGGAA
TCAAACTATAATTTGTGTGT
SLQKPKNGPKKVVMATSTTP






CAGTCTCGACCAAAAAACCG
TTTCTTTTACTTGACCCGGG
EKKISELESKIQTREVAKQL






GGCCTGGCAACCCACCGAAT
CAACACATTATACCACGTCC
SALKESAQKNQQGNKTKNVK






CCGGATGTCGGAGGGATTTG
ACAAGGACGAATTCATAATG
SSLKTIAENTNETKKISARK






GCAAGAAATGTTGGAAATAA
GCCCCTCCCTAAATAAACTC
SLINYLKPEDVLNHIPKEPK






CGAAATTTCGTTATTTTCAG
CCTAGCAACTGGTGGTCCGG
PASAKXGLQELTGAQRLQET






CACAATTGTCAAACCGGCAA
CGAAGCCGGTTCTTGCCACT
RRRFMAGNRRDSIARRESLS






GAAAACTGGATGGACAAGAC
ATTGCGCCCCAGGCTCGCCC
LGKISNSFKIELKNAPEKTT






ACACAATTTACCGGAAATTG
(SEQ ID NO: 1293)
LKKPAVTQKQNTSQNVSSST






TGCTTGTTACGTCGAATTTC

VVKENKTGNDVITIDDTETV






CCAATTTTGAAAAAATTCCT

KRKINTWCLDHESTENAWMA






CGTTCCACTGGTCGGGACGC

DDIIFWYIQKQIEISLDNKK






GAGGTCAGACGATCTGCACG

FKVIDPLIWTTYRIYGVECV






TCTGAAACCCAAAATCTTCG

QDELVGFEKYFFPICENGHW






GATTTTATGCAGTAG

VLLIIDDKRVWYSDSLADKP






(SEQ ID NO: 1170)

IEVIEDLINKLNRTQGKFNQ








TVPKQKDGFNCGVHVCLVAK








SVITENFWYTEKDVNDFRKT








VKLWLFSEGFELYSEPYKQI








QNKNISVNSEKNQISDNEKN








WGDKTQTVNESTLKERDEDI








FLLRPHISVGVALKTEDEKN








QKAENLKAPQKLKAIRRLKI








LKTCLKKLTAVKGKPEETER








AAIPNLMAIKLKTPPKVEPV








RRNPEKGENYXKSQPNKKRQ








IPTGKPDELVKKVREWFEIQ








FQAYFEDGKSFQRLEWXTGL








LTAAIHKASAGDEQAVGKII








KRCPPLEIEEGEMATQTETK








QKPKNQKSTKGANSSSSIRE








AYAENRARTFNKIIGKDDKC








EIPIEKIEKFFENTTSNTNV








PTETLARITSDLPKLEIGSW








IEEEFREKEVAEALKKTKDT








APGVDGLRYHHLSWFDPKXK








LLTKLYNECREHKKIPGHWK








EAETVLLYKGGDETQAENWR








PISLMPTICKLYSSLWNKRI








KSVTGVLSKCQRGFQEREGC








NESIAILRTAIEAAKGTKKS








LSIAWLDLTNAFGSVPHESI








EATLIAYGFPGMVTEVIKDM








YNGASIRVKTKNEKSKQILI








KSGVKQGDPISPTLFNICLE








SVIXRHLKSADGHKCIXSNI








KLLAFADDMAILSDSKTKLQ








QELQKMDDDCTPLNLIFKPA








KCASLIIEWGKVQKDQKIKL








KGQFIRSLAEQDTYKYLGVQ








TGIETRVSAMQLMKKTVSEL








DKINCSALAXWQKLDAVKTF








VLPKMTYMYANTVPKLSELK








EFANITMRAIKVMQNIPVKG








SPLEYVOLPIGKGGLGVACP








KTTALITYLVSTMKKLWSTD








DYIRKLHTDYLKMVAIKETK








TKEVTLEDLASYLSDDKTVC








KKAVGYNSFTRVREICKTLS








KNKGALLSQLKIIAKDGKLA








ILVQAXKDGKTKIFTHDHVK








TLQKXLKKEINEALLHRFTT








EKRVKSEVVRVVQEYPQCNS








FVRDGGKVSIGAHRFVHKAR








LNLLACNYNTWQDAATKQCR








RCGYEKETQWHILSSCPKSM








GGKITERHDSVLKTVKEMIQ








TGSLKNWKLKLDHELPGSTR








LRPDIYLRSPNGSEIILGDV








TIPYEHGIEAMQTAWQKKIE








KYEEGFKYLRSTGKKLTIVP








IVVGALGSWWKPTTDSLVSL








GIDKNTVKRAIPEICSTVLE








YSKNIYWNHIFGDSYQKVPM








FFGGEKPKGQSWKKVKPPEG








KTASNHEPPG








(SEQ ID NO: 1415)





NeS
NeSL-
.

Caenor

CGCGAACCAGTCAT
TAGCCGATCGTAAAAGAAAC
MTVFIDRGIGERGQMAVCSL


L
1_CRe


habditis

(SEQ ID NO: 1171)
CGAGCCGTAACAACAAGCAA
HRYFSFSPFSPIPPYVNNGS



m


remanei


AGTAAACAAAAGAAAAATCA
FGENGCGTDKSLLPVIEVVV







ATAAAAAGGAAGGTTGACCT
REVKINWSENILVVECLIMV







CAGACCCCGAGGAGGGAAGA
KSGERVVVKRQNLEKVIQNL







GAGACACCSAGAAAAAGAGA
ARINSTLFSNLGNQIFCVVP







GACGCAGAGAAAAGGAGAGA
RIKDSTNKEQGYRKEKQXKF







CACCTCTCATAAGGAGAGGT
HVSFRSIKSQVPPYLRGGGD







AGGTCAATCCAAATGTAAAC
VMEDTEIRGIRKLEPEAQLD







AGAAAAAACCAGTGGGGAGG
SSKPLICRVLYPTQGYMYKC







AAAGAAAGACTGATTTCACC
FYPKCKGHSNGSTDLRSLKK







CACTAAAATGAATTTGGAAA
HMVDKHFTNIEFAYKCATCM







CAGAATTTGGAAGAGAAAAG
FLTTGKSATALKSIKAHMAS







AGAAAGGGAAACCTAAAGAA
HHKVTMEPGKKSLVQKLNAR







AATAGTTCTCTTGCCAAAAT
LEEAAPSLPMPRNRSKVIQL







TCTGTAGAGGAATACTTTGT
TPEKSISELEKKKQTRSVAK







CAAAACATGATAGAAACCAG
QLSTLKESAQKKEEEVKIAE







TAATCTGGTACGAAAGACAA
VKKREPRLSIIPESNVRRSL







GTAAGACCTGAACTGACAAG
AAGLEQCINPEQSVAQRIRE







AAGGAAGTCAGAAAGAAATA
KREEYAKASREAAAKRRSSL







CCGCTCACAAAGCCTGTGAT
AMKPARLPDKENEITLQETK







CGATTCTCTTACCTACTGAA
KIDDPIVIDLEKECILTTVL







CTTGTTCTCTTGGCCTCGTA
QVPRNQFNSWCLEHETTIDA







ACCGGCTAAAGGGAGAAGGA
WLTDEVIHMYMCTITENRKY







ATGTTAATTGGAGATAGACA
FMAIDPVLWPVYVRNGAEDL







TAAAGATAGGTGGAGTGAAG
LRRTSCPGTFFFPICESNHW







GTCCTGTTCTTGAAACTAGG
VLLVIEHDVYWYLDPKGEEP







AGGAATGTGGAAAGAGCAGA
KGNVEILLESMKRKRQYYEF







AGGCCGCGAGGCTTTAGACG
PPPSQRDNVNCGVHVCLMAK







GGTAACTCAGTCAGTTGCTA
SIVDECGYNWYSEEDVRSFR







GTGGTCTTCGGATCCAACGG
TNMKDILKSKGYELCPEPYN







CTTCGGACATAGTGAGGAAC
RQNLLKTEKQKEVILEEMID







CCTGGGTACGGAGAAGAAAT
SFVVEDDMTFTVHRDSDHGD







GGAAAAGAGATAGGGCGGGC
DEVEHLKTIEQEPENEISEI







AAAGGCTAAGTTCATACACT
ENVEGSVDSVIPKLMEMRVQ







GTCATGCAACCACTAAACCA
TPPVINEKRGKKRVSAKEKP







GTGGGATCTGCGGGTGAATC
RKQKEKEQKVPTGKPDELVK







ACTTTCGAAAAGAAGTGAAT
RVRVWFEKEFKSYVEDGKSF







GGACGTGCTGATGTCTGACT
QRLEWXTDVLTAAIQKASAG







TTAAAGAAGTCTGAAATTAA
DEKAVELIEKRCPPLEXEEG







AAAAACAGATATAAAGGCCC
EMCTQTEKKKKPKSGKGNGG







CTCACTATAAACTCCACAGC
QESMKSLMASYSENRAKTYN







AACAGGTGGTCCGGCGAGGC
RIIGKHSKQCEIPIAKVQKF







CGGTTCTTGCCACCATTGCA
FEGTTAETNVPKETLKEMCS







CCCCAGGCTCGTC
RLPKVEVGTWIEGEFSESEV







(SEQ ID NO: 1294)
TEALKKTKDTAPGVDGLRYH








HLKWFDPELKMLSQIYNECR








EHRKIPKHWKEAETILLYKG








GDESKXDNWRPISLMPTIYK








LYSSLWNRRIRAVKGVMSKC








QRGFQEREGCNESIGILRTA








IDVAKGKKRNIAVAWLDLTN








AFGSVPHELIKETLESYGFP








EIVVDVVEDMYRDASIRVTT








RTEKSDQIMIKSGVKQGDPI








SPTLFNMCLESVIRRHLDRS








VGHRCLKTKIKVLAFADDMA








VLAESSEQLQKELTAMDADC








SALNLLFKPAKCASLILEKG








IVNRLNEVVLRGKPIRNLME








NETYKYLGVQTGTETRVSIM








DHITEVSREIDLVNMSQLAM








HQKLDILKAFILPKMTYMYQ








NTTPKLSELKVFANLVMRSV








KEFHNIPLKGSPLEYVQLPV








GKGGLGVACPKNTALLTFLV








TIMKKLWSSDSYIRKLYTDY








LEEVAKVEIGKFEVNLNDLA








EFLSDERAVDSKLFGFNAFT








RVREVVRSLCKNKDSPLHSL








KIIEREGKLAISVQATEESI








EKIFTEDQEKKLMYLLKGEL








NTALQHRFFTQKVFKSEVMR








VVQQHPQSNSFVRNGGKMSF








SAQRFVHPGRLNQLPCNYNT








WAKGRTKLCRRCAKNENETQ








SHILQVCDYSIGNIIKERHD








AVLYKFRELIKRGSKGHWLE








RTDRTVPNTGSQLKPDLYLE








SPDGKHVILADVTVPYERGI








EGMQKAWNEKINKYTDGYKE








IFRRQGKSLVVLPLVVGSLG








TWWKPTEESLIKLGVEKTTV








RRIIPETCGMVAEYSKNCYW








RHIYGEKYVQTPMINGGKKP








EGNDWKKCEKGIEVPKVTN








(SEQ ID NO: 1416)





NeS
NeSL-
.

Trichomonas

GGGTGAGTAGTCTAGTGGT
TAAGAAGAGATAAGACGAGT
MIPVLGTGGPEKLPLQSYVY


L
1_TV


vaginalis

(SEQ ID NO: 1172)
GAGAAGAACAGAAGCATAGT
CGNTAITDSFTPTAKTILKP







AGGATTGGCAGAGCTTAAGC
EEQNLDIVLKNIAALNPENY







GATGTCACTCGGTACGAAAC
SDLIRSLSKMEFRLDYPKEI







GTGTACCAAACACCGGATTC
ENYWISEKLFSQSIASLPIS







CGTGCTAGGAATCACAAGCC
LLVASMFSPEDRDLSTEPFH







AAAATAAAAGAGACACCACG
CNADGCNFHCDNCERMVEHI







AAAATTACTCACCCTCCCTC
REHHNTDPMINTFETTEDTF







AAACAGATAATAATATTAAC
RRITAIKIDKTGIEELNPLK







CTCCCATCCATCAGTCCGTA
YRCSYCDELFTEAEDHAIHM







TGGTCTGATAACAGACTAGC
ISHLTEKLSPDISFFFNDIL







ACCACATCCATGATACACTC
RLYKTIDKPTVQNLFPETQV







ATTGGAGTGAAAACCACCAA
AIFDTLEETNRFRLIVGREA







CAACAAATCCACCTAGACCA
IETIEEAFPPSPPGTDRKPS







AATCCTGCCCCACCTCCACC
IIITDTCQLRFVPCMDEPPK







CAAGTAGCTCGCTTCGCTCG
GDLGILTLLLRDFSAHNIPI







CTCACCTAAAACTTTGCTCG
KSLNNKELIADKDIDYSPDF







CTCGCTTCGCTCGCTCGTCT
VEGALANAEEHDTTNSQNNN







TAACCCTTTCCGAATAAACA
GRYINSAEKLTEFLIQCEDY







CTTACAATTCCCGGCTCGCC
LTNIKTLEDLERFYTTIKDY







CCATTTTTT
RVNKEVIAEDTPIFVYFLVE







(SEQ ID NO: 1295)
EGKLPKPGLRCPLESYEGHE








DKAFESLRKLCDHFKGEIAK








TSFDPKVHTIDIWVEFLAQA








YGTGTFVYKDENGNIDLDTH








VFKCPYADCSYTNNDRSKLM








DHMKTKKHAKNVYIERYGFF








WGIVIEGVNRPKGIVYPTLK








DIKEHACRKCPEAGCNTYVT








ELSDIKEHLKKKHKSTTAGV








DGEIAHTDATYCWITKEELD








ALHAERARERAEQVDNTPVQ








QIINADNNEENNENQEDNGN








NEEADALDPPNNTTETEDEA








VHAVIINPPATEEEEVAIIA








EARRNIPELQQAEERGCVTP








KMTSLVRLKLLKGGGELFNK








KLTPLATRYAATGNTEADKI








KVDYLTLKCNAALREMIYTN








NHSESKFMTAENGEDTAPPP








RISEDTRDRIQKAANEIKGT








LIKVVKHISHARCLKDSTRD








DEHNKFVEMIAKIKNDLRDN








KFEQYNIEEIFQGPISDQSI








LNIVNTEDNNEFIKKMDYIN








RILGTPQDASPYARKKLQAC








FADNPTKTLRNIILADKVPQ








QSLKPSEYLDYYGPQWANEA








EGYENFLHHDYALPERYGQV








FANDFLDFMTNESKIIEVIR








NKNHLSAHGLDGIPNSVYML








FPVSAAKFLSILFRSIIISG








HIPDCWKLSKTVMLFKKDDP








SLAKNWRPIGITSCTYRIFM








TLVNKALQMIPMFHAMQKGF








VRGATLSEHIAVANEVLCQS








TRTQSEMFQTAIDFTNAFGT








VPHQLIFDSLEAKKVPDSII








NLLKDLYKGARTAIYTRHAH








SEIVPVRRGVIQGCPLSPIL








FNCCLDPLLYAVQRRHFEDG








YRFQDKAGQYSIAIQAYADD








VLVISPTHEGMQRILNTVDE








FQKIAKLKVAPQKCVTLAKT








STAIQPFRIGPDEIPIKTSM








DNITYLGIPISGTKTSRFAA








ATGILEKVKAQIRVVFASHL








ALSQKIIALRVFILPQLDFY








MFHNVFRVNDLKATDQMIRG








LIDKEAPTSNIPVSFFYMPK








NKGGFGLVKLELRQPQLVLT








KFARLWLSQQAETKAFFHTM








AQEEKSFRKVVEDQENGFLG








IKMENGKIVQKNERSKRTNC








FITQAAKAADKLEVRFKEWD








KGGIQVRGVGENATDWYRSK








HIGQISPLIGRVIQQRQYEE








FKKDETHSHTFCEPAALAES








HDIMKRPQAVPNNLYSAAIA








LRTNTAPTPANMHFHNPEVL








ANCPLCGCQSCTLFHTLNMC








RNRFSLYKWRHNIICDDIYQ








FIHDHYPGVTIKCSARITSD








GYQTTGPELDDTVKDLLPDL








VVYDEANKMIKIIEVTCPYG








TDNNVGNSLDAAYDKKVNKY








KSLAEQTERLFNWTTTLSII








VVSSLGVIPLRTKLDALRIS








PADHIQLLKRLSMHAIAASA








CIVFEKVPEFFGMRCRPLPG








RVTAPNAAIPPNNNENNNDT








DHGQENQQATSEEQPTNNGN








AQEDNGQGEQINNSTEQTIS








VDQIIEEDAENNAIEQALDQ








PDEDEFLN








(SEQ ID NO: 1417)





Nes
NeSL-
.

Caenor

GACTCGCCTTGGGGAAGGTW
TAAACCGGCTCCTCTGGGAG
MRYHXSNXPAXRTSDNXWRS


L
2_CBr


habditis

TTTCAGGGGKSAATTGCCGM
GAGGTATGTCAGAGGACATT
IXKDVRRPDPSTIEEKSRYN



e


brenneri

AGGCAAGGCAGCCCCCSMMT
CTCCGTGGGCGGATGGGAGG
RSIGIPDSLKXRSSAVRSXS






AGCTTACAAAGTAAGTACMC
AGTAGGGTAACGACCCGTCA
SXPPSGPQDVRLXNSPSLDD






ATTTTCATTTCTTGTGAATT
TTCTGGATGCCTAAACCACC
RRRLVDCETTLGSYREWTDK






CTTTAAACATATTTTTCTTG
ACAATCTGTCAAGGCAAAGT
PMMGKMTYAAVTKRAPPRPQ






TTTTTTGATTTCTTTTTTCT
GCCCCAAAAGCACACGCGTG
TGGARLSTNLLADEMEIKYR






CTACCTTCCCCCAATTCTTC
GATCGGTTTGGATGCCGACT
DTNDIRLVIDLPNPHLIKCP






CCCTCATCTTGTGTATACAT
GAGCCAGAGGGCAAAGTCGA
LCKSCISARGRGANALKYMK






CCCCCTCCTCCAACCAATCA
AGGCCGGTAGGCTCCCGGCG
RHIADAHHLNADFVYKCSRC






ATACATTGACCTCTCTCTTC
GGTTGTCCGTCATAGTCAGT
QEHEPENVCGAKWIVNHLKR






TGTCAAAAAATCAATACTAG
GGTGCGCCTACACCCAACTG
VHGYTLEDAVSTAKPSTRQQ






TATATTGTCCCTTGTATAGT
CTATGACACACAAGGACAAC
IANAFNDSAPFIDARKTSDV






ATTATTTGACGTCGTCTTTG
CCAAAATAAATAAGCCAAGG
PEKKSREAGLEKFLAPTKSE






TATTAGGAGTAGGTAACAAW
CGGCGTTAGCTTCGAGCTAA
DTREKTPPSTRKSSESSEAS






CTGTGTATGGCTTCAAAAAG
CAAGCTCCCCGAGAGGATGG
IQSTIQETLSESSDTLTVQE






CATGCACAAACWCCTGTCAA
TTGCCACAGGGCACCATCCT
IINISSEDEMDEEPPKRRVN






AAAGTAWTTCCCATCMTGTG
GGGGAACGACCCGATCTTTC
VWALIHENGKDAWIDSDLMV






AATAGCTCAACGACWKGAAG
GGATGCCCAACCACCGCCAA
IFLESRARGYESCSIIDPLN






MCCAATGAT
TCTGTCAGGCAACGTGCCCC
FICTDMSYLTTIVRRRMEEG






(SEQ ID NO: 1173)
AAAAGCACACGTGCGGAGCG
YKKIIFPLCANDHWTLVTIT







GTTGGATGCCGACTGAGCCA
GSTATFYDPMGNEPTETVKK







GAGGGCAAAGTCGTAGGCCG
MIDELDLEMQLAPSNSPRQR







GTAGGCTCCCGGCGGGTTCT
DSWNCGVFVMKMAEAYIKDT







CCGTCGTAGTCAGTGGTGTG
QWDLTDVDTDVKTFRRSLLT







CCTACACCTAACTGCTATGA
ELKAKFNIFAEDIQTYRPPS







CAAGCGTATAGGAGGCCCGG
RKALTRNSQSPVVVCHKCSR







AAAAACAAGCCAAGGCGGCG
PATPIQDVSRMEVEEAPVLV







TTAGCTGAGAGCTAACAAGC
PTPEEPPQEWTFVGKNRKRG







TTCTCGTGGATGGGTGCCAG
VTSRTPNTSPEAKRPAFPPV







AGGGCACCATCCTGGTGGGT
PLKPSANRWHFPEEETEKME







GGATGGGGGGAGCTTGGGAA
VSSADEVKNSTPPKPPKIPN







CGTCCCGATCCTTCGGATGC
LLAMKIASPVPLKRGNPSKK







CCAGACCACCGCAATCTGTC
HGKGHMMNTARKGPTKKEMP







AAGGCACCGTGCTCCAAAAG
KGEPANLIVKIRSWFDEQLK







CACACGCGCGGGTTGGTTTG
MYKDEGSNLQRLTWLSDSLT







GATGCCGACTGAGCCAGAGG
AAIGKAFNGNKYIVDQIIKR







GCAAAGTCGTAGGCCGGTAG
NPPPLVEKGAMSTQTSRKRD







GCTCCCGGCGGGCTCTCCGT
EFKPRERMAQEPNEPLRIQY







CATAGTCAGTGGTGTGCCTT
AKNRQKTFFKIIGKQSEQCT







CACCCAACTGCTATGACATG
INIETVEQHFRKTLKAPVVS







CGTACAGGAGGCCCGGAAAA
ENAIKTVCGSIKKVLMPKTI







ATAAGCCAAGGCGGCGTTAG
EDPISSVEVKSILTKVKDTS







CATAGGGCTAACAAGCTTCT
PGTDGVKYSNLRWFDPEGER







CGTGGATGGGTGCCAGAGGG
LAKLFEECRKHREIPSHWKE







CACCATCCTGGTGGGTGGAT
AETILLPKDCSDEEKKKPEN







GGGGGGAGCTTGGGAACGTC
WRPIALMATIYKLYSAVWSR







CCGATCGTTCGGATGCCCAA
RISGVQGVISPCQRGFQSLD







CCACCGCAATCTGCCAGGCA
GCNESIGILRMCIDTASVLN







ACGTGCTTCGGAWGGTCATT
RNLSCSWLDLTNAFGSVPHE







GGTTCTAGACTTGTAATAGA
LIRRSLESFGYPQSVIQIVT







CCATTGGCCGGAAGAGCACA
DMYKGATMKVKTADQKTQSI







CGCGCGGTTGGTTGGATGCC
KIEAGVKQGDPISPTLFNIC







GACCGAGCCTAGAGGGTGCA
LEGIIRMHQMREKGYDCVGH







AACCTGAAGGGCGAGGTCGA
KVRCLAFADDLAILTNNKDE







AGGCCGTGAGGCTCCCGGCG
MQEVIDKLDADCRSVSLIFK







GGAAACTCCGTCATAGTTAG
PRKCASLTIVRGAVDKYAKI







TGGTGTGCCTACACCCGACG
RINGDAIRTMADRDTYRYLG







ACTATGACACATAGGAGGAA
VKTGVGGRASETEALIQVVK







TCCTGATCTGATATGATCAT
ELQKVHETDLAPHQKLDILK







GTATATAGGGAGGGCGAAGG
TFLLPRLQHLYRNATPKLSE







TAAATAGTCAGKGTCAAAGT
LREFENVVMKSVKRYHNIPI







CCACGTGGCAGCTACTCCCC
KGSPVEYVQIPVKKGGLGVL







AGCATAGTAGTGATGCGAGT
SPRLTCLITFLTSTLCKLWS







GGAWCCAACTTTGACACTGA
DDPFISSIHKDALSRITVKA







TGTTCCCTGAGCCTGACCCA
MGLTTQSATIKETCEYLNTR







TCTGCACAAATCCAACAGTG
KAVTKGGYSLFCRMNESLRT







TATGATGGCCCACACACTGA
LSVIQGAPLKSMEFIPVNNE







GGACGAGTATCACTTGTGAT
IGIAVQATKDSEIKVFTKAD







ACTCAGAGGTGTCCCCCATG
SLKLMSKLKDLVRSAMLKRF







ATCAACCAATATCACAGCTA
LEEKSVKSRVTQVLQHHPQS







GCGGACCTACCGTGAGGTAG
NRFVRDGRNCSIAAQRFVHP







ACCCCCGCCGCTGTAGCAGG
ARLNLLSCNANTYDVNHPKG







CTCGCCTC
CRRCQADFESQQHILQNCHY







(SEQ ID NO: 1296)
SLAGGITQRHDRVMNRILQE








IGNGRKAHYKIMVDMETGAT








RERPDIIMEERDGPEVLLAD








VTVPYENGVQAVERAWDKKI








EKYKHFLDYYRKIGKKATIL








PLVVGSLGTYWPDTSHSLKM








LGLSDGQIRNVIPEICQIAL








ESSKNIYWKHILGDSYKTVE








GLFCQRNNKEVRFEGKGEKH








HVSQRFQPLKCEKVRTMKST








KEEGRSRSNAKKGPNWRRSK








SESDGRSVSKGRYWRDPSNK








PPHSKMTQSALAKR








(SEQ ID NO: 1418)





NeS
NeSL-
.

Caenor

CCAACTCTCATCGTATTAAC
TGAATACCGTCAGATAAGCC
MTNVYLKPVNDNQTNKTGDN


L
2_CRe


habditis

CTACGGTATTCACTCCTAGT
CCCAACATAAAAATAAAAGT
SRNTMSNSQCEMTWKPVART



m


remanei

GAGTGTAATAAAGGTTAATT
CGGCGTTAGCTAACCACTAA
YAQAASTNPADDKTVTVLGC






ACGTTTTCTCTTGCMAGAGA
ACCGGCTCCTCATTGGGGGA
KYNLLKLGNTPQTSKRSPPK






AAAAGAAAATTCGAATCCTT
GAGTATCATTCCGGTGCTCT
PSRGGARISSVYTLTDELEI






TTTGTGTAACTCACAAACTG
CCGTTTGGGCGGTAGGGAGG
THREEGKITFAIDLPNKNNI






ACAGAGACCTATCGAATTTC
AGTTGGGTAGCGACCCGGAA
LCPLCRECTQTRGRGSSFTK






CTTTGTTTCGTATATAGGAA
GTATGGATGCCCAACCACCG
HMKLHVKEKHQLDATFIYKC






TAGTCACTCTGGACCACGAA
CAATCTGATCTGGCATTGTG
SMCNEYEPEKKCGTKWIQTH






GTGGACAGTTGTCGGCGGAC
TTTCGGATGGTCTCTGTCTC
LQKVHNYKYDESAIVVPVPP






TTCCAGAGTGGAGAGAAAAG
TAGATCTGAAATAGAGCTCT
NTRQQIANELNNAAPFVDIR






GTGTGAAGAGAGGAGGTCTA
GGCCTGAAGAACACACGCGC
KPKAAAVEEKKTENGALLKF






GAAACACTTCGGCTGTCTAG
GGACCGGTTGGATGCCGACT
LTKSNKDDQVKSPSXDIPDA






GACCAGTTCCTGAGTGGAAA
CGATCTGGAGGGTGCAAACC
ESPEKETQALTIDPKGNNSP






GAGGAAGGTCTAGAAACACT
TGAAAGGGAAAGTTGAAGGC
SKSSIRSSQSSASSVCQEIQ






TCGGCTGTCTAGGACCAGTT
CGTGAGGCTCCTGGCGGGAA
EIITLSEDEDPKGARPKPGI






CGTGAGATCTCTCGTGGAGA
ACTCCGTCAT
NVWSUNETGKDAYIDTDIMM






GTTGAAAACAGTCAGCTGAG
AGTCAGTGGTGTGCCAACAC
AFLKMRVENCDSVNIIDPLN






GCTACTGTATTTCTTGATAG
CCGACGACTATGACATAGTT
YQFPARVDLVPUQRNLEDGK






CCCCGCCCCCAATCCCCCTC
GGAGGAATCCTGATCTGATA
KRWFPICADEHWTLLTISNG






CCCCCCCCCTCGACAGATTT
ATAATCATTGTTCATATAAG
IAAFYDPTGSRMSSYIEELV






TTCTGTTTGACCTCCTGGAA
GGAGGGGGATGGTAAATACC
NELGLIIPKEQDEQPRQRDS






TTTGCGAGGAGTGCGCGAGA
CAGGGTCCGAAACCATCAAA
YNCGVFVMKMAEAFIQDTEW






ATTTTCGAATTCTTCGCGCG
GCAGCTACTGACCAGCATAG
EMEEVEEDVKNFRRNLLEEL






TTTTCTCGAAATTTTCCAGA
TAGTGATGAACACATAGACC
KPNYEIFAEKIKYYNSPGKS






AGATTCGAGCGGAGAATCTT
CTGGGGTTCCCTGAACTCGA
FAQSRPTSRSSQCAVCPTCS






CGAGAAAGTGAGCTGAATTT
CCCATCTGCACAAACCCACT
RSATPMMDVGNMEVDPVPQQ






CGCGCGAATTTTCCGCGATT
TTGTACAAATGAACCAAACT
QETPKSREPEQDEGWKVVGK






TTCAAATTATCGATTTTTGT
GATGAAGAGTTTAATGATTT
ARKRGWTERSPNISPEAKRQ






CGGAAAATTTATTTTCTGGC
CTTACATCACAGCTAGCGGA
FTGPEIKWSPGKFHPLVGET






AAAATTTGATTGAGTTCACG
CCTACCGTGAGGTAGACTCC
EEMEVTCDSPPTKEPTTEPK






CGGGAGAGAAGGAATTGTTG
CGCCGCTGTAGCAGGCTCGC
VTPSLPAMKIASPEVTKKQT






GAAAAGGGTATTGATTTTTG
CATTG
SKKKGKYGKKKQXTKKAQPP






TGGCGGAGGAAACTCCCACT
(SEQ ID NO: 1297)
KGEPTKKAQPKGEPAKLIEQ






GAATCAATAACTCTCAAAGG

VRTWFDKQMKSYQEQGSNIQ






AGAACTCATCGAACAACCTC

TLTWIADSLTAAIFKANSGN






GGGTGACCTGAATCTTGGGC

KYLVDKITARCPPPLLNEGE






GAAATTTTCGCATTGACACA

MATQTSRRTEAVKPKDRFVK






AGATAAMACAAATTACTGTK

ESNEPLRIQYAKNRAKTFNV






GAAAATAAATCAGAACAAAC

IIGKHSARCEIDINVVENHF






TGTCAAAAAGAGAGACAAAA

RQTLKAQPVTEEALNTVCSG






AGTATTGATTAACAACATC

IKKAKVDPSIEGPISSGEVK






(SEQ ID NO: 1174)

AILAKIKDTSPGTDGVKYSD








LKWFDPEGERLALLFDECRQ








HGKIPSHWKEAETVLLPKDC








TEEERKKPENWRPISLMATV








YKLYSSVWNRRISSVKGVIS








DCQRGFQAIDGCNESIGILR








MCIDTATVINRNLSCSWLDL








TNAFGSVPHELIRRSLAAFG








YPESVINIISDMYNGSSMRV








KTAEQKTQNIMIEAGVKQGD








PISPTLFNICLEGIIRRHQT








RKTGYNCVGNDVRCLAFADD








LAILTNNQDEMQDVLNQLDK








DCRSVALIFKPKKCASLTIK








KGSVDQYARIKIHGMPIRTM








SDGDTYKYLGVQTGNGGRAS








ESESLTQIAAELQMVHDTDL








APNQKLDVLKAFILPRLQHM








YRNATPKLTELKEFENTVMK








SVKMYHNIPIKGSPLEYVQI








PVKNGGLGVMSPRFTCLITF








LASTLFKLWSDDEYISSIHK








KALSRITAKVMGLKTQKATL








QEQCEYLNTKKAITKGGYSL








FSRMNEAIRTLSVNLGAPLK








SMQFIPENGEIALEVQASEN








SQIKVFSKADSMKLVTKLKD








LVKSAMLKNFLENKKVKSKV








VQVLQHHPQSNKFVNDGKNX








SISSQKFVHPARLSQLVCNG








NSYSKDLPKNCRWCGYECES








QAHILQHCTYSLSSGITQRF








IDRVLNRILXEVIKGRKNND








YYDIMVDTEPGPTRERPDII








MIQKDGPEVLLADVTVPYEN








GWAIEAAWDWKMEKYSHFID








YFARLGKRAVILPLWGSLGT








YWPDTSNSLRMLGLSDGQIR








NLIPDISMIALESSKQIYWR








HIFGDSYRIVSDLYCRKDQQ








EIRFGDEPMENVQVSDRFQP








FKTREREKKSEEEKKRRSKS








KKGKTWRGSKKQTDSRQSGK








SNQNQGFQRSVGQGVSR








(SEQ ID NO: 1419)





NeS
NeSL-
chrUn

Caenor

CCCTTTTCTATCGTATTAAC
TAACATGCCTTGGAAGGCAC
MTKTEWSWRHRSRSRSVGIV


L
4_CRe


habditis

TACGATAACCGCTCATTTGA
CACGCCAAAAGTCCTGGCAA
VKIDTSDYANVRVHVAADLS



m


remanei

GTGTAAAAAAGGTTCCCCCC
CTGATTTGAATAATGTATAA
NEDGHTSHNNGIILPIPMKP






TCCTCGCCTGCCTTACCCAC
AAGTAACTGGAACCAAATGC
SVDRFCQIQYPPRGYYVPHP






GCATCTCTGCCTCTGGGAAG
CCGATAGGTAGGGCGGGAGA
QSQKGHDAKPSRHWNEEAQP






GCGGAGGGTCAACTTGCGGG
AAATGACCTAGAAAACACAA
PYYHNNNHGRRGRSAKPSGR






TCTGTGGATTTCCTTTCCTA
AGTCCCAAGCCCCCGGATTC
RPPRKPILQEESLAAHPQIP






TCCACCGCCCATATTCTCTG
GAAAGACCTATAGGAAGTCA
GDTASAVPLYSDVVNNENKS






TCGAAAGCCTACCTAGATCA
GTGAATAGAGAGAAATATCA
QGKPPQGSHRRSGRPGTKPS






GCCGGGAGTTTTTCCTATCC
AACAAATCTCACCCATTCAC
VPVGEAEQETNSRPIAPEPI






CATTCAGGCGATCGCTCAAG
AAGGACTTACTGGTCGAGTA
VKFKHDKHGWTTVQGSHSSG






GCTGTTTTATCGACACTCCT
GAAAACAAGCCAAAACATCA
RPVPKPSVPVVSEANRFQLL






TCTTGACAAGTATTTATTTC
AGCACGACGCAAAAAGGGGT
QEGDFPPLTTSESSQEEIKV






TTGACAAATTCTATTTTTCC
AACTTTGGGCAACTAATTAA
PNYQRIVSPIPLPSEEDSKL






TTTTATCGATTTTCTCTTAT
CGGATACCTCCGTGTATCAG
PTKSNYRAPKGRKSRNYKKP






TTATCGATTCTTGTGAAAAT
GCAAAGCCGCCACCAACAGC
QQQNPKKYQQRLPYQPKVNN






(SEQ ID NO: 1175)
AAATTACTGCCCGATAGGTA
APTDRMAPEQLKGGGGKTAH







GGGCGTGAGAAAATGACCTA
NDIEEMEIEEDTDEKIIQVK







CAACCTCCAAGACCCGAGCC
RIKIVNKLTPHHFVCMMTYP







CACGGAATCGAAAGACCTAT
TDNIYRCFVKGCTATSQGGW







AGGAAGTCAGTGAATTGATG
GAEDLKYLTVHIRQEHKIKV







GAAATACAAAACCAAATTTC
EWTYECGICGDLSGGAGKHI







TTCCATTCACAAGGACTTAC
SKWIKPHMRKKHNRDAPTNF







TGGTCGAGTAGAGCACAAGC
KMGSRSSGKPKITELLEESA







CAAAATATCAAGTATGACGC
PSCSNPRRKTLNQKKTAIIT







AAAAATGGGTAACCTTGGGC
QVTPEKLKTGYQTRSVTKAL







ATCCAATCAACGGATACCTC
SVLKESRQKELEVLREEEKA







TGCGTATCAGGCAAAGTCGC
NAKQKSKLHPFFTKAPHIDG







CACCAAACTGTACTACTCCG
VKPTVRRELSKMITPGGEHK







AAAAAACCAAGAAACATGAT
GTKIPMVHTKRGLIQKINRK







TTTCCCACTCCGTTAAAGCA
AKKAKPMHLDESTIIEASQL







TCTCAACCAAGCTAAAGCGG
DVITIDDDDEDDNMTPMRRR







TAAGGTTATCATGTCAAAAG
FNTWCLDHETTQEAWLTDDV







GTGTAGCTACAGCAACCTAA
INWYLKDLCFGNEQYMLVDP







AGCCCGAAAGGTAGGGCCGT
LVWLIYKMGGMAGVEQRFKS







ATAAAAAGACCTACACCCTC
KKTCLFPICEADHWILLVFD







CAAGACCTAAACCCACGAAC
ETNLCYANSLGSQPNGQVKN







TCGAACGACCTACAGGAAGT
FIQQLNRKLCSFEKEVPLQK







CCGTGAATGGAGAGAAATAT
DSVNCGVHVCLIAKSIVNGQ







CTCACCAAATCTCTTCCATT
FWYDDSDVRTFRTNAKAALK







CACAAAGGCTAACTGGTCAA
AQGYELFSEAPKQIENPDSS







GTAGAGCACAAGCTAAGCCT
HREDIKENSMEMCSESLMIV







CCAAGCACGAAGTGATATGG
ATPQRSEAPMELVDTEPSDL







GTAATTTAGGCAACCAATCA
ESPKSDRVVYEDCITALSDV







ACGGATACCTCCGTGTATCA
SEPRMTPEKSETPEVPVVEE







GGCAAAGTCGCCACAAACAC
RDLDWPKLESPKSDRVVYED







TGTACTACTCCGTTACTCCC
CITDLSDVSEQRMTPEKCET







AAACACATGGATCTCCTTCT
PEAPLVVECVELERLPKDLP







CTCACCAAAAAGCTTTATAA
VTDRSTVVAIPEAVKLEEKS







CCAAGCTAACGGTGGAAAGG
EVVIPRLMELSYTVPPEPSP







ACATCATGTCACGAGGAGTA
VVEYTQPYTHTHTKPKVKAT







GCTACAGTAACCTCTCTCTT
CQMGKKRKVPTGKPDELIQI







GAGACTGCAAAGTCGAGGAT
VRQWFEKEFNDYVTEGRNFQ







GGATTGGGAAGGCCGCGAGG
RLEWLTNLLTAAIQKASAGD







CAAAAGGCGGGTAACTCGGC
EETIEKIRKRCPPPEVRENE







CAGACGCTAGTGATCTTCGG
MSTQTSQRQKPTTTNQKKRS







ATCCGACAGCCCTGGCCTTA
RNTTQSDTQANTYWRNRAKT







GAGGAACCCTGGGATAAGGA
YNQIIGQDFKQCDIPIAILE







GCACGACGGGAAGGATGTTC
EFYKKTTSVTNVPQETLVKV







CGCAAGGATTTCCCTTCCCA
TSRLPRLDIGKWIEDPFTEQ







TTAGTCAGGGCTGGCAGTTG
EVFGALKKTKDTAPGTDGLR







GTAATATAGCCTTTCTACAC
YYHLQWFDPDCKMLSSIYNE







ACCACCGTCTTGCACCCACT
CQHHLKIPAQWKEAETILLF







AAACCAGTGGGATATGCGGG
KSGDESKPDNWRPISLMPTI







TGGACTCAATGTAGAAAGGT
YKLYSSLWNRRIRTVKGIMS







GTTCCCACTGCCTGACTCGC
KCQRGFQEREGCNESIGILR







CAACTTTATATGTCTTGTCA
SAIDVAKGKRSHLSVAWLDL







ACATAATGGCCCCTCACTAT
TNAFGSVPHELIESTLSAYG







AAACTCCCTAGCAACTGGTG
FPEMVVHIVKDMYKDASIRV







GTCCGGCGAAGCCGGTTCTT
KNRTEKSEQIMIKSGVKQGD







GCCACTATTGCGCCCCAGGC
PISPTLFNMCLETVIRRHLK







TCGCC
ESSGHKCIDTRIKLLAFADD







(SEQ ID NO: 1298)
MAVLAESKEQLQKELTEMDE








DCTPLNLIFKPAKCASLIIE








FGKVRTHEQIMLKREPIRNL








NDDGTYKYLGVHTGADARTS








EEELIISVTKEVDLVNRSAL








TPPQKLDCLKTFTLPKMTYM








YANAIPKLTELSAFANMVMR








GVKIIHYIPVRGSPLEYIQI








PTGKGGLGVPCPRITALITF








LVSTMKKLWSDDEYIRKLYN








SYLKKVVEAETGIVEVSTKD








LAEYLSNKVPSRKHEFGYNC








YSRIREVCNGLALNQAAPLY








KLEFIEQDNELAVVVQPTEE








SKERIFTKDHVKKLQSLLKA








SVNDALLHRFLTTKPVKSEV








VQVLQQHPQSNSFVRMGGKV








SISVHVWIHRSRLNQLTCNY








NIFDPKQPKNCRRCGYKNET








QWHILQDCTYGWAKLIRERH








DAVHHKVVTMICAGAKKNWG








RKIDQELPGFTSLRPDICLT








SPDGKEVIFADVCVPYSRTR








NIEFAWKEKIRKYTEGYSHL








VAQGIKVTVLPIAIGSLGTW








WTPTNESLYQLGISKSDIRS








AIPLLCSTVMEYSKNAYWNH








IYGNSYTSVPLRYGHQKPDG








DDWKKELSCEPVLALQQ








(SEQ ID NO: 1420)





NeS
NeSL-
.

Schmidtea

TTAAATCATTTTTAAATGTG
TGAGTGTGCTACGAGGCAGC
MNVDLDATIKSIGMNTKETT


L
4_SM


mediterranea

TTTGAATATCTTAAATTATC
GCTGGTAATTGCATCGGCGT
YPNSQLRVETTPCTSTTIMH






AAATCATATTAATATCAATG
TGCAGATTTGTGTACGATAG
ASCNTTSTISYSPLPSAVSL






CTAAAAAAAAATCGTGCKCA
ATAAAAACCAATAGTAATAA
PESPASSITITTTDDNCDII






TCAGGCGCACGAAAATAATG
ATGCTGAGCCTAGCTCGCAT
ETPYPLPQTNGDLSEILKDI






GACACAACTCGTCGACCTGC
ATCTAAGCCGAAAGGCAGCA
EANKDTTMSNKVLDCDSDSG






TGTCGACTCACAGAGAACCT
TATATATGAGACAATTTAAA
DDRDMIIENDRESDMDLFSQ






CAATTTGGAAGAATGGGAAG
AAAAAA
SLLNTNQSDERREKNLTENA






CCTATAATGCTACAATTCCG
(SEQ ID NO: 1299)
PTEITTEKSYFDIISKASDN






CCAACCCCTATTTGAATGAC

TTSKKLLNVKNELTAGLPPM






AGATAGTCAAATATCAAAAA

PPVTNTAKFIRNVRPEDIAD






ATATACAAACTGCTGTCAAG

PTLYRLDSRGKLGCRTQYKK






CGTGACTCACTTCCTTCCAA

PGCGDIAVYDYEAIVEHAAF






TCGAAAAATAGGAAKATGTA

IHTIPFNEQNNVDCQPCHPK






AGAAACATGAAAGTCAAGCT

KGKDVHTIVLIKYADIFNHI






GAAAAACCAATAATATGTCC

EAHSHVVQTAITDNMKTYLR






TAAAATAAAACAATTTGAAA

LTKENXFYCSYRNNKKKNKC






ATATGCAAAAAATACCTATA

KKAFNLESNMMDITEHMKTH






AAATCACAGCCGAATAAATT

TGYSFDXNLNILCYCGIWKP






CCCATCCGTTCTAAGCAGAA

FTELIAHIKTEHLQEYINSI






ACCGCTACGAACTACTGCAA

PNKENIHNTTTIVSPLNFAG






GAATCGGATCAAGTATATTA

ILASGETQNIPDEEIIKPRD






ATTTCCCCCCMGGGGGAAAT

LPENLAFNRNIENELSWSQH






TAATATACTTGTTAKAAAAT

LVKAYIFSYAVKTSTIFINP






TAATTTTTTAATAAAAATAA

YTCNALIQCNYKTFFETFPF






ATAAATCGAATAAATATAAA

KDFAKWNEIVLPIHNNTSSW






ATAAAAATAAATCAAATTAA

SFFFLNKKKRVAMIIDPTAD






ACTTTTATTAACAATAAAAT

DSHTLHFELATDILRTILNV






CGCAGTAAGTAAATTTCCAC

QNIFEDLNFPLTEVEYPVCH






TGTTATTAAATTTAAAACAA

EANLSAFXVCHFLKCLMSDL






AATTCCTTTAAAAATGCCTC

PIDIPDIDHMKETMRPIIRK






TCTTTTTCAGTAATAACACC

YNCAKFPESDVRNYRVLIED






TTTTCTTGCTTTTATTACTA

LIYQLNLDTITCEEILCEIE






TTTCTTGTGTACTGTACAAA

RINGRLNPKRYFKESKPKTD






TCGAGCACAGTTATTGCAAA

IIHLQKKKSAELLCVKRLKF






TAGGACATAGAAATTCCTTT

QISQKTEIGKIWENDDVDHR






TTAAGTAAATTTAAATCCAT

PPMARFLKTFASQDCPVSNT






GAGAAATAAAATAAAATCCT

SSINLPYYMDTDTDXCTDCE






TTTGATTCAAAGTTTCTATG

NLSHIMKNLDSSAPGMDLIT






TTGCTTTCTAATAGAATGGT

GGDWKKISPKHELITAICNC






GTAAGCATTAATGGGTCTTG

ILRNKVCPEKWKLFRTVLIL






ATTTTTATAAATTAAATATA

KPGKMSESFRANSWRPLAIM






TTTAATCTATTAAATTAATA

DTAYRIFTTLLNNRLLQWIR






TGTTTTTATTAATTATTAAT

NGNLISPNQKAIGIPDGCAE






TTTTATAGTGGGGGGAAATT

HNATLHFAIDRAKRCKTELH






AATATACTTGATCCCAAGAA

IVWLDIADXFGSLPHDLIWY






TCAACTGATGATGAAGAATA

TLANMGLKNETLTLIKELYK






TGTTATTTCAAAATACATAC

DVKTIFDCQGTLSEPVPITK






AAGAAGCTGGAAAAAACAAA

GVKQGCPLSMTLFCLSIDYI






TCAATCGCTACA

LKSILTNYPFLLHDLNISIL






(SEQ ID NO: 1176)

AYADDLVLLSDSYLEIKKSL








ESTVELAAFANLKFKPSKSG








YLSINNVNSDILKLHLYNEE








IPTISENNKYRYLGVDFSYK








RNQDVDGRLGSALALTRSLF








KSYLHPAQKLNAYKTFIHSK








LIFSLRNCVIGHRILDCDRN








RVTQGREKQLGFDQEIKALL








KTMIGDKFQAXNNYFPYTHC








KLGGLGITSAIDEYLIQSIT








GITRLFHSSNLSFRKMLITE








LAHSRGGKNFEAGLKWLNCE








VNKAFPNTSFFVKFQKSALA








LKRKFCICVNLKFVEDNFSL








EMTYKKRTSYVNHQNLSTLS








KELHDFVGLYYAEQXCQMRV








QGHIATAIGDSITAKYLIAS








DILNDAQYYFLVRARNNLLN








LNYNAYRLKYNIGTKCRLCH








LDEETQAHXFNHCRAKPNAR








RVKHENVLVSIVAFLEKIGF








EIDVEKSPKYISIPTKLKPD








MVIRSKRNKDIHVLDLKVPY








DSGEGFEKAREDNYVKYKDL








AIEIGKAFNQKATISAVVIG








CLGTWDKKNNAALSKIGLTK








TEIISLARIACPNAVIACYH








IYREHVSFTKSAMALPFSLA








(SEQ ID NO: 1421)





NeS
R5
AY216701

Girardia

GTAGGTAACTATGACTGCAA
TGATCCGTGTGTTTGTGTCG
TTGRNLGQWSCYSRSIQQSN


L



tigrina

AATAATAATTCTACACCTAT
TATGATTGTTTCCGTGTGTG
YSFKLSSTEVGELVEQSPAP






TGTTGATAACTCATCTCGTG
TCTATATTTTTCTTTTTTAT
LQSPQFSNNYNNLNINNNLY






CGCAAACGGAGCATGTTATT
ACTTTCAATTACCTCGTTGT
YSLNTFNQSNNLCCLVNIEF






TCTAATCATTTCGTCACACA
AATGTTATAACTTCATATGG
FPTQHLLGDIVNSGCINYMN






GGATTCTTCTAATTCTGATA
AATATATGTAATTTAGTTTA
NYNNFDNINLYINSNVLSYN






GTAATATTATAGATAGAGAT
GTTTAGTTAGTTTAGTTTAG
NYNHSFLASPYTTNITEHAD






AGGAACCTTGTTGATTTAGA
TTTAGTTTAGTTTAGTTAGT
INMHVQEVNMQQDNNTQHAI






TGCGTCAATAACTTCTCCTA
TTAGTTAGTTTAGTTAGT
TQQVSLQATSLQHTLDEMIV






CTATTATACAGCCAGAGGAT
(SEQ ID NO: 1300)
QFNTAVRLKKKHKVAKIFRG






AGTAAGATATCTGAGGATGA

HNHRKDLPTLPAREQYKTKP






GGACTTCATCTTAGTCAATA

KLAIREVLHRKTTATSSPSE






GGAAAAAGAGCAAAAATAAG

NAIKAFFSSYSRPAELFTGQ






AAAAAATCTAAGAAAACAAC

ELLESSWFPVHPEDDFEFRI






TGAAAATAAAAATGAAATTC

PGRDQIAKYIKFASKSAAGL






CTATTCAAAAGAGTAAAGAT

DWITYEDIKLGDPSGEILQP






AAGAAAAAGAAGTCTAAAAT

IFEYIVQNNICPSEGKASRT






TAATACCGAAAAACTAACTG

IMIPKPGKSDYSDPSSWRPI






AAAATATTACTACTTCTGAA

TITSAVYRLLMKYLTWELYN






ATACCACTTGAAATTGCTCC

WILLNQMLSRSQKSLGKFEG






TTCCATACCTTTACCTTCAG

CHDHNAMLNMLIQDVRRQTN






CAAGTACCTCGGGTTCTCAA

PSNPINKNKRLYIVFLDFTN






CAACCGGCCAATCCTCCAGA

AFGSVPLDTLMYVPQRFGLG






AGACGCTACTCTAAGTGATA

TSALTLIKNLYLDNYTNVTC






CGGATCTCTTCCTTACACAG

GESKIENVKLNKGVKQGCPL






GATGATCCCGATAGTCTTAT

SMLLFNIFINIIIRAIEAMP






TCTTTCTGGAAGTACTCAAC

DVHGYPLGDMDIRILAYADD






CAACCTTTGTTGACCTCAAC

IALISDSHKDLQEMVYKAEY






CCTTCACAGCAATCGGAACT

IGRILGLLFNPSKCALMDIP






TCCTTCAAATACTGACAGCC

HDKKRTPPILVNGEMIKCVG






AAAGATTTGAGGCGGGTGAA

KADPYKYLGTFRSWFRKLDI






ACACCCAAAATCATAACTTC

KELLQMMMDETKLITESNLH






TTACAGGGATGACCTTTTCT

PHQKIHAYETFIHSQLPFHL






ACTCTACAGTCCTTCACTAC

RHSRIPFSDFITNRKTNKTT






AACTCAGATACAGGTTACGG

NNSNDSEKSIQKAYDPESGQ






TATAAGTGTTGACAATGGGG

LFLNTFALPSGCAKDFFYIT






AGCAGAGGTTTCGAATTCTT

KDAGGPQLTSGLDEYLIQSI






GCTAGGAATCTTGTCAGGAA

MYIFRLLGSEDPTLNSAIKH






AACCAAGGATAAGTTCCCCT

DLISHLNLKGFVNINFSQAI






CTTTATATGCTGGACAAGTA

SIFNSNFTDRTDHFSHLSRT






ATTAGACACACAGTCTTCTT

EWARLQLARKKLKSTLAIQT






CAATCACTTCAACCAGGCAT

NVCLINGHLVLTLSLENNVL






ACTACGCCAATAATATAACT

LIDSKEKGDVKKIHASLMGF






GATAGTAAAGGTAATCTAAT

LRLAHLIRLQKHGWSKLLFS






TGAGTTTTCTGATGATAAGC

ATTHHEILNKRILNGHVPYK






CTTTTCAAAGTATACCGACT

IWYFIHRARLGLLPTKLFSV






GACCCAAAAACTGAACTAGA

SNLCRKCGGKKETMSHALVN






GCAAATTAGGAGAGAGAGAC

CPMMQTLINERHDALEISLV






AACATCTAGTTGATAGAGCT

QILSSKFQGTVIRQKTYVNE






CTTAGACATAATCAGTTACG

LRPDITMESDTQYYLVEVKC






GGAAACTTATATTTTAAATA

PFDTKMSFELRTQQTTDKYN






AACTTAATAATAATAATGGG

IIIEILEDVHPGKEVRLVTF






GGGGGTGGCGAACATTTGAA

IVGTLGSWGPQNSDFLRDLG






AAGGAAAAAGATCAAAGTCA

FSKDEIDQVKTRLMLQNINS






ATACGGATGATGTCTCCAGC

SCEQWKRFVQYAPTITPGPI






AATGATGGAGACAGAAAACA

PDAESEDDQGTSDNGPTAAT






TAG

VQGPVIGDEEEELQIYDSGL






(SEQ ID NO: 1177)

DESSDDEPDPDDAELLFTID








IEQYLNSVITD








(SEQ ID NO: 1422)





NeS
Utopia-
.

Chrysemys

GTTTAATTCCTTCTGATGGA
TAACCGAGACCGCCGACCAG
MESPAXIFEKIDAALXIYSA


L
1B_CP


picta

CATCTGCAACACCCGTCCTG
GGAAATAACCCACTTCCTTC
AAXLXXNSLSLSPXXAXXSX



B


bellii

AAG
CCTGACGAACCAAGGGACGC
XAAPASSTPQKTQXKPIPXT






(SEQ ID NO: 1178)
ACCCCACCCATGTACTTATT
TLGASRKXRTTXKDEXIXXW







CGCTACACCGATACTGACTT
XKKAPVDTSXGRXSTRRTAL







GGACTCCTTATACATTCCAT
RDLTSRSXNIXXALQEEDPR







GGGTGGCGTACCCGAGCCCA
RTPPXSRDQDAERRPAAPEK







CTTATCCACTGACACTTTAA
AATRGAPPTIQDQDADRCPA







AAACTCTTGCACCCCAATCT
GRDATGGAPRRPRTRMLXAA







GGGTCTATGCCGGTTATGCG
PLGRMPPEEPPPTTRDQDAD







ATATGTATGTATCTCTTCAT
RRPAAPERDAPEGTTSSTPD







CCTTGCAACCGATACCTGTA
PETTYHPPVRRRAAPRGTHS







ATCCCTCATAACCCAAGCCT
XAXDLDAARCPSGQRDIVAS







GACCCCAGATGTACAGTACC
ESSTPPGATSPPQASLPDXE







TTCCCTCTTAACTCGTGTAT
ESPAESAGTTEVRPTEGEAG







ATTTAATTTTAAACATTAAC
EDDCIYLQYPXPTGLLLCPF







TTTAATAAAATTTTTAAA
CLPXHGVQTLGALSKHVRKA







(SEQ ID NO: 1301)
HNKRIAFRCSRCDAPFETQK








KCKXHXATCKGPLTTAKVNP








TDTLRVPTPTPTDGPASAPQ








PASPEPQXVRGDQPPTEGSA








TPASRTDDATKRTSPASRIP








TLDPAVRGITATSQVSDLTR








CLSDLIKTIRHNTDTRRXSA








PPQVTSCRPAVGATSTAPQA








ARRDPANGGASRSPQIPRPD








PAPGRPNTSSKVTQRDSDRQ








KPHAPPRTPQPDTTRRRTRT








IPSASKHDRAPTKPNTGVSR








TPLPPGRSSAASETPRAAPP








PPHQDPRLKTHLNTAPQSEG








QQGHRLSPQHLNPRRQRSRR








NDGREQRVATPWQSAWMEEL








AKAEDFETFDTLMDRLTAEL








SAEITARRREPQEASRATRR








FPAPTRNNTAREGRRGDVGR








RYDPAAASRIQKLYRTNRTK








AMREILDGTSSYCAIQPERL








YSYFKDVFDHEAQTNLQRPE








CLLPLPRINLTEDLERDFSP








QEVQARLMRTKNTAPGKDGI








RYHLLKKRDPGCLVLAAIFT








KCKQFHRVPRSWKKSMTVLI








HKKGERDDPGNWRPISLCST








IYKLYASCLAARITDWSVCG








GAVSSVQKGFMSCEGCYEHN








FLLQTAIQEARRSKRQCAVA








WLDLTNAFGSIPHHHIFATL








GEFGMPETFIQILRDLYKDC








TTTIRATDGETDAIPIRRGV








KQGCPLSPIIFNLAMEPLIR








AISSGPTGFDLHGKKISILA








YADDLALVADSSESLQQMLD








VTSQAAEWMGLRFNPKKCAS








LHVDGGARALVRPSRFLIQG








EPMASLEEGEVYQHLGTPTG








VRVRQTPEDTIAEILRDAAQ








IDSSLLAPWQKINALNTFLI








PRISFVLRGSAVAKVPLNKA








DSTIRQLVKKWLYLPQRAST








DIIYISHRQGGANVPRMGDL








CDVAVMTHAFRLLTCPDPTV








RSIAQEAVRDVVRKRIARAP








SEQDIATYLSGSLEAEFGRE








GGDLSSLWSRARNASRRLGK








RIGCCWKWCEERRELGILVP








RIKTPDHTIVTPTARAMLER








TLKDAIRCHYAENLKRKPDQ








GKVFEVSSKWDASNHFLPGG








SFTRFADWRFVHRARLNCVP








LNGAIRHGNRDKRCRKCGYA








NETLPHVLCGCKQHSGAWRH








RHNAIQNRLVKAIPPSLGKI








TLDSAIPGTDSRLRPDIVVT








DAEKKKVLMVDVTVPFENRS








PAFHEARARKALKYTPLAET








LRAQGYEVQIHALIVGALGS








WDPHNEPVLRACGVGRRYAR








LMRQLMVSDTIRWSRDIYTE








HITGHRQYHTE








(SEQ ID NO: 1423)





NeS
Utopia-
.

Acanthamoeba

CCCGTCAAGGGTGCTCCACG
TAACAACCATGTATGGTGAA
MAAKSVACPHDGCANKYASE


L
1_ACa


castellanii

AGATCCCTGTCGCTAGCCGA
CCACACCTCTCTCGATCTTG
ASLRRHIKNKHATDEEGDET






CCGGTTTTACCACCCCACCC
TATTCTGTGATTGGACATCA
SHSCPHCHRPFSTARGLSVH






CGCCCGGACAACCACGGACC
GAGTTCCTGCGAAGGGATAC
IGKSHRQAPPEPTRPPPAPA






CTGCTCCGCAGCAGGACCCC
ACTCTGCCAATCTCGTGGGT
PADPGLDPDPGPTVTPPSRD






ACGCACG
TGTAATAAATCCACACCTTC
DEDREEPDDDPVEIADLSCP






(SEQ ID NO: 1179)
AACA
HCAQALPSAHGLANHLRACK







(SEQ ID NO: 1302)
DHRVPAPGAPRSGPPSSRYW








TAVEHHRYVEAMARFADHPD








LLARAAAHIGTRTYKQVDSH








RTKVIAAEREGRPVRTLDPT








MDWRMRPYCASTTARWLAEQ








GRSPVAPRSPCPEPHAPPPA








AALLYIPATPPAPTPRAPVA








PPKLAPPAESTVPATPDGNP








EAPAPPFSAPGPPTPKALPP








PPPSRRNLRPHLVPKDAWQG








VADAVAPAASRLLRTPLAHL








STEQWATFEAALAGLEATLH








HAARSAEAVPTRCASRARED








AERQLREARKTREIFGKAAA








LYAAGKDPTATIERIPPEVR








LHLPTPGSAEWPARAAAARR








VIRRAVARADRLRKRMGILD








SDRDLQRLFNANQKKAVRQI








LAPSTKAPRCQLDPAAVEEA








YIQTLAKPPPIDPSPPWKNS








VQWPRPPTAADDGGSPFSVA








EVRAQLRRLPNGSAPGIDGI








PYEAYKRTKLDATLAHVFEV








VRLNARLPARWDVARTVLLY








KKGDPNDTGNWRPISLQVTI








YKIFTAALSKRLISWAGKHN








TFSASQKGFLPAEGCHEHAF








VLRSVLDDARRHKQNVYLAW








YDLRNAFGSVSHDLIAWCAA








MLGLPRYLRDAIGAIYRHSA








LFVQVGDQETTGVIPMRCGV








KQGCPLSPLLFNLCVEPALR








CLRRTTGYKFYGTSITVEGQ








AYADDLLTAAPSAYHAARQV








ATIEEWANWAGVSFVVQALS








LDAPAGKCAALAINFEGGLM








HSIDPALKVQGAAIPAMSRN








NVYRYLGVHVGLTDALGQAN








ELLEKASRDARTICASGLEP








WQKVVAIKTFILSRLPFFFH








NGKIQRGRCQQFDRELRENL








RAALRLPVCTTNAFFHSRVA








SGGLGILPIAEEQQVYLAAH








VFKLLTSPDLSIRAIARHQL








AEVTHARHTTPVQDGEASPF








FGWLMRGQEVASTTPSGDVS








SIWFAAAGAYSRMGWSVRDA








LHPTLTVGPGVQFEGRFORA








NVIPALRASAFSRHAVEWSA








LRTQGRAAAYQHAVHPATHH








WVHNSAGLTTKEYRFAIKCR








LGLLPTRAAPHHRNGPTACR








ACSYARETANHVLGHCPATK








AEVIARHNRICRALAQAAEA








SWTSVLEDVPIPGVDSPLRP








DIYCSRPGQCAIIEVAVSYE








DAFNASMEGRAKQKTDKYAG








LAATVEEQLRLQTRHAAFVV








GFSGVVLPASVTATATSLDL








PPKTWNVLLKRCVAASIKGS








YTAWRRFRRSTP








(SEQ ID NO: 1424)





NeS
Utopia-
.

Acromyrmex

GGTGCACAACGGATGCATCA
TAAATTATTTTGTCTTTGTC
VCSVRGCRREDSRRFYKFKF


L
1_AEc


echinatior

TACGTGTACCGGAGCATACG
TTGGCCCCCCCTTTTTAAAC
PLNFVKVPKTIVIGSAFQKS






GGCTGTCACGGCGGCTGCAT
CAAGCAGGAGAGAGTGGCCC
SVSARSQNHSRSTRVPKTRQ






GCGCGATCTAGCTCGGAGAT
AATGCCCAACTATTATATAT
PRTSNTIGRYTAASANNYLT






TTTATTTATTTATTTATTAA
TAACTATTTACTGTGATATT
VIITGNYTVFAQWICYRECT






TTTATTTATTTATTCATCGA
TATTATTTGACTGTTGGGCG
WLLSKFVNFFLTIIGYFFQL






GTGTGAGTGTTCGCGTTTTG
GGCCCCTCTCTGCTGGTTTT
RLVVIYEGPVILDTFSNCGS






CCGAGAAGCGATTTTCGTTA
ATTTATATATATTTTTTACT
SLFMRGQXSKALLVRLNRSA






AGTGATACGCGCCGCGTTCA
CGCGTACTTTTTGTACTACT
LAMADPQVHYIDYPLPPRVK






TAGGTTAG
CTATTTTTCTTTTTATTTTA
CVKCFGAEGAGKVKGEYSDP






(SEQ ID NO: 1180)
GCTATGCTATTTTTATCTCT
PHLAKHLKKCHPGDTLNYKC







TTCTTTGTCTCTATTTTCTT
SICDLRGTGKYPLRDVKAHY







TCTTTTTTTCTTTCCTTTTC
AECHVSPAVDAAGPSTRGSL







TTTTCTTTCTTTTATTCTTC
GECSGAGQPTASRAAKATTR







TTTTATTTATCTTTTTTTCT
LAETVGGTDKRRAATSGSRQ







TTTCTGTTGTGGGGCCCTGA
LTLPFAATPSPSTAAGEARA







CCGTCCGAGTGTGAATGCCG
PRSXSTTPTSRSPSYAAVTA







CGAAAAACAATATTATGTTT
GPPSMRSTTTSTTARSKTVA







TATACGAGTGTGCATGTGCG
KGAAPNTTTTTTARRSGEAA







TGATATATTTATCTATTTTA
ATRKPPTTATVSKPRVLSVE







TTTTATTTATTTATTATAAT
TVRLPVDDIQRAGVQNAAKP







TTATTGCCGCGCGCGCTCCT
ARAPSRPPQRTSPEAGGPRT







CCGGGACTTTTATTCGTTGA
TGAKEKCGEGAYKKLPANSG







CAATACTGTGATATTTTTCT
NPISTRTRRATSVPVEKSEG







GCKCAGGCTGGGGGGGCTTG
TARRERVSPHPPPKGIDIIL







CCCCCCAGCCCCTTAGTTTT
SSTSEEEGTPYQPGGVGRLR







AATTGCCTATGCGGGGGGGG
LRRKKVTGPPPKMTPREGVV







CTTTTGTCCCCCGCAAATGT
TRARRSTSAPVEKSALDARL







ATATATATATATATTTAGCG
TALDRTSSRATGNPTSQIAG







CGCGGCTTAGCCGCTTTTGT
GLYTSRGQPERTPPARLPSL







TTGTATTACCCCAGAGGGGA
SPTTRGSPSGSLGEIRTPIS







ATTGTCCCTCTGGGGAAAAA
PATSLPATLTTCTVTTTTCG







AAATGATTGGAAAAATAAAG
SPITSTGFTGGVGRLITPPS







TGAGCTAA
LPQTNILPTIGEEGTSPCVA







(SEQ ID NO: 1303)
VVTTHPRPTGEDAPCEAPQP








VSDHQRQSIGEPRRDTDTHL








ACDVATGNAHHLHGDDDHLW








KPHNIHGLHRWRGEADNTXE








PPPNEHPPDHRGGRNVTVRG








GRHHPSLGGRSTSPLILPRP








TTPEPERGQEERRLEGAAQP








PTTPVVEGDNQWDGQWTVSV








RRRARRQQLNDTSPSNSESP








PTAGPSRSPRIAPLSALIAA








STSRHETSLNLNCTNGNICM








DRTPPRNILPVXAERRRETS








PQDRVEGDIGYGAGKVSAEH








PSAPVNVRGVMSRGRATASS








IVPPRANRGEGGRQHHSRRR








PDAPVGQPSRDHPAPATVAR








QRRRERVAARDALLDRAKDV








ATIADLEAFAASVAAFFGED








ASATGAAARARDRSVRSREA








GARRGVKGGERPEREGAGRP








GSAPADPGASGEARGDWVRE








AKRLQALYRANRRKAVREVL








QGPADQCQVPKRQVQEYFER








LYSGGEDLAGAGVEAERPDP








SSPREVSAVLGPLAEREVDR








RLRRMNNSAPGPDGVSYRDL








RGADRGARLLTALYNICLRL








EAVPASWKTSNTVLIHKKGD








RGMLENWRPLALGDTVPKLF








AALLADRLTDWAVTRGKLCS








AQKGFLRDEGCYEHNFVLQE








VLTHAKRSKRQAVVAWLDLS








NAFGSIPHATIRRALIRSAV








PRGLIAIWDSMYDGCTTRVR








TAEGHTAPIPIRSGVRQGCP








LSPIIFNLAIDSVVRVAAEX








NDGYSLHGNTWSALAYADDI








ALLAQTPEGMERMLASVEAE








AASVGLRFNPAKCATLHVGA








GNGGRVLPTSFQIQGETINP








LAQGESYTHLGVPTGFSVDQ








TPYAAVGDIVSDLRAVDRSL








LAPWQKIEMLGTFILSRLDF








LLRGARVFKGPLTAVDLNIR








RHVKSWLNLPQRASAEGVYM








PPRWGGCGLLPLSDLADVLT








VAHAYRMLTVRDGAVRELAW








ESLRGVVGRRIGHAPSCEDI








ASFLSGSLDGRMRGGGEASL








WSSARNAALRQSERLSLRWR








WVEATEEMTLECRGPRGAAI








KIPPEARGQVVNRLRSAVAE








HYASRLLSKPDQGKVFEVSS








RSRVSNHFIRGGSFTRFADW








RFIHKARLDVLPLNGARRWE








ANDKRCRRCGEVSETLPHVL








CHCGIHSAAIQLRHDAVLHR








LWKATRLPGVVRVNQRVEGV








SDELGALRPDLVVRHEPSKS








VVICDVTVPFENRWTAFEDA








RARKIAKYSPLAEELQRRGY








RVVVTAFVVGALGSWDPRNE








AVLRLLRVGNQYAAMMRRLI








VSDTIRWSRDIYVEHVSGTR








QYLAPSRPSGDLATPPRAVR








RRWLAEERSAQDAARRGSDS








VSVA








(SEQ ID NO: 1425)





NeS
Utopia
.

Alligator

TGCTGGAAAGACGGAGAACC
TGAACCCCCCCTCTGCACCA
CHHAGLRPGTPNRTRRPDQT


L
-


mississip-

GCTTCCTTTTTCCCTGCGCC
GATGGACCTTCACTTCGAGA
APLPDPRGHPMPPNRRGSRS



1_AMi


piensis

TGGCCTGGTATTGCAGTACC
GGATTCTTCAGCAATGGACG
RPEEPSRREPPXPRACQGLR






TCCAGGATTAGCGCCAACTA
ACCCCGCTCCACCCGAAGAG
VWSPPQQRMPTPWQTLWLEE






GTCCGGCAGACTGTCGGAAT
GACCCCCGCGATGAGACTCT
LSRATTFKAFEASVARLTEE






ACAGCAATAGAAAGWGAGCT
ATATGGACTGAGACACTTTT
LSAAARPGQPRGGNNRPATR






GACTAGCAGCTTGCTTTCCT
TCTTCGAACCACTTCCTCCA
RDHRLQPQRRPRRQRYDPAA






TCCTCCGGTGCAGCATGGGT
CCATTGCGGACCATTGTAAC
ASRIQKLYRANRPKAVREIL






TCTCGTCAGTCMTGACGGGC
GGGTTTGTGTGTATCTATCT
EGPSAFCQVPRETLFNYFSR






TAGGGAAGGCGGTGCTGCCA
TCTTTCTCTCTCAGCGTCGC
VFNPPAEAAAPRPATVEALT






GTACGTCCGAAAGAGTGCCG
GAACCCCCTCCCTCCCCTTC
PVPPAEGFEDAFTPQEVEAR






GTTGCGCAAGCGACCGCGCC
CCCTCCCCCTCCCCCCCACC
LKRTRDTAPGRDGIRYSLLK






ACTCAGGTGAGTAGCCAAGG
CCCGGGCTTAGTTGGCTAAC
KRDPGCLVLSVLFNRCREFR






GTCTTACAGTTCACCGGACC
ATTGTATCTCCTGTAACCTA
RTPTTWKRAMTVLIHKKGDP






CGAWAACGCGAAAACCCCAA
GTTGCGTTCCCCTCCTCACC
TDPGNWRPIALCSTVAKLYA






CTCGGGCTAGTAGCCGAAGA
CCCATCCCTCTATTGTTAGT
SCLAARITDWAVTGGAVSRS






CCTGGGTCCCCCCCMGGTCA
CCCTCGCTCGGGCGCTCTGT
QKGFMSTEGCYEHNFTLQMA






GAGTAGGCGAACGCCWGKGC
ATTTCCCTACCGGCTTTGTC
LDNARRTRKQCAVAWLDISN






TCAGAGGACGGAACGCGGAA
ATCTTTTTTGGATTCACAAT
AFGSVPHRHIFGTLRELGLP






AACACCCCCAGGTCCCAAGG
CCTAAACATCTACTAATAAA
DGVIDLVRELYHGCTTTVRA






ACGCCCTGATCCACTGACAA
AGTCAATC
TDGETAEIPIRSGVRQGCPL






GAACGCTCGAGGCACGCCAG
(SEQ ID NO: 1304)
SPIIFNLAMEPLLRAVAGGP






GAGACCCCCAGCTAGGGTGG

GGLDLYGQKLSVLAYADDLV






ACCGCCGACTGCAGGTCCGG

LLAPDATQLQQMLDVTSEAA






AGGACCCTCCCAGGAGGGTG

RWMGLRFNVAKCASLHIDGR






GACCAGCGAACCCAAGTTGG

QKSRVLDSTLTIQGQAMRHL






CGACGAACCCTGACGCACCC

RDGEAYCHLGTPTGHRAKQT






CCCACGATGTCAGGACCCCG

PEETINGIVQDAHKLDSSLL






ACAGGCGGCGGTGGACCACT

APWQKIDAVNTFLIPRVAFV






GACCATCGACCGACCCCCAG

LRGSAVPKTPLKKADAEIRR






AGGCAGAGAGACTCTCAGAG

LLKKWLHLPLRASNEVLHIP






CCCGGAACCCCGGCTGACGA

YRQGGANVPRMGDLCDIAVV






GAGCCGCCTCCCGGCGGAGG

THAFRLLTCPDXTVSIIAAS






ACCCCGGAGCCTGAGGATGC

ALEETARKRIGRQPTRRDLA






CCCCCGGATGACGGCGGAGC

TFLSGSLEGEFSRDGGDFAS






GCCCCGAGCGACAGCGGACC

LWSRARNATRRLGKRIGCAW






CCTCCGGACCCCCACGGCCC

TWTEERRELGVSLQPAPHAD






CTCGGTGACGATGGCGGGCC

RVTVTPRTRTFLERFLKDAV






CCGAACGACGACGACCCCCG

RNKYAGDLRAKPDQGKVFDV






GACCCCGGCGGTCCCGAGGA

TSKWDSSNHFMPSGSFTRFA






CGCCCCCCCCGAGGGTCTCC

DWRFLHRARLNCLPLNGAVR






CCACGCTGGTGGAGGAGCCC

FGHRDKRCRRCGYVAETLPH






CGGACCCCCCCGACACCGGA

VLCSCKPHARAWQLCHNAVQ






CCCCCCCACGGACGACCCAG

DRLVRAIPAAAGEISVNRTV






GCGAAGGCGTAGACATGACA

PGCESQMRPDIVITNEEAKK






GCACTCACGTTCCTCCCCTT

VVIVDVTIPFENRRQAFTDA






CCCCCTCCCGGCGAAGCTGT

RARKREKYAPLADILRGRGY






TCTGCCCGACCTGCCACCCG

DVTVDALIVGTLGAWDPSNE






CCAAGACAGTACAGGTCGCA

SVLHACRVSRRYAKLMRCLM






CGGCGACATGAACAAGCACC

VSDTIRWSRDIYVEHITGHR






TACGGCGCTTCCACCAGCTG

QYTDPTRRTAAGPDPEGTA






CGCCTAGCCTTCTACTGCGC

(SEQ ID NO: 1426)






CCTCTGCGGCACCGAGTACG








AGGCCCTGAAGCTCCTGAAG








AACCACCAGAAGGGATGCGA








GGGCCACGGAGCCGAGAGGA








GACCCGGCACGCTGGTGAGG








TCCGCTGCCCCGGCCCGCCG








GACCCAGGCCGCGGTGCGAA








GGCCCGCCAGACTGGCCACC








CCGCCGACAACCCCACCGGA








CCAGACCTCCAGGGACCACC








CGACGGAGAGACCTGCCCCA








GTGA








(SEQ ID NO: 1181)







NeS
Utopia-
.

Chelonia

CTCTTCTTATGAATACTTGC
TGAGCCGGTACGACATCGTG
MTTKKVLGASTTLQTSSTKG


L
1_CMy


mydas

AACACCTGCACTGAAGATGG
CATCAACTATGAGAAAGGGA
KNSGCSKDPLRDAVPGRSWI






ATTCTCCGGCTGCTATTTTT
CTGAGAGACTTTTTCCATTG
LRPACRDITTRRNIPPAPQQ






GAAAAACTGATGCTGCTTTG
GACCATATGAACTGGAACCA
QQPPMESPPTLQLQDALRRP






AAGGTGTATTCTGCTGCTGC
TAAACTCACTGAACATTAAA
SPTPAAAQVADAGGALAALH






TACCTTGGAAGGAAATTCTC
TCTCACCAAATGAGGGTAAA
TIKRGISVDWTSISPKXXQR






TCTCTGCTCCTGAGACATCC
TCCATCCTCATCATCGTATC
XTSASPDACPASETTQRDXR






CCAGCTGCACCGTGTACCAC
CACTCATTATACTCCACACC
XLLDARPAGPLDPTRPHQDE






CACCACCACTGCTGCTGCTC
TGAACATAGCCATTATATGA
PASDTADAAGTPLLQGNEDT






CACAGAAGGTTTCTCGGACA
ACAACATACCCCCATATCTC
IYLQYPLAADMLICPICSPP






(SEQ ID NO: 1182)
AATGTCTGTACTTTGACCCG
QSFHLLGVVTRHLKRCHSKR







TTAACCTTTTACCCCCAATC
VAFSCALCSLPFETQKQCKM







GGGGATATTGCAGATTATGT
HQVACRKCLKGTTQSPAPAP







ATTCCTTACGCCACCCGATC
SPPAARRPAAPEPQRRKXTS







CTAAACCGAATTTCGCACCC
QAAVKKPAPVARPAERDAAI







CTTGATAATCTGTACCTTAT
EKVPAASGNITQVLASRRPV







TCCCTGATAACCAGAAACTT
SPSHVAKXISMLRRLSAASP







CTATGCTTAAACTCTGTACC
PVQHVPVPRRISAPPRIAAR







GTTTTTTTTTATTTCAACAT
DPVAGRASAAPQTALRTPAA







CATCTTAATAAAATTATTAA
GGASTTPQTALRTPTAGGAS







A
AMPQTTLPXPRRPDWRNQPR







(SEQ ID NO: 1305)
SHSKAPGLHRQTDQHGPQVH








SAGHCLREISRSSSNRLGSS








HSAAATHRRTGGVPATPEPD








RVSPTTSNAXIPPEIPPQHP








TEGNPDPRDRRQADHTAGSE








PAPDEVEDXEGQRPMVRAAT








PWQTAWTEELQAAASFDDFD








LLVDRLTRELSAEIAPRRSS








NQENAPPAHRTPAPNHNTTT








RGARSRDASRRYDPAAASRI








QKLYRANRSKAMREILDGPS








PYCTIPSERLYSYFKDVFDR








IARNDAQRPECLRPLPRVDE








AGVLETDXTPKEVMARLSKT








KNTAPGKDGIPYSLLKKRDP








GCLVLATLFNQCKRFCRTPS








SWKKAMTVLVYKKGERDDPS








NWRPISLCSTMYKLYASCLA








SRITEWSVSGGAISSIQKGF








MSCEGCYEHNFVLQTTIETA








RRARRQCAVAWLDLANAFGS








MPHHHIFATLQEFGMPENFL








RVIREVYEGCSTTIRSVEGE








TAEIPIRSGVKQGCPLSPII








FNLAMEPLLRAISNGTDGFN








LHGERVSVLAYADDLVLTAD








DPESLQGMLDATSRAADWMG








LRFNAKKCATLHIDGSKRDS








VQTTGFQIQGEPVIPLAEGQ








AYQHLGTPTGFRVRQTPEDT








IQEILQDAAKIDASLLAPWQ








KINALNTFLIPRISFVLRGS








AVAKVPLNKADKIVRQLVKK








WLFLPQRASNELVYIAHRHG








GANVPRMGDLCDIAVITHAF








RLLTCPDAMVRNIAANALHD








ATKKRIGRAPSNQDIATFLS








GSLDGEFGRDGRDIASLWSR








ARNATRRLGKRIGCRWEWCE








ERQELGVLVPQIRSNDNTIV








TPSARGMLERTLKAAIHSLY








VETLKRKPDQGKAFELTSKW








DASQPLPRRGRLHPFRRLAV








HPPCPAQLRPAQRSRPPREP








RQALQEVRLLQRDPAPRPVQ








LQAPLQSLAAAPQCHPEPPG








ESHRTAPGGGRRELRHPRYP








ASGTPANHFLAGGGFTRFAD








WRFIHRARLNCVPLNGAVRH








GNRDKRCRKCGYSNETLPHV








LCSCKPHSRAWQLRHNAIQN








RLVKAIAPRLGEVAVNCAIP








GTDSQLRPDVVVTDEAQKKI








ILVDVTVSFENRTPAFREAR








ARKLEKYAPLADTLRAKGYE








VQMDALIVGALGAWDPCNER








VLRTCGIGRRYARLMRRLMV








SDTIRWSRDIYIEHITGHRQ








YQEV








(SEQ ID NO: 1427)





NeS
Utopia-
.

Chrysemys

TTTTTTCTGATGCTTGACTG
TGAGCCAGAGTGACATCGTT
MTQDQDADCCPAGKDATRGA


L
1_CPB


picta

CAAACACCCATCCAGAAGAT
CTCCCACTACGAGAAAGGGA
PPMTQDQDADRCPAAPERDA






bellii

GGAATCTCCTGCAGCCATTT
CCAAGTGACCTTCTCCGTTG
PEGTTSSTPDPKTTYHPAVR






TTGAAAAAATTGATGCTGCT
GATCATATGAACTGGAACCA
RRAARRGMHLRAQDLDAARC






TTAAAGATATACTCCATTCT
TAAACTCCCTGAACATTAAA
PSGQRDNVASESSAPPRATS






CCTWKTTTGKAAGAAAACTC
TCTCACCAAATGAGGGTCAA
PPQASLPDPEESPGESAGTT






TTTTTCAGCTTCAGCTATTC
TCCATCCTCATCATCATATC
EIRPTEGEAGEEDRIYLQYP






TGTCATCGGCTGCTGCTGTT
CACTCATTATAMTCCACACC
LPTGLLLCPFCLPVHGVQTL






CCTGCTTCCCAGAAAGCTCA
CGAACACAGCCACTCTATGA
AALSKHVRKTYNKRIAFRCS






GCMAAAACCTATCCTGAAGA
ACTTCATACCCTCATATCTC
RCDLPFETQKKCKFHQATCR






CCWCCCTTGGTGCCTCACGG
AATGTCTGTACTTTGACCCA
GPPTTAKVNPTDILRVPTLT






AAGACCCGGASCACCTGCAA
TCAACCTTTTACCCCCAATC
PTDDLASAPQPASPESQQIR






GAACCAAAACATTAGGAGCT
GGGGATATTGCAGATTATGT
GDQPPTEGSVTPASRTDDAT






GGCTGAAGAAACCCCCCGTG
ATTCCTCATGCCACCTGATC
KRTSPVSRIPTLDPAVRGTT






GATACCTCWGCAGGGAGACC
TTAAACCAAACTTTGCACCC
ATSQVNNLTRRLSDLIKTIR






TGGSTCCAGMAGGACAKCTC
TCGATAATCTGTATGTTATT
HNTDTRRCSAPPQVTSCRPA






TTCGGGACCTCMCATCSAGG
CCCTGATAACCAGAAACTTC
VGATSIVPQAARRDPANGGA






AGCAAGAATATCTCAACAGC
TATGCTCAAACTCTGTTCAC
SRSPQIPQPDPAPGRPNTSS






TCTTCAGGAGGGGGACCCCC
TATTTTTTTTAACATCATCT
KVTQRASDRQKPHAPPRTHQ






GGAGAACCCTGCCCGCTTCC
TAATAAAATTTTTAAATCTG
PDAARRRTRTIPSASKHDRA






CAGAACCAGGATGCTGATCG
TT
PTKPSTGASRTPLPPGRSSA






CCGCCCCACCGGGAAGGATG
(SEQ ID NO: 1306)
ASETPRAALPTTPGPPPQDP






CCACCGCAGGAGCCCCCCCA

PEHRSTVRGTTRPQTVPAAP






(SEQ ID NO: 1183)

EPAETTQQEERRPRARVATP








WQSAWMEELAKAEDFENFDT








LMDRLTAELSAEITARRREP








QEAARATRRFPAPSRNNTAR








EGRRGDVGRRYDPAAASRIQ








KLYRMNRTKAMREILDGTSS








YCAIQPERLYSYFKDVFDHE








AQTNLRRPECLSPLPRIDLT








EDLERDFSPQEVQARLSRTK








NTAPGKDGIRYPLLKKRDPG








CLVLAAIFNKCKQFHRVPRS








WKKSMTVLIHKKGXRDDPGN








WRPISLCSTIYKLYASCLAA








RITDWSVCGGAVSSVQKGFM








SCEGCYEHNFLLQTAIQEAR








RSKRQCAVAWLDLTNAFGSI








PHHHIFATLGEFGMPETFIQ








ILRDLYKDCTTTIRATDGET








DAIPIRRGVKQGCPLSPIIF








NLAMEPLIRAISSGPTGFDL








HGKKLSILAYADDLVLTADD








PESLQGMLDATSRATDWMGL








RFNAKKCATLHIDGSKRDSV








QTTGFQIQGEPVIPLAEGQA








YQHLGTPTGFRVRQTPEDTI








QEILQDAAKIDASLLAPWQK








INALNTFLIPRISFTLRGSA








VAKVPLNKADKIIRKLVKKW








LFLPQRASNELVYIAHRHGG








ANVPRMGDLCDVAVITHAFR








LLTCPDATVRNIAANALRDA








TEKRIGRAPSNQDIATFLSG








SLDGEFGRDGRDIASLWSRT








RNATRRLGKRIGCRWEWCEE








RQELGIRVPQIRSDDNTIVT








PTARGLLERTLKAAIRSLYV








ETLKRKPDQGKAFELTSKWD








ASNHFLDGGGFTRFADWRFI








HRARLNCVPLNGAVRHGNRD








KRCRKCGYPNETLPHVLCSC








KPHSRAWQLRHNAIQNRLVK








AIAPRLGEISVNCTIAGTDS








QLRPDVVVTDEAQKKIILVD








VTVSFENRTPAFREARARKL








EKYAPLADTLRAKGYEVQMD








ALIVGALGAWDPCNERVLRT








CGIGRRYARLMRRLMVSDAI








RWSRDIYIEHITGHRQYQEA








(SEQ ID NO: 1428)





NeS
Utopia-
.

Drosophila

AAAGTGTAGTTCTTTTCTGT
TAAAAAATTAAAATGCCTTA
YAPGYEAAQSPCGREPPRDH


L
1_DYa


yakuba

TTTAGTGTAGTGGGAAGTCT
AAAATAAATAAATATATCAA
HRRPRDACGSSHSPEPCLTT



k


GTTTCTTTTTATTATGTTTT
AATTTAAAAAAAAAAACGAG
PRLLPETVSAEPCDDESQRT






TTACGAAAAAGTCCTGGTCT
GAACAAATAAACACAAATTC
RYASPHKQARTLHDAEPRDA






TTGAAATTCATTGTCTAAAT
TGAAAGATTTATATAATTTA
SREHAPSCAEPRCHRCQWTH






TTTAAATAAAATTATAAAAT
AAAWATAAATCGAAAATAAA
WKDCCPHSTNTTDGPEGTDR






TTAAAAAGAAAATTAATTAA
TGTTGAAAACAAAAAAAAAA
CADTITSPATAACPQRSPCP






AGAAGCGATGAAATATCTCT
TAATAATAATAATAAAAACA
LGSSNGCDETAPEKRQPAAD






GAAATTCAATCAATCAATTA
CAATAACACTCACCCGGCCT
LVHTAPFAVLVRAGPFADLV






ATCATGGCGTCTCAGCGAGT
GCCCCAGAGGCAGGTAAACA
RAGPFADHHQDDDPLPHRSG






GCACGTATTTGCCTACCCCT
TTTACTGGCCATATGGCTTT
SLGPLCSKQKDPRKTHQHRH






TCGTGGGACCATTCCGGTGC
TTTTTTAA
SGQAGNQTHTDIPRAAPSRR






TCCGTATGCATGGATGCGTC
(SEQ ID NO: 1307)
AAICLMANAAATREDLLRAA






CGGGATGCATCCCACTAGGT

TSLSEMAAANQPTRSPTGGG






CGCTGGGCGAATACGGCACA

EPTSQGRRGPQALADAAKRI






TACGCTGCGGCATATCAGCA

QQIYRTNIPRAMRKVLRTLL






CATAACCCGGCGCCACCCAC

TAVFSACLRTGHVPDLCKKS






AAGTGGTTATTACATACCGT

RTVLIHKKGDRTDLSNWRPL






TGCCGGGTCTGTGGCGCTGA

SMGDTIPKLFAAVMADRLTA






(SEQ ID NO: 1184)

FLTNGGRLSEEQKGFLQHEG








CHEHNFVLGQVLEESRRQGK








DLVMGWLDLSNAFGSIPHAT








IMDAVAGMGIPSRIRTIIHQ








LATGAATTAKTIDGMSEEIP








IEAGVRQGCPASPILFNIAI








ERVLRKIKTVNAGYLLYGSR








ISPLAYADDLVLIASSPEEM








RSLLRAADDAAIEAGLHFNP








KKCATLHLTGKKSSRRAVQT








GFLVRGTPIPAMTEGDAYEY








LGIPLGLKKNQTPRAAMEAI








VGDIAKIDDSLLAPWQKIDA








ARTFVAPKLDFVLRSGATLR








APLRHLDTVIKKHIKKWLYL








PQRASAEVVYTPLKKGGAGI








LPSSILADVLTIAQAHRMVS








CPGEVVSRIASEGLREAVKR








KINREPSGDEMAHFLSGSTL








SGETASFGDAGFWSRVRMAT








KRQAVHLGVRWAWRGGELLV








ESRGQRNRPVATDSNSRSQL








IQRLRCAAQDEFLTILINKP








DQGKVAKLSTLTPVSNAFIR








DGSFTRFADWRFIHRARLGV








LPLNGAIRWGSGDKRCRVCG








YQLESVPHVLCHCMHHSNAM








QQRHNAVMDRLAKAGSRLGT








PRVNCRVEGVAEDMAALRPD








LVWRDERSRKIVIVDVTVPF








ENGAEAFDNARGEKEEKYRP








LAEALRAMGYQVKLEAFIVG








ALGSWDPKNERVLKTLGVSR








FYAGLMRRLMVADTIRWSRD








IYVEHVSGIRQFTLPSGAPS








N








(SEQ ID NO: 1429)





NeS
Utopia-
.

Gavialis

CGCTGGAAAGACGGAGAACC
TGAACCGCCCCCCCTCCGCG
MSGPRQAAADPRPSTDPRRQ


L
1_Gav


gangeticus

GCTTCTTTTTCCTGCGCCCG
CCAGACGGACCTTCACTTCA
RDSQSPEPRLTRAASRRRTP






GCCTGGTATTGCACTTCCTC
CTCCGAGAGGATTCTTCGAC
DPEDAPRTTAEHPERRRTPP






CAGGACCAGCGCCAACCTAG
CACGGACGACCCCGCTCCAC
DPRGPSATTAGPERRRPPDP






TCCGGCAGACTGCCGGAATA
CCGAAGAGGACCCCCGCGAT
GGPEDDPPEGLPTLVEEPRT






ATAGCCTCAGAAAGAGAGCT
GAGACTCTATACGGACTGAG
PPTPDPPDGRPRRGCRRGSA






GGCTAGCAGCCCTCTTTTCT
GCACTTCCTTCGAACCACTT
HVPPLPPPCEAAVPDLPPAK






TTCCTCCGGTGCAGCGTGGG
CCTCCACCATTGCGGACCAT
AVQVAQRHEQTPTALPPAAP






TTCTTGTCAGTCCTGATGGG
TGTAACGGGTTTGTGTGTAT
SVLLLPLRHRVRGPEAPEEP






CTAGGGAAGGCGGTGCCGCC
CTATCTCCTTTCTCTCTCAG
PQGMPGPRGREETRHAGEVR






AGTACGTCCGAAAGAGCGCC
CGTCGCGAACCCCCTCCCCC
RPTTRAAARRPARPAAPPAT






GGTTGCGCGAGCGACCGCGC
ACCCCCCACCCCCGGGCTTA
PPDQTSGDRPTERPAPATPP






CGCTCAGGCGAGTAGCCCAA
GTTGGCTAACATTGTATCTC
RRSAPRDPRPDVTPRPDGPP






GGGTCTTACGGTTCGCCGGA
CTGTAACCTAGTCGCGTTCC
PGPPGPPDAPDPPRIPEPPG






CCCGATAACGCGAAAGCCCC
CCTCCTCACCCCCATCCCTC
EPEPPGALQLPSVPGSPGAE






GACTCGGGCCAGTAGCCGAA
TATTGTTAGTCCCTCGCTCG
TSAQQRMPTPRQALWLEELS






GACCNTGGGCCTCCCTCCCC
GGCGATCTGTATTTCCCTAT
RATAFEAFEASVARLTEELS






AGGTCGGAGTAGGCGAACGC
CGGCTTTGTCATCTTTTTTC
AAARPGQPRRGADNGPTTRR






CCGTGCTCGGAGGACGGAAC
TGGATTCCCGATCCTAAACA
DHRPQPQRRPRRQRYDPAAA






GTGGACAAAACACCCCCAGG
TTTACTAATAAAAGTCAATC
SRIQKLYRANRPKAAREILE






TCCCAATGACGCCCTGATCC
TGTTCTTT
GPSAFCQVPRETLFNYFSRV






ACTGACAAGAACGCTCGAGG
(SEQ ID NO: 1308)
FNPPAEAAAPRPATVEALTP






CACNCCAGGAGACCCCCAGC

VPPAEGFEEAFTPREVEARL






TAGGGCAGACCGCCGACCAC

KRTRDTAPGRDGIRYGLLKK






GGGTCGCGGAGGACCCTCCC

RDPGCLVLSVLFNRCREFRR






AGGAGGGTGGACCAGCGAAC

TPAAWKRAMTVLIHKKGDPT






CCGAGTCGGCGACGAACCCC

DPGNWRPIALCSTVAKLYAS






GACGCACCCCCCCCGCG

CLAARITDWAVTGGAVSRSQ






(SEQ ID NO: 1185)

KGFMSTEGCYEHNFTLQMAL








DNARRTRKQCAVAWLDISNA








FGSVPHRRIFGTLRELGLPD








GVIDLVRELYHGCTTTVRAT








DGETAEIPIRSGVRQGCPLS








PIIFNLAMEPLLRAVAGGPG








GLDLYGQKLSVLAYADDLVL








LAPDATQLQQMLDVTSEAAR








WMGLRFNVAKCASLHIDGRQ








KSRVLDSTLTIQGQAMRHLR








DGEAYCHLGTPTGHRAKQTP








EETINGIVQDAHKLDSSLLA








PWQKIDAANTFLIPRVAFVL








RGSAVPKTPLKKADAEIRRL








LKKWLHLPLRASNEVLHIPY








RQGGANVPRMGDLCDIAVVT








HAFRLLTCPDATVSIIAASA








LEETARKRIARQPTGRDLAT








FLSGSLEGEFGRDGGDFASL








WSRARNATRRLGKRIGCAWT








WTEECRELGVSLQPAPHADR








VTVTPRTRTFLERFLKDAVR








NKYAGDLRAKPDQGKVFDVT








SKWDASNHFMPSGSFTRFAD








WRFLHRARLNCLPLNGAVRF








GHRDKRCRRCGYAAETLPHV








LCSCKPHARAWQLRHNAVQD








RLVRAIPAAAGEISVNRTVP








GCESQMRPDIVITNEEAKKV








VIVDVTIPFENRRQAFTDAR








ARKREKYAPLADTLRGRGYD








VTVDALIVGTLGAWDPSNES








VLRACRVSRRYAKLMRCLMV








SDTIRWSRDIYVEHITGHRQ








YSDPTRRAAAGPDPEGTA








(SEQ ID NO: 1430)





NeS
Utopa
AGCV0

Lytechinus

ATCTACTATC
TGAATAGCATTTATATTGTG
MSCPREGSDHLGPDPETPAL


L
-1_LV
1358106

variegatus

(SEQ ID NO: 1186)
TTCCAAACAACATACTCATT
HQGSDIRVTSSRLRGSRGKS







ATTATATCTAAACATTTTTT
SRQPSSRHQVPASEASATAQ







TTTCTGTTCCTGACAATCTA
QTAANECQVCGSSFATSSGL







CGTAAAGTCTGCTAACCAAC
RRHMARLHRAASADPEGAAP







TGGCATGATGAAATAAGATA
ASITEIFDYPLPSRWKCSAC







AAATCCCCTTACACATTAAT
SENFFNQQTLKRHQTRHHPA







TTCTTGTCACATCATAATGC
TTFAYAFRCSSCRSEFDSAR







TTTGTCAAAGCAATGTCCTA
RAANHWQVHKKERSQLSGTE







CATAATATCTCGATGTCACC
PQASSQARVSMAHSPPPLPN







CCAATTAATTTTACATCCTT
TSWAELASNPAEIPSFVWES







CGGTAACCTTTATACCGTTG
PPKNRPSVEEFGSSLPTDVT







GATCAACATATATGATTTGT
MMSQSPPPQVQSSPVPALTP







AAAACTGTTATTTCTGAGTT
LSPAATASSSPPGAARQLTP







TTTTCTATGCTAATAAA
PTQTNTPVTQRARLQPEADV







(SEQ ID NO: 1309)
VPELPPSVTEHPVSDAQHWV








DAVSSASDWSEFEAVCDQFV








IHAVAVSRPNLARPQQQDRQ








RSGDHPPRQQRGQHRPTFDV








REASRIQKLYRTSKKRAIRH








ILKEKSPSFSGSESDVLDFF








REVYSAKEVDEEAVGKLASS








LFDVPQGDDSATSLSLPTSA








KEIGARLSRMTNSAPGKDRL








EYRHIRRADGSFSISEAIFN








KCLAEGRIPAPWKTASTILL








HKAGPTDDPANFRPIALQSC








LYKLFMAVLADRLTKWACEN








QYLSPEQKSARPCEGCFEHS








FLLSAALKDCRRNQKTICIG








WLDLRNAFGSIPHPVIKIVL








SSLGVPDSLVTLLMDAYNGA








STSFTLTGGQTDTVPIRSGV








KQGCPMSPILFNLAIELIIR








AVKKNASDNHLGVTVQGKNL








SILAYADDLVLLSRDTEGLQ








SLLQVAGSSASTLQMQFKPQ








KCATLTLDCKRGTNVRQSAH








HIQGAAIPSLTEEERYRYLG








VPIGLPRLTSLQESSRKLSS








DIETISSSLLAPWQKLDAIK








TFVIPILQYTLRATEYLKSD








LKPLRAAIIKHVKKICHLPV








RSSNAFVFASRPSGGLAFVD








PGVDADILVVTQAVRTLASD








DDTVRAVALGQLTSVVHRTV








HSAPSDDCIDKFLSGSSEGP








LANSGNSGQASSLWSRTRAA








SRRLKIRIVGASSGDIKVES








GGRAIPSKKVTAGLRSDHHN








EMSEKLRSLPDQGKVARALS








LDSFANATSWLTSGSFIRFC








DWRFIHRARLNCLPTNAAVR








RWKQNANTKCRRCDHQLETL








PHIINNCRPNMVPIRRRHNS








IQERLVKAIHYGDIYQDQHV








PGDPNPRERPDITVVEGNKV








TIVDITIPFDNGPDALSTAA








NAKVMKYDTLRQELASRGMD








VEVHAFVIGSLGSWHGDNER








VLGRLGISRRYRTLMRRLCC








IDAIKGSRDIYIEHVTGHRQ








Y








(SEQ ID NO: 1431)





NeS
Utopa-
.

Nasonia

CCATTCCTTCGTACGGGTTT
TAGTGGGGCCATAACACCTA
SGXTGREVKCITVNVLMEQQ


L
1_NVit


vitripennis

TCGTGCCGGCATAGCCGGGT
GGCCCCACAGTGTGGCGATG
PHTKAIREGDFIVILLPQSD






GGGAGACTCGCGCGGGGGAG
TCCATGTGTGTCGTCCTTAC
DETLCCPLCVGRGRYSGKTR






GTCATATCTCACCACCATCC
TTATTTATTTATTTGTCCTG
VECLNRHVKEVHPDLTTTFR






TCGAGCTTTTTGCTGCACCT
TCGAGCTGTTTAATCTATTC
CWGCGFAAPGDKKYPRKIVT






GA
ATATGTATGTGTTGTGTGTG
QHCATCVPEVSSAPSGRVDG






(SEQ ID NO: 1187)
TCGTCCCTCGTAGCAGTTTT
ERRVNTRRRLGIAAATEASP







ATTCCGTCCAACCAGAGGTC
VRRTRRNGLASPPVEQNISQ







GACCAATATTAAAATAAGCA
SAAPPEPARVPQHPEIVALG







TGGCTTGAAGCAGGCCAAGC
ESADDEVFRSPVNSPPRDWR







GCCGTGTTCTAACCCCGTTT
AAAPQQAASSSPXAVPGITA







TAGGGGAAGTTACTTAACCT
ATPSNTTRTGNGSAXSILAE







AAAAATACAACTTTTCC
HPIPAPPPTNTTEANGRADI







(SEQ ID NO: 1310)
PRSGRAPPPGXQAARRRAPT








TEQRRIVGLLEAATGREQLE








EATTQAMLFLARLTGRRPEP








RNAIRPGXRQRHPAQGDVQA








QAPDRIXEAKKLQRLYRTSK








KRAVQKILAGPXMNCQIDKN








TITAHFVELAARRDGGEDWP








DVFDREEPTAASGEALCTPI








TREEVFRRLKGRNNTSPGPD








GITYRDLAKAXPGAHVLAAL








YNXIWRIEATPALWGVSNTT








LIYKKGDAMDISNWRPISLG








DTVPKLFAAILADRIKRWAV








ANGRYSASQKGFLEFEGCYE








HNFVLQEAIREAKGGRKELV








VAWLDLASAFTSVPHSSILQ








ALEGHGLPSKARNIISSLYT








GMTTRFHTAEGPTDPILIQS








GVRQGCPLSPDVFNLTLEVV








LREIQRTGEGYTIEGRRISH








LAYADDVAILADSPAGMRRL








LFAAERGARAVGLTFNPAKC








ATLHIAGRGEEAVRPTEFSV








QGTPVRALASGEAYEHLGIP








TGYQVRQTPINTLRDLLADI








GSIDRSLLAXWQKLDAVGTF








LLPRLDFTMQGAHIDKGFLT








EADKIIKKAAKSWLSLPQRA








SAELVFLPPSQGGGGLLTVA








HSYKMLYSSDVTVSTIAGST








LRRTVSERLKKRASNIDIAR








FLSGDLDLPRSTSPSTFWTK








VRSAALRIKTKLGLRWSWCQ








GGEVLLMACGDPRAPGTRVS








PQTKHLVTTSLRRCLNRHYA








ESLLAKKDQGKVFEVTRRSG








QSNHFLRSGSFTRFCDWRFI








HRARLDVLPLNAAKRWQRGM








DKRCRRCGSDLETLPHVLSH








CGPHSAARQKRHNNIQDRLV








KAASRCPGTISVNQTVVGVR








GPDAALRPDIVVRDDVNRRV








TIVDVAVPFENRLEAFDGVR








EAKIAKYTPLARQLTDSGYT








VTVEAFVVGALGAWDPRNER








VLSLLSISRYYAILMRRLMV








SDTIRWSRDIYVEHVSGIRQ








YRE








(SEQ ID NO: 1432)





NeS
Utopia-
.

Phytophthora

GCCCGCCGGTGGAGTAGCCA
TAGACGGCACAGTTCTGGCC
MVVSRITARLEATPAPRWDP


L
1_PCa


capsici

TGTTGGCCACCACCGCCCAA
CACGTAGGCCGAAAGGGCCC
PLPRRVIASRIADRLVPATA






GTCTCCGCCGCAGCTGCGAC
CACCCATGTAGGGAACCGCC
PCRSALNAAFPSPSRDTVTE






TGCTGCTGCTCATCGAGTCG
CTCGGGAAATCCATTCCGGT
SFTQEDRQLEPLTRHVDEET






CAGTAGTCGCAGCTGCACAA
GTTCGACTGAAGAGATGCTC
KDSELPGRAPTVLDEESKDN






GCACCACCTCCTGCCGGACG
CTTCGCCTTGACGGAGGTAC
DATAGEWLLRFDGACRANPG






CGCCGCCGTGGAGCACCACG
ATCTCGACAGTCGAACTTCA
PGGAGAVLFNPSGAASWTCS






CGCGCGCTGAGCCGTACCAA
ACTCGCAACATATCCGATAC
HFMPGATETNNTAEYTAFLL






GACCAAGGNTTCCAGGCTCG
AGTTACAAACCACAGTTAGA
GARAAADHGATLLRVQGDSQ






CGCGCGCGTGGMGCTCCCAG
TATCAGATAGGAACCTTCCT
LVLRQVKGIYGAKSTRLRRL






CCAGCGACGCGTTCAGCAGC
TTAGGAAGCTAACGGGTACA
RDAVRAELARVGQFSLHHID






AGGGTCGGCTGCGGCAGCTC
CTGGATGGTAAATACACATA
RQDNAHADRLANRALDMKST






GACGCTGCACGGGGACGGCA
CATTTC
LVECATHPGRNACTTTLTTS






GCGACGGCAGGCACCAAGAC
(SEQ ID NO: 1311)
AAAESPASPPPVGARDTPMA






CACGACGACCAAGCCGTCGC

DAGEERLADVDDGEVYAAMR






TGCCGTCCCCATGGACGTCG

LGPGEVPERRPRLRLRQLSD






ACCAAGGTGCTCGGTGGCCG

EELEAASEMVERLGAALSAK






ACAGCGGATCAMCACCCGCT

ITDAEDWASAEGYITALPYM






GTCGCCGCCACCGGAGCTCC

LYDKLQSYSQAPRGPQQPVL






GAGTCGGCGGCAAGCGCCGC

TRSPRGDDRPASSEPNASST






CGCCTGAACGACGGCGACGA

TGGVASEHQPRRRRRRGRRK






CGAAGACATCCGCGAGCTGG

GRRQRRNPRRSGREGATGGH






CCGAGCTTCTGCTGCCCGAC

QQHKKHKPRPPRETQHHREH






GAGGAGGAGGCCGACGACCA

RLDEALDELHALERTDPHNR






CAAACCAGCGCCCAGGTTAC

PAIAKARRRVGRIRSAINQQ






CCGCGACCAGCGCTCATCCG

LLRHKFDTDEKACVDGILST






GCCTCCGTCCTCGCTGTGTA

ARAERAARAATPSPPASGAP






CGCGCACAACGCGCAGCGCT

TTTVSAPGAIVTNDDGTCPI






TCAACTGCACGTTGTGCGTG

PSDKLWRHFDAVNTPRLDFD






TACACGGCTGCCAGCTTCGC

AEAPGSAAFRAAMDHLPAAT






TGCTCTTACGCGACACAGGG

RLLDLLKEAPSTDEIETQLQ






ACTCTCGGCACCGGCGCGTG

HVKASSSPGLDGVGYDVYKR






ACCTTCCTGGACAGGTTCTC

FTIQLLPVLRAAFRCCWLYK






GGCGGGTTGCGCGTGCGGCA

KVPQSWKLGVVRLLHKKGPR






AACCTTTTGCCTCGAGGCTG

EDPANWRPICLQQAIYKLYT






GCCGCAGCAAGACACGCACA

GILARRLTRWMDANDRHAPG






AACGTGCGCCAGCCTCAGCA

QKGFRAVNGCGEHNFLAATL






CCACACTGGTCGCGGTTTCG

IDNARRKHRPLYEVWYDFRN






ACGACAGGAGGAGCATCAAG

AFGSVPFALLWDSLQRLGVP






CCACACTGTCGTCGGAGCCA

PDYVDMCKGLYNQASFVVGN






ACACCACCGTCGCCACGGCG

AVDGSTAPVEQRVGVFQGCP






GTCACCGCCGAACCCCCCCT

LSPQLFNAAISPLLYALRRL






GCTCCACCATCAAGCCTCGG

PDTGVQLSSVDRPGASAYAD






AACTCACTGTGCCCCCCCCC

DLKIFSGTKAGITQQHELVA






ACGTGTGAGTTCCCCCGACG

TFLRWTGMQANPAKCRSMGV






TCGATGTGCAGCTGCACAGT

RRNTNGAVEADNVHLELDDT






CCGCCACAGGAAGATCAGCA

PIPSMTHMQSYTYLGIGDGF






CGAGGACGCCACCCANCACC

DHVRRRVELAPKLKTLKHDV






CGGAGAGCACGCAACACCAA

TALVESGLAPWQVVKAVKVY






CCTCCTGAGGCAACCCGCTG

LYPRVEYALRHLRPDDQHLE






GGGTTCGCCGCTCGCGCCCA

SFDLHLRRGLRHLLRLPKNA






CGCTCGTTGCCTCCAGGATT

TNEFFYAPVSRGGLGLLPLV






GCTCAGCGACTCGGCGAGCT

ELHAALQIAHGWQTLHSPDP






GGAACCTCCACGCTGGGGCC

AIRRVAREQLYQIADARHRL






CACCATTACCCCGCGCG

DKDHWPHRREELCELLLNGE






(SEQ ID NO: 1188)

LGTSAHAPPKRRNGDIGSLW








VDVRKNLKTFGLKVATAPAN








QETGVPAQPLQLRVPHHAEW








LDHGNVLRHVKLHIKNLHWQ








TWCALSDQGKTARVHGGVGS








AFLTRPRGMWESDYRFAVAA








RLNVVDTVNTLSRRRLRAHD








RCRYPACRWKETLAHVLNHC








PGTMDAVRGRHDDALKEIEH








TLRASSGDRRELRVNQTVPG








LPGPPLRPDIQVYNHDKRTV








AVVDLAVAFDEQPSEDPESS








GLAKAVQIKKAKYAGIKEHL








ENQGWKVHLSAIVYGSLGSV








AASNHKVYTEHLGLLKRDAK








RLDRQLSSACIQSSRRIWNF








HCAKHRARQHEHQAPPSQAT








RGRRVTETGGNPSRTDRR








(SEQ ID NO: 1433)





Nes
Utopia
.

Phytophthora

AGCTCGGCCTCGCGGCTGCC
TAAGCTGGTCATCATGACCG
MLADPAALAAGLARAPPPPS


N
-1_PI


infestans

TTCCCAGGCGCCGCCGACTT
ACAGGGCACTACCCAGGTAG
APQDPSPAFPAGPAGQNPRA


L



CGCGCTCTGGCGCGGCCCAC
GGAACCGCCCTTAAAAAACC
AAPARVEVHTVVAPPGRAGG






ACGCCGCCGCCGAGCCTCCA
CAGGAAGACACAAACACCCT
MLPDPGLVEEPIQATYAHDA






AGCGCGCCCGTTGGCTTTCG
CCACTTAGTGACATACATAT
AQFECALCPYVAESMAVLVQ






CAGACGCAGGGCTCGCGGCG
TTTAGCCTAGATTTCAGTTA
HRRSAHRGTRFKDIFTSGCQ






ACGGCCCTCGCCAGCCCCCA
CGGAGAGGTTACTAACTGGT
CSLVFYARIVAASHAVACAR






AGACCCCCCCTACGATGTGG
AAATACGAACACATATTCTG
RNQRAVPPAPTPVAPTRPEA






CACCACCCGGCAGGGCGGCC
TTCTAATCAGTGTGAAAACT
TPQPTGYLAAAMTAAAAAAS






GGCAGGCTGCCCGACTCGGT
GGTTTTCGCCTTTTGGCGGA
SDTVVAAATNMQSAVPAAAK






ATCGCCGGGTGCTACACTCT
CTTTTTCACTCGCATTTTTG
TTGLQLVPPELEPALPQRAS






CAGCCGCCACAGCTCGGGCC
GGCAATCGTCTGCGGCTAGC
CHAGKRRRLNADEAVTPCTP






TTGGCGGTCCGCCATTGGCC
TTGCTAGCGGCGGACGAGCG
TARVSPQTEVAMAPHDAPQD






CTTGGAGCTCGACAGCGACA
GTCTCCGGGGGCGTTCACCT
DTVLQREAAEPQPDPAATPG






GCAGCGACGACGAGGACGCT
TTCCCCCGCGAGGCCAACTA
AQVQRVEDTTAAQDDTVQQD






CAAGACCCCCACGCCGCCGC
CACCGATCTTCTCTACACTT
HDADTAQVSPPRRTPTRWGP






CCCAGAACCCCCAGAAGACG
TTCTAATTCGCCTCCGTCTT
RPSSTQEPSPMTGEPAATLA






TCGCGAGTGTGCTTGCCCCA
CGGTCTTCGGTTGTCGGGCT
ARRPLTPAATGTRATRWGPC






CCCGGCAGGGCAGGCAGC
TTTTTCTTTTTGACCAATCA
HRAIGAAAIARLVTGLSTEP






(SEQ ID NO: 1189)
GAGCGCGCCATGCGCCTCTT
AQPQRRQPPPPQEPPSQPEP







CTGGCCAATCAGAGACCGGG
LAAAATAAADIAATVAADIA







CCCTGTCCTCGGACAGCGAG
AAAANAAMDVDGGPAADETW







GCCTCCACGGCCAGCCAATC
LLRFDGACRRNPGPGGAGAA







GAGTCTCGGCAGCGACGCGT
LFAPSGAVVWTCSHYMPSRS







CTTTCTATAGCGCAGCTGAC
ETNNTAEYTALLLGVQSAVH







GAGGCCGATCTGGCGGCCCC
HGASHLEVEGDSSLVIAQVK







CGATTGGTCCGACTTTCGGC
GTFACRNARLRQLRNRVRHA







CAATCAGCGACGACGAGGGG
LRSVDTHKLRHIDRQANAHA







GCAGGGGTTTACACTTTTGC
DRLANRALDQRRTSSECGTH







CCCCGTTTCGACTTCAACTT
GSCMDSCLAVPTALAAQETP







CAGGCCAAAATGGCGATTTG
PAAPPSTSATPAEGNAMDDI







GACCCTCCACGCGCCGTGCC
AAEIAARDEGETFPVLPIGP







ACTGCTCGGCACCGGCGGCG
GSAPERQPRLRLRQLSDEER







ATTCAGCGGGTGCAACTTCG
DAAADALQELADTMASKIED







GGCACGTGTGCAACACATGC
ADSWTSGEGYISSIPERIRE







AGCGCCCATTGCACGCCAAG
VLQPYATAPPQPGRSRRQQR







CGGCATCGCGGGACGACGCC
RRPPRVTRNQREHRLDEALD







TCGGCCGCCCAAGCGCAGCC
DMAATQQATPRDQRAVRRAR







CCGCCCTTCCAGCACGACCT
RRVGRVRASMAQQELRHEFA







CGCGCCGTTTGGCGGATCGC
KDESKCVAKILKTASTETAA







CATCAAGACGTGCGAGAGCC
EDEHPETCPIDAATLHAHFT







AGGCGGGGTCGGGCAAAATA
GVNAPRTDFDYDATSGREFR







TACTTACTCTAAGTATGCCC
AAMSDLPPATVEIDAFDAEL







GAATCCCTGCCCTCTCAGGC
TIDEVEDQLTRAAKTSSPGH







TGAACGCGGCCCCATACTTG
DGIDYGIYSRFAAQLVPLLH







ATCTAAGTATGGGAGGATCC
AVFQFCWRHRRVPRLWKVGI







CTGGCCTCTCAGGCTGTACG
VRLIHKKGDPRQPTNWRPIC







CGAGACCC
LQPTIYKLYSGLLAHRLSRW







(SEQ ID NO: 1312)
LEGNDRLPMAQKGFRAFNGC








HEHNFMATTLLDQTRRQHRK








LYQVWYDLRNAFGSLPQQLM








WRVLRHLGVDSGFIDRCRDI








YRDSAFVVANAADGATDPVR








QEVGVYQGCPLSPLLFVAAL








VPLVRRLEKLDGVGVPLADG








VRPCTTAYADDLKVFSDSAA








GIRKCHDTVAGFLAWTGLRA








NPGKCASLAVTTNARGNPTR








DSSMRLEVHDAAITTLSLHE








SYRYLGVGDGYDHVRHRLQL








EPKLKQLKREAVALLTSGLA








PWQVVRALKVYVYPKVEYAL








RHLRPLQSQLQAFDRVVSKG








LRHLLSLPRSATSEVLYAPT








SSGGLGLQPLVELHRALQLA








HAWQMLHSKDPAIQAVARAQ








ACQVVRKRYRLQEDHWRGRD








DELVRSFLNSELAASPHAEV








LRRNGDIASLWSDVQRWLRI








YHLRFEHCDETEAHGPLSFR








VPHHNKWLTHKTVLRHVKLH








LKIRHQTRWKGMVDQGKTVR








VHGGVGAKFMTTGAGLSDDD








YRFGVKGRLNQVDTNSVLKR








KRLRAHTTCRDPTCSSAETL








AHVLNHCESNMDAIRQRHDD








ALEQIGSKIRGALDRAKSPT








ELRLNQTVPEYTGAALRPDI








VLRNVAAKTMVIADLAVTFE








DQAARARHSSLQLSHDHKTL








KYQPIVAELQHKGWRVQTAA








IVYGTLGSVQPSNFKAYTEK








FKLHKREARQLDLQLSSHCI








RASHRIWGWHCRQHRDRQRS








GTASRASRGSGGAPRRTSQA








PARR








(SEQ ID NO: 1434)





NeS
Utopia-
.

Patiria

CTGATGTGGATACCTTGGAA
GATTAGCGAACACTAATATC
MCLKSFSSTSGLRRHMARLH


L
1_PMi


miniata

TTACTCAACCGTGTCGGAGT
CTGCCMTAGACGTGATTGCT
RQPSPDASTPSTMTEVFPYP






CTTTTGTCTTTTGCGCCCAA
AATCCGCAAACCAACCGGAT
LPKVWPCVVCRENFYHNQTL






CACCTCATGGATACCATGCT
CTACAGCCTGAACACTGAAC
KRHQKNFHPNVDLTTVYQCS






TGTCGCTGGAGCGACGTTAC
TTTAATCTTCACCCATGTCA
VCGQEFVTGRKASFHFKVHR






AAGCGTGAGGGCGCCCTCCA
CATCTGGACACTAGGTTTTT
RMSASAIPTPSAMPSSPMDL






TGCCGGACAGCTGGTCTGTG
GCTCTGTTTGTGTTTTCCTG
IRGLVGEPLPPSPARTPPPL






CC
CCTTTWCMTTGGAWCTTTCG
ARYISPAPRSSFSPPWNPSP






(SEQ ID NO: 1190)
CMCTGGAATTTATTTGTCGC
PPRSPTPLPRPLTPPPRSPS







TTGGATTATTTTTTTTCTCA
PPPRSPTPPPPVTLTTAPVT







CAATTTGGATCTATTTTCGT
EPAVPVALTTAQVTEPSAPA







TTGTTCACTTCGAACTCTAG
VHTAAPVTLSNAPVTEPATP







CTGCCCTTTCTTCGGACACT
ATDPATPVTRLHSPVTHISC







GAACTTTAATCTTCGCCATG
SISFTASHAPYSCAAPTSPS







GCTGTCAGTCGCCGGTTCAC
VYACSPRRRQCSSTIAAVCN







TTGCTGCGGTGGGATCCTGT
SEASSGNPCLLALPVHRHHL







TGTGATAATCCCCGTGCATT
PDTSPQRPGLLFHHPGIPPH







GCCCATGGATTTATTTCCGC
RPGRPPHCHGHSLHRHDHRA







CTTAGTTGTTCCTAACCTTG
RRPGRQHHLPRSPSPPPRSP







GATTTATTGCTGTGGGTGAT
SPPSRSPSPPPRSPSPPPRP







GCCCGGGTTTTGTTTACATC
STPPPRSPSPTPRSPSPPPR







GGGATCCCGCTGCGGCTCGG
PPILPPRSPDSTHRSLTSHA







TGTTCCATGCGACTGGCAGC
RSPSRPATPPIPVQPRHLRP







CCCTTTGTTTACTCTSGACT
STPAHPGVDNAVPPSQQSAI







CTATTCATTGTTGTATTTCT
DVWLAELSRSADFESFEDVC







TCCACACTGGCCAGTGATCA
DRFVEFAAAEGRNNGRPARP







CACTGCTGTGTTTCCCGGGA
AHQPPRDRGNQGPRPQRPPR







AGATATCCTCTGCGGTTTTC
PHRPGLGPEFDAQEASRLQK







ACGCTCTGGGTGTCTCCCCG
LYRTSKKRAIRTILTGSDVR







GGCAACGCACTGGTTGCTTG
YSGYRGPHPALMSDTAATEV







CTGCGCCATCACCCTTTTCG
LTSGFLSLEVFSAREVDTDT







TTTATATTCATTTTCAGTCT
IATDTSLLFPNSAQARESGQ







GCCGTTATCTTGGCCAGCGC
DLLRPVTQREVSLRLGRMSN







TCATTCTTTTGTGATGGCCG
SAPGKDRLEYRHIRQVDGAF







TGGACTGACCCTCTGCGGTT
RVTLEIFNRCLRESRVPSSW







TTCTGCGGTCASCACTCTCG
KTATTVLIHKKGDATDPANF







GTGAATGCTGTGCCACCATT
RPIALQSCLYKLLMAILSDR







TTTCATTTGTTTACTTTTTC
VTTWALDNDLISSSQKSARP







AGCTACAATTATCCTGGCCA
GEGCYEHTFLLSTVVKDARR







GCTTTCACTCTTTTGTGATG
NQKNMYAAWLDLQNAFGSIP







GCCGTGGACGGACCCTCTGC
HDAMFTVLTSIGAPEGLVSL







TGGTTTTCACGCTCCGGGTT
VRDVYTDASTDFVTPTGRTA







TGTCTGCGGTCAGCACCCTT
AVPIHSGVKQGCPISPVLFN







GGTGAGTGCTGGTCGTTTGT
LTLELIIRAVNASATRDRSA







TTGCTCATTTTGCTTAGTTC
PVVHGQAVPILAYADDLVIL







ACCATTATCTTTMTTCTCTT
SRSSDGLQSLLTTASIMATK







TTGTWTGGTTTTCCTAGCGG
IQLKFKPAKCASLSLECRRG







TTGTCTGGGAGTTGAGCTGC
TKVRPLEFNVQDKIIPALTE







AGTTGTCTGGTCTTGGTTTT
EQHYRYLGVPIGLYRTDDSL







ACCCCCATTTGTTTTCTTTT
ETLVAKMTDDIQRIDSSLLA







AACGCGGGGCGTATTGCCTT
PWQKLDAIRTFVQPCLAYTL







GACCGGCCGTCTCAGCTTTT
RAGDCAKKHLKRLRGQLVKT







CTCCTAGAGCAACCTTCCGT
ARKVCNLPTRATTNYIFADR







TCATCCAACTTTTAGTTTTC
RAGGLGFIDPNVDADIQIIT







TCAGTTCTTGGCCATTCCGG
QAVRMLSSPDDITRAIATGQ







TTGGTTAATTTTTATTTATA
LSSVVHRTIHRAPTQEETDE







CTTAATTTTATGTTTACATT
FLSASMEGDFANSGNSGQAS







TTCTGGTTGGAGACCATTTT
SLWSRARAAARRLKVTISGS







AGCTTGTTTTAATAGCTTTT
LSGSVITKSTENREMAAKSI







CTTCTTTAATTAATACCCTC
TTALRAQSRAHYTHRLLSLP







TGCCATTGAGGGTTTTTATT
DQGKVGQSLNQDQYMNSSSW







ACTATTAATTTTGTTTACTC
MSSGSYILFCDWRFIHRARL







TTTGTAACTTGTTTGATTGA
NTLPTNATAQRWKPNTSPAC







ATATTTTAATAAACCAC
RRCQHPQETLPHILNHCPPN







(SEQ ID NO: 1313)
MVPIRRRHNLVQQRIVSAVR








HGRVFVDQHVPEDPNPRERP








DITVVEGDKVTIIDVCCPFD








NGRDALMTAAAAKETKYADL








KQALVAAGKDVEVFGFAVGS








LGSWLPSNERALRRLGIAKR








FRTLMRKLLRIDAIKGSRDV








YIEHMCGHRQYT








(SEQ ID NO: 1435)





NeS
Utopia-
.

Phytophthora

AGACGAGCAACGCGCTGGGG
TGAAGCTGCACAAGCGCGAG
MDVDGGPAMPEPWVLRFDGA


L
1_PS


sojae

CCCAAGACCTGGCACGAACG
GCTCGACAGCTGGACCTTCA
CRRNPGPGGAGAALFKPCGT






ACACTGCCCAGGCTGTGAAC
GCTGTCGAGCCACTGCATCC
VAWTCSHYMPNSSETNNTAE






GACGAGCACGCTGCTAACCC
GCGCCAGCCACCGCACCTGG
YTALLLGVQSAVHHGASHLE






GGCAGCGCACCGGCCTCTGG
GGCTGGTACTGCCGGCGCCA
IEGDSHLVVAQVKGTFACRN






GCTCCGCTGCACCGGTTACC
CCGCGAAGGACAACGGAGCG
PRLRQLRNRVRHALRAVTSL






GGTGCAACACGGTGGGGTCC
GCAACGCCTCGCGAGCGCCG
TLKHIDRKANAHADRLANRA






ACGTCACGGCGCGATCGGGG
CGTGGGTCTGGGGGGGGCCC
LDLKRSLAECGEHQGAMESC






CAGCGGCTGTAGCTCGCCTG
GCGGCGCACATCGCAGGCTC
LHMNPAAQRQREQPAPPARP






CTCACAGGCCTACCCACGGC
GGGCACGGCGGTAAGCTGGT
ACAPTRAESASDHDEDIDAE






ACCAGCACCAGCTACGCGCC
CATTTGACCAACAGGGCACT
IAARDGGEAFPTLPIGPGTA






GGCCTGCTTCGGCTCGGCGC
ACCCAGGTAGGGAACCGCCC
PARQPRLRLRQLTEDEQEAA






TGCCCAGACCCGCCCGCTCC
TTCAAAAACCCAGGAAGACA
ASALQAMAEELACKIEDADS






CCCCGCGGCCACGACGACAG
CAAACACCCTCCCTTTAGTG
WTSGDGYISAIPSRIRQLLQ






CCCCCGATGCG
ACATGCATATTTTAGCCTAC
PFTAAQPHPRPPLQQQRQRP






(SEQ ID NO: 1191)
ATTTCAGTTACGGAGAGGTT
PRVTRTQREHRLDEALDEMA







ACTAACTGGTAAACACGAAC
AVQQERPTSRSAVRRARRRV







ACACAT
GRIRASMRQQQLRHDFARNE







(SEQ ID NO: 1314)
SKCVEDILRAASAETAAEEH








PETCPIDSGTLHEHFTAVNS








PRINFLPDEACGALFREAMA








DVGTPQERRSALTDELTMDE








VEDQLMQAATNSSPGHDGVG








YDIYKKFAAQLVPLLHAAFQ








SCWRHHRVPALWKVGFVRLI








HKKGDPNDPANWRPICLQTA








IYKLYSGLLARRLSAYLEAN








GLLLMAQKGFRAYNGCHEHN








FVATTLLDQTRRMRRRLYQV








WYDLRNAFGSVHQDMLWYVL








RLLGVERAFVERCDDIYEDS








YFVVGNAADGATEPVRQEVG








VYQGCPLSPLLFIAALVPLL








RALEKLDGVGVALADGVRPC








TTAYADDLKVFSDSAAGITR








CHAVVEKFLEWTVLOANPGK








CAFLAVTRNARGNPAHDKDM








KLSLHDEEVSSIKLHDSYRY








LGVGDGFDHVRHRLQLEPKL








QQIKREAVALMQSGLAPWQV








VKALKTYVYPKVEYALRHLR








PLQSQLQGFDRVVAKGLRHL








LRLPRSATNEVLYAPTSSGG








LGLQPLVEMHRALQIAHAWQ








MLHSKDPAIREVARAQVWQV








ARKRHRLREEHWRERDDELV








RCFLNSELAASPHAEALRRH








GDIGSLWSDVQRWLRIYHLS








LVVQDDRNGLDPLGLRVPHH








AKWLDHKSVLRHVKLHLKIR








HQTRWKGLADQGKTVRAHGG








VGAKFMSTWAGLSDDDYRFG








VKARLNQIDTNAVLKRKRLR








SHKTCRDPTCSSAETLAHVL








NHCESNMDAIRQRHDDALEQ








IGSKIRNALKRGKSTAELRL








NQTVPEYTGAALRPDIVLRI








VAAKKMVIADLAVTFEEHAA








GARHSSLQLSHDHKTLKYQP








IVAELQLKGWQVQTAAIVYG








SLGSVQPSNSTPTRKS








(SEQ ID NO: 1436)





NeS
Utopia-
ADOS01001

Pythium

GCGGTGTACGCGCACAACGC
TGATGCGGGTCATATTGACC
MGTQSARERGAPSAPHSHTL


N
1_PU
321

ultimum

CGCGCTCTTCGAGTGCACGT
GAAAGGGCACCATCCACGTA
GPRTPPRPPACSKHGELESA


L



TGTGCGCGCACACCGCGCGG
GGACACCGCCCTCAAAAACC
AGGRDGQCSDGAERERDAER






GATCTCGCCGCGCTCCAGCA
CAGTTCAGTTTATTGACACC
DIRANERDCNGDGDGDDADS






GCATCGGCGCTCCGCGCACC
CTCCACTTAGTGACATGCAT
DSDDRNDARRRSRRPRATAT






GCAGCGTCCGCTTTGTGGAT
ATTCAAACCGATACATATTC
TTTSAPTTTTTTTTSATTSA






CACTTCCACAGCGGATGCGC
GTTAGGAGAGGTTACTAACT
TTPATDSSPWVLRFDGACRR






GTGCGGCGTGAGCTTCCACT
GGTAATATATCACCATTTC
NPGPGGAGAALFEPGGAVVW






CGCGTGCGGCGGCAACCAAG
(SEQ ID NO: 1315)
TVSHYLPGSETNNTAEYSAM






CACGCGCGCGAATGTCCAGA

LLGVRSAIHHGATRLRVEGD






GAGCGCGTTCTCGGTCGCCG

SHLALSQVRGTFACTNRRLR






CCGCCGCGCGCACTGCAGCG

KLRNRVQAALRELGDYRLVH






GCCAACACCGCAGGTATGTC

IDRQANAHADRLANRALDLR






TCTCGGCGCCGACGAACGCG

KTKVDCGPHATTTDACVQPA






ACCACCTCGCGTCCGTCGGC

EILAPTARLSSSSSSSSSSS






GCCTTGCATGATGTTGCATC

SDEPMPGLEEPAADDETDAD






CCCCGCTTTTGGCAACATTT

AEADIAMRDGGEIFPTLQIG






TGCCGGTTGCGTTTGCGACC

PGSAPAQQPRLRLRQLSDDE






GCGGCAGACGCATCAAGCGC

SEAAARTLEHFANDMASKIA






CACCGTGATCGCAGACGCAG

DADDWRSGEGYISAIPVRLR






CCATGCAGCACAGTGCTGTG

ELLAPYAVPIRSPPRNASSR






CCCTCTGCTGCCGCCCAATC

PPRPQSRPPRPPRVTRHQRE






CCCTCGGCGTGCGCACGTCC

HRLDEALDDLAAAQRSTSTD






CCCCCGTGCCGCGCGCCACC

QRSIRNARRRVGRIRTAQAQ






ACGACACCATCCGCGCTGCG

SDLRSQFATNERACVESILR






GATTGGTGGCAAACGCCGCC

AAKPDGTEPQASAGTCPIDR






GCCTGAACGACGACGGCGAC

ATLHAHFAGVNTPRERFDFD






AACGAAAACAGCGACGGCCG

DALGADFRAALDVLPPPDQA






CGACGCCGACATCGAGATGC

ADAFADELSLGEVEDQLDRV






GCGCCGACGACACCGACGCA

VASSSPGLDGVGYDVFKRFR






CCAGCGCCGACCAACCCCGC

LQLLPLLHAAYQCCWRHRRV






GACCAGTGCGGCTGCAACGC

PATWKVGLVRLLHKKGDRAE






CCGCGCGCACAGCACCAGCA

PNNWRPICLQQAIYKVYSGL






CCAACGGATGCCGCGACCGC

LARRLSRWLEANERFTTAQK






GCGCCGCCGCCACACGCG

GFREFNGCHEHNFVASSLLD






(SEQ ID NO: 1192)

QTRRLHRKLYAVWYDLRNAF








GSMPQPLMWRVLARLGVDTA








FLQRCEDIYADSFFVVGNAA








DGATDPVRQEVGVYQGCPLS








PLLFISALIPLLRALQRLPG








VGVPLADGVRPCTTAYADDL








KVFSDSAAGIQQCHGTVARF








LRWTGLRANASKCALLPVTT








TARGNPAIDDTLQLELHGDA








IARLTLQSSYAYLGVGDGFD








HVQHRVQLAPKLAELKRDAV








ALLRSGLAPWQVLKAIKVYL








YPRIEYALRHLRPLQSQLEG








FDRAVAKGFRHLLRLPANAT








NELLYAPVSSGGLGLLPLVE








LHKALQIAHGWQMLHSKDAA








VQAIARAQVRQVVQKRYTLD








ADHWQGRDDELVQLFLNSEL








AASPHATIKRRNGDIGSLWS








DVQRHLKTLQLRLETREPTA








DAPDSPNGLLHLRVPHHRKW








LSHKTVLRHMKLHIRLCHKH








KWQSMSDQGRTVRAHGQAGS








HFVSRGVGLWDADYRFALQA








RLNQLDTNSTLKRRRQRTNA








TCRAPNCSRTETLAHVLNHC








ETNMDVIRQRHDGALEQIGA








AINAAIKGRRTDTEVRLNQT








VPEFNGPAWRPDIQVRDARS








KTMVIADLAITFEDQPNDQS








ASSSLQHSREHKIAKYQPIA








AALERQGWRVHTSAIVYGSL








RSVHPSNFTVYTELLGLLKR








DARRLNTTLSCHCIRSSRRV








WNWHCGQHRARQHQRCQEGR








AHGSGGNQRAEGGTATT








(SEQ ID NO: 1437)





NeS
Utopia
.

Strigamia

GGAGTGTTCTTTTCGGAGAC
TGATGGGAGAGTGAGGAATC
MATVRLKYPYPPEGILCGPC


L
-1_SM


maritima

GCCGCCTACTTTAGAGGAGA
TTCTCCACTGTGCAAAACCA
AANTNAPQTRPYSDKSGLAK






GAATCCCCACGGGCATCCTC
TACAGTCAGAAGATGCTAAC
HLKLYHKATLWECRHCGHEE






ATTTGATCTGATCCATCGAG
TACTAGTTTGATACCCTGTG
SDLRKMKKHISTNHPVAAAA






TATCTGCGAATAGTCGGCGC
CCCCCTGCAATGTCCCGCGT
APTVPPRLGPTAPPPPRVIL






ACTCCTTTTGCCATGATCCC
GTCGTACCCAAGCCCGGCTG
RPRFIPRPRTPSPSSSSSSA






GGGGGTCTCATGGTAAAAAG
GCATTGAGACACATTAGGCT
SSPASSRRSVSLPPASPPVS






GTTTGTGGCACGGCTTAGTT
CTCGCTCCCCCGTATACTCT
SASSPAARSGRNSPDSQGTA






GACGCCCCTCTTCCACGTCA
CATAATTTCGTGTACGCTAA
PVTPIGTVRNSPAGSPALSY






CTCGGCCTGCATCGATCGAC
TCCTACCCTACCCCTCCCTT
STASPIASTITTPRHLSPAS






TGCTCTCTCCTCTTCCTCCC
TGACCACTCACCCAACCATG
PALSAGPGSLGASPPVSPTA






TCCCTCCTCTTCACGCTCTC
TGTATAGCTGTGCTGGTGAT
ATVPPAPPATVPAVMAATVP






TTCACGTGACCCCTTCCCAT
CCCGGGGCGGTTATTCACTG
FVAATTVPSVGSSTVPQRPA






CCCGCCCCTCGGCTTTTGGC
GTTATCATATCATTCTAAAA
GPRRPPPFPIDDWIGRIARV






AAAGATCTGTGTGTCCTCCA
TGATCTTTGATCTCTCAATT
SSLPELDAVSRLLEDEWKRR






AAGCACCCATCTACCATTTG
AACAACTAACTTATTTCTGT
PPDPNARPASLHPTRRPPPP






CTCGAGTTGCGATTGGTCGA
TTGTTTCATTGTTTCACCTC
STRPRPCHGTGGTSVSLAAL






AGCTGCCACGCCACTCGTCT
GTAAGAGGAAGTTCATTGTG
TSSCIREDHRGSLPLLCTSG






ACTCTGCCTCTCTGACCCCT
CGATAAATCAA
WDSPWPLSPSPSSHCPSSNP






TCCCCCCTCTCTCCCTCTGC
(SEQ ID NO: 1316)
CPSSSTPPSSLLGPPRHSHL






CGCCCTCGCCTCTCGGGTCC

TWRGSGSTTPSHRHCSRARY






CTTCCCATCTCCCTCCTCCC

HHLLWRLPYPSSPRLPYPPS






ATCCACGTGCTCCCTCCTCT

ALARYPSVPPALDDPLPSLS






CGTCACGTGATCGTTGCGGC

TIGSEGSPECHLCRNWTPSQ






TCGAC

GCSRMKWSRDAPLTPTPDPP






(SEQ ID NO: 1193)

HSIRHAALPLHLLALVPAMG








PAELQSLWRRSRPRAFAKIT








EGASPFCALPVGTVHGHFLQ








VHQATAHLPTPVPLPPLRPP








RSSDPLVTPISPGEVLDRLR








RATDTAPGPDTIIYSEWRAI








DPTGRLLSSLFQKVQTFGAP








TRWKESTTTLIHKGGDHTAM








SSWRPIALLSTVAKIYGSIL








SHRLTTWAVQNGRLSLSQKG








FLPFRGCLDQNYLVQSCLQD








ARRNKKTLSLAFLDLKNAFG








SIPHLTIRHSLEWLGLAPSS








IDILEASFLGSSTRVRTETG








LTPPISLDTGWQGAPLSPIL








FNLAIEPLLRTVPSAHSGFS








LHGHWSWAYADDLAILAPST








PALQSQLDAISGMADWAGLS








FNPAKCATVTLTGKDNSRDT








LSLQGSPVPSISDGDAYKHL








GVPTGTTTFPSGTDAIKKMT








TDLQAIDHSDLAPAQKLDAL








RTFIMPQLSFHLSHGSVPKA








PLTQLDKKIKRAAKHWLFLP








QRASNEILYMSHLHGGQSLL








PLSVLADIGQVTHAVALLQS








RDPAVADLALRTCREVASKR








AKKTVNGPELAQYLSGSTDG








IYCTPTSDIPSLWTTARAAT








RRLSSTLPLTWTSPLPSGVP








FLSINGSPLSPFRVQSTLTN








AIRHNHLSTLIAKRDQGNSY








RTSHDPDPSNYWVKGGDFLR








FCDWRFIHRARLNLLPVNGA








RRWDANSIKTCRRCGAPNET








LAHVLNVCPVGLPEMKKRHD








AIHARIKKALRPSPHTWHHD








RTVPGCGPLRPDILRISERD








KSVAIVDIHVPFDNGTDAVE








RAHETKRAKYELIRRHYEHQ








GYRVTFDSLVVTALGRLWRG








SEAALQALQISSQYSKLLRK








LLVADAIHGSRNVYAHHMTG








MVM








(SEQ ID NO: 1438)





NeS
Utopia
AAGJ0

Strongy

AAGGCTCAAACCAGGCTGCC
TGACGAAATGTTCAATATAT
MSHSITEVFDYPLPSRWKCT


L
-1_SP
21405

locentrotus

AACCAAGCTGCCAGCTTAGG
GACATTCTAATGTTCATGTA
VCLENFFNQQTLKRHQARHH




37

purpuratus

CACCAACCAAGCTGCCAGTC
TTTTTTGTTTGCTTGACAAA
QTTSFLYVFRCSACQAEFDS






CAGGCTCCAACCAGGCTGCC
GCTAGATGAATATATTCCCT
ARKASNHWQSHKRKPILSQP






CACCAATCTGCCAGCTTAGG
GTCCTACTACATACATCTCG
AVNEIPSSGLDPSPPRSRPP






CTCCAACCAAGCTGCCCACC
ATGTGCCCTTGCTAGACGAT
VEVIGSSFPDDVSMLSEPST






TAGCTGCCAGCTTAGGCACC
CCATTGGTAACCATGATATC
PSTSLQMDPEVVHPPSRSIS






AACCAAGCTGCCAGCCGAGG
AATGGATTAACACATGATCT
FSPMHLSPTQPASPIQIGVE






ATCCAACCAGGCTGCCCACC
GTAAACAATATATGATTTAC
VSFNSSSSLQMDPEVVQPPS






TAGCTGCCAGCTTAGGCTCC
ACAATGTATTTATTGTTTCA
PSISFSPMHLSPTQPASPIQ






AACCAAGCCGCCAGCCCAGG
ATAAATCTGTTCTTTTCACT
IGVEVSFNSSGSLQMDPEVV






CTCCAACCAGGTTGCCACCC
TTAATACATGAAACATGACT
QPPSPSISFSPMHLSPTQPV






GAAAGTCTGCCAGTTTAGGC
GTGCCTTCTCCAACTGGAGA
SPIQIGIEVSFTSSSPLQMD






ACCAACCAAGCTGCCAGCCG
CCTACATAATTTGTTAAAAT
PEVVQPPSPSMSYSPMHLSL






AGGCTCCAACCAGGCTGCCA
GATATAAATATTTCGAAGAT
TQPDSPIPVDIDVIPAAEVP






CTCGAGGCTCCAACCAGGCT
GAAATTATTATTAATAA
LPDIEIPPSPDRHPAAEVPL






TTCACCCGAAGCATTACCCA
(SEQ ID NO: 1317)
PDIEIPPSPDRHPVAEVPLP






GGCTGCCCGTTTAGGCACAA

DIEIPPSPDRHPQSPPRPVM






ACCAGGCTGCCAGCCGAGGC

MEQPVHTPPPADTQQANGPQ






TCCAACCAAGCAACCAGCCC

HWVTVLANATNWEDFGRVCV






AGGTTCCAATCAAGCTGCCA

EFANHAVEAARSRQDAPQVR






GCCGAGGCTCCAAGTAAGCT

PAAQRQPRRPTRPRQPTFDV






GCCAGCCGAGGCTCCAACCA

REASRLQKLYKRSKKRAVRH






GGCTGCCACTCGAGGCTCCA

ILRDDAPSFSGSNEQLLDYF






ACCAGGCTTTCACCCGAAGC

KEIYAPPEIDENRAQQLAES






ATTACCCAGGCTGCCCGTTT

LFTDLEEAKESAAALMSPIS






AGGCACAAACCAAGCTGCCA

QQEISTRLSRMSNSAPGKDR






GCCGAGGCTCCAACCAAGCA

LEYRHIRQADGACRVTHIMF






ACCAGCCCAGGTTCCAATCA

NRCLQEHRIPSAWKEATTIL






AGCTGCCAGCCGAGGCTCCA

IHKSGTTDDPANFRPIALQS






ACCAAGCTGCCAGCCGAGGC

CLYKLFMGILSDRMTQWACN






TCCAACCAGGCTGCCCACCA

HNLLSPEQKSARPCEGCHEH






AGCTGCCAACTTAGGCTCCA

TFLLSSVIKDTKRNQKTANI






ACCAAGCTGCCAGCCCAGGC

AWLDLRNAFGSIPHQAIHAV






TCCAACCAGGTTGCCACCCG

LTTIGAPVSLVMLLKDTYTG






AATCTCTGCCAGTTTAGGCA

ASTSFLSTSGETDPIQIQSG






CCAACCAAGCTGCCAGCCTA

VKQGCPMSAILFNLTIELII






GGCTCCAACCAGGCTGCCAC

RAVKKKATDDGLGLVVHGQR






TCGAGGCTCCAACCAGGCTT

LSIMAYADDLVLMSKTPEGL






TCACCCGAAGCATCACCCAG

DAILSVASEQAETLRLAFKP






GCTGCCCTTTTAGGCACAAA

TKCASLSLSCRHGTSVLPRE






TCAAGCTGCCAGCCGAGGCT

YTVQGHLMPALDEEEQYRYL






CCAACCAAGCAACCAGCCCA

GVPFGLPRFTNLKDLIGKLK






GGTTCCAATGAAGCTACCAG

GNIETIASSLLAPWQKLDAI






CCGAGGCTCCAACCAAGCTG

KTFVQPGLSFVLRAADYLKS






TCAGCCGAGGCTCCAACCAG

DLRSLKSAITTNVKKICQLP






GCTGCCCACCAAGCTGCCAG

LRAANAYIFAAKESGGLAFI






CTTAGGTACCAACCAAGCTG

DPNVDADIQVITQAVRVLSS






CCAGCCCAGGCTCCAACCAG

DDEVVQTIATSQLKSVVHRT






GTTGCCACCCGAAACTCTGC

IHAVPTEEDIDNYLSGSNEG






CAGTTTAGGCACCAACCAAG

LLANSGNSGQASSLWSRTRS






CTGCCAGCCGAGGCTCCAAC

AARRLHLTLRATTSGTVVVN






CAGGCTGCCACTCGAGGCTC

QQADIDHTRDILPASITRGL






CAACCAGGCTTTCACCCCAA

RLIQRTTNAEKLKSLPDQGK






GCATCAACCAGGCTGCCCGT

VARSLSNDPFANGSSWHATG






TTAGGCACAAACCAAGCTGC

KFIRFCDWRFIHRARLNCLP






CAGCCGAGGCTCCAACCAGG

TNVATKRWKANANGKNGHQQ






CCTCCAACCAAGCTACCAGC

ETLPHVLNHCLPNMVPIRRR






CGAAGCTCTGCCAGATTAGG

HDNIQQRLVTAIRHGDVFVN






CACCAACCAAGCTGCCACCG

QHVPGDPNPRERPDITVIEG






TTAGAGGCCCAGAGCCCACC

NKVTVIDISVPFDNGPNACT






AATCCCATAACGTGTGAGAT

TAAQAKVEKYSALRQALRDM






ATGTGAAGCTTCCTTCCACA

GRDVEVHGFIVGALGTWHQG






CCTCCGCCGGTCTCCGTCGC

NERALGRLGVSRWYRTLMRK






CACACGGCCAGGCTTCATCG

LCCIDAIQASRDIWVEHVTG






CACCACTGCTGAACACACTG

HRQYE






ACGACAGC

(SEQ ID NO: 1439)






(SEQ ID NO: 1194)







NeS
Utopia
.

Trichinella

TTTCTGGTATGAATCCCAAG
TGATCCGTCCCGAACCAACG
MCSAKTPALKSGRRRGKEVN


L
-1_TSP


spiralis

CGGATTCGTTACGAAATTTG
GAACCACATTGCCGCATGAC
YEGQIVRVERRRGSRSSTSA






CATAAGTTTTTGAAAAAATA
TTCGATTTCGCTTTTGCTCT
TDLGTRMVTRGRKKLMEASV






GGCATTTGGTCGAGTGCTCG
TTTTGTATTTAATTTTGCTA
REAGHHGGESASTVDVDVVE






CACCACCATTTGTCGCGGGT
TTAACAATTCAGTTTGTTAA
SKKITGKTARRNRRAPSGDG






CGTCCTGATATTGCACTACA
CTGTTTTGTATTCATTTGAA
KRRESCGAECGQAVCGNAVA






TTCAGGAACGGCCTATTCCC
GATCCAAATAAAAC
DRSEASSPRTPNVSKSGRDK






TTCGGGGAATTGTGTTTTAG
(SEQ ID NO: 1318)
CGQPTIKASTPSPPKRKPTT






GAATTGGAATCGGTTTGGTT

SSSPRTPCLSKRGARSKIPS






ACGATCGGTCGAGTGGTTCG

TPDTPSTSGGSGKQRVLVSP






TGAGATCGAGTGACAGCCGG

LLRTEKLPDLEVLQRTEEQV






GTGGCAGCGACA

TVRATFPIAQAVVCPLGCEK






(SEQ ID NO: 1195)

PYTAVRPDGQFAHQTLTRHF








MRVHNCHSVQWHYRCRNCNT








DFLPADHRYPLRVVNTHVRS








CVSRWEITRKLGESEDLHGV








RCDLCDYVGVSKRAVGLHRR








RHANENIMQNTGTAAQIEAL








SKQVGEIRVAGDYSQFKFGK








RVRQYVAPTQRRDGLDEEEV








HEAEEEEVPAESRTILGEPS








TATAIGAEEISATGPVRADT








AAQQMICRIGQWCVWPQDYH








SIPAPQCWTDTLMDLMIEQI








VLQRYPDGAGVSVMSCSAVS








AAIHHEISAEFAAQVMSSHD








ASLYCIIPVNVRNHWQMIVL








DVAERVVHYYCSLREHNTVV








LSSLLSLVELSGKHTGCTSW








KIETHDGAPVQTNAFDCGPF








SCLFLKHLLHGIDMNFGDRE








SAALRTDLKFMIDAVSTPVV








PATDKLKKKPDGSATQLTQF








QQKFLSASDNWQSPDVDLQA








VYDEVVESIVSGHNEPSSRN








TSRLQKKSPGKGGKGQVRRR








SAVTRDPAWLKSASAVQKAF








NSAPARTVNAILRRPNACPS








FTATQVADHYFNLRPAVTSL








APEVIDILPPPATDHSMLVA








ELSESEVWEKMQKAPNSAPG








ADRITIRMVRMADPGAMILT








RFYRACLLRKWVPLQWKQSV








CKLLYKDGDKERLANWRPIA








LEPVLQRVLSAVVASRVTNW








ARANGLISLEAQKGFQPADG








TSEHNFVMEVAIQEARRTNA








QLAISWLDISNAFGTVSHQL








LFSLLERYGLDPTFTSFIQN








LYKDATIVVKGANGTHVTAR








WSVGVRQGDPCSGILFCLFV








EPLLRSVLPSLPCEAETTAV








NVLGQPITALAYADDIALFA








PSIGVMQQQLCKIQGMASAM








GFRFNPKKCASLYLNRAVVN








AATFTISGEEIPALVHGDTF








RYLGVAAGLGKPQTPFSLLR








ENLREAELIFRSKLAPWQKM








DAYRTYVLPRLTFQLMIAKF








NNIKQSAGQYDRAILRLVKR








CFQLPVETSTDFIRAPRQCG








GLGVPSLRELYATAKVSRAL








KMLWSPCRVVSSLAASQLQR








VASAYFAKRLRDVEAADLST








FMNAARSTPLDRSGYPTCLW








MDARKQMSYLTKVAGVDCYF








LVGEAGTSFFIRNGLGQTVS








VLSPLRKNKVMSVLGGAIQT








RHLDAWLQCKRQGKTASCIV








LDRSSSRFITTGRYTSFAAM








RFALPARLDLLPCRARSSMR








SYQNCRRCGYDRETLPHILQ








HCRQFSAPAYQARHDAVQGR








LETVMRRRFPNLRVNRALPE








IGSNKRPDLVVVDEEKRLVI








LLDIAIVFENTAAAFVDART








RKWAHYEKEILAYRLRGYSV








TYDAIVVGALGTWDPKNDAI








LKRIGVVSQRYLRLMKVLVV








SEMLEHSSRIYRKHLGLRDL








LPDTGTKRRPVGTTETDPPG








GDLRQKKRNTISARASGGKC








LERRFTSPVGTPSQRGELQC








QPCPGPRRPALAGIAPNPPS








LQPRKPPPRQHQKPVTKSTA








H








(SEQ ID NO: 1440)





NeS
Utopia-
.

Chelonia

GGCAGAAACTGCACSTTCTA
TGAGCTGGAGTGCCGATGAG
MLQLRLPTPQTLRLLHPSQL


L
2_CMy


mydas

GAAGACTCACTGCCTATCCT
AAGCGCAGTCGGGAAAGTAA
PQSHSTKRWSNIYEERARAP






GAGGAAGACTACCGCTTTGG
CTGAAATACTTTCCTCATGG
KDTTIERVSAASKIPKLDPA






AGATGGATTCTACTGCCGCT
ATTGTATTTTCTAAATGGAC
KRRIGAPLQLMQGNSISRQL






GCTTCAAAAGAAAATCTTCA
AACCTACCTAATTCTCAATT
SASSQYVQHNAWRRVSAPPH






TGCTGCTTCGGAGGCTCCAG
ACTGAGGGACAATCTCCACT
TGNFTGSCRRKLALPHRPPS






GACAGASTGAGAAGATGATC
CATTGATATATTTTGCTT
ETQPAESYLHCWTTQRDPTL






GCTCCCTCGCCGATTCCACA
TCCACAACCAAATCTC
ADQHGLQDHPTGLPPEGSHC






GAAACCTTCAACTGCCGCTC
TGTACAACTTTTCATGAGTG
IKDPRQNSSTEGQRRSGNSQ






GGACCGCTGCTGCTCCACGG
ATGTACCCGAGTACTTGGAT
ARRIPKRASTAASKTVLPKR






AGCGCCCATCGGGGAACAGC
TCTAATATCTAAACTGTATT
TSAALKSVREDTALVLEDPA






CTCTAAGAGACCCTCAGCGC
GTTAAATCTATTCACCTAAA
KWSSQHREGXRQQANPTAVF






TTCCAGGACATCGCAGATGA
TTTGGGTTATTGCTGATTAT
QPEPAEIEQQPXVRAATPWQ






GCAGCATCGATTGGAAAAGC
GTACTCTATGTATCATATGA
AAWMEELARTASFXDFDLLV






AGCGCCTCCCTGAAGAAAAC
CTTTTAAAAACAAACTTTGT
DRLTKDLSAEIVSGRKGTQE






CCGTGGAGATGCTGCTGCAG
ATTTGTGGATAATCTAAGCA
NTPTAHRQNQNNMREARRRN






GGAWATCTTGCACCTCGAGG
CTATACCCAGATGTACAGAC
ISRCYDPAAASRIQKLYRSN






ACGGCCTCCCAGGACATCGC
ACTCTTTTCCCAACCTATGT
RPKAMREILDGPSSYCAIPS






AGCCAGGACAAACATCATCT
ATTATATTTTTTTAACATTA
ERLFLYFKGVFDRVAQNDMQ






CGCCTGCTCTTCAGGASAAG
GCTTTAATAAAATTTTTAAA
RPECLXPXPRVDYAEDXEQD






GATGCCMSAAGAACTTMTCC
(SEQ ID NO: 1319)
FTSWEVEARLTKTKNTAPGK






CACCTCCTCMACTGCCCAGG

DGIRYNFLKKRDPGCLVLTA






ATCCTSATGCTGGTCGTCGT

IFNKCKQFRRTPSSWKKSMM






CCTGCTGTGCTGGAAGMKAC

VLVYKKGKQDNPNTRRPISL






CCCAACTGGCGAGACCTCAG

CSTMYKLYASCLAARITDWS






AAACCACCCAGMWGGACCKC

VNGGAISSIQKGFMSCKGCY






AACATCTCACTAGATGCCTG

EHNFVLQTAIHMARRAWRQC






CCCAGCCGAACCTCTCCATG

AIAWLDLANAFGSMPHQHIF






CWACTCTCCCAGAGCAACSA

DMLREFGMPENFLQLVRELY






GAACCATCCAGMGAATCCGC

EGCTTTICSMEGETPEIPIR






TGATATGACTGAAGCCMATC

SGVKQGCPLSPIVFNLAMEP






CAACAGAGGGAGAAGGAAAG

LIRAISSGLGGFDLYDNRVN






GAGAATGACTGCATCTATCT

ILAYADDLVLIADNPESLQQ






CCAGTATCCCCTCCCTACGG

MLDITSQAANWMGLRFNARK






ACACGCTCCTCTKCCCCTTC

CASLHIDGSRRDSVQATSFQ






CGCTATCCGAGGGTTCCAGT

IQGEPMIFLEDGQAYQHLGT






ACATTGGCAGTCTCAGCAMA

PTGFRVQQTPEDTIAEILRD






CACCTCAAGAGAATCCATAS

VARIDSSLLAPWQKINALNT






CAAGCGGATCACCTTCCGGT

FLIPRISFVLRGSAMVKVPL






GTGCCCTCTSCGACCTGCCT

NKADNTIRQLVKKWMFLPQR






TTCGAGACGCAGATGAAATG

ASNELVYISHRQGGANVPRM






TAAGTCTCATCAAGTCACCT

GDLCDVAVITHAFRLLTCPD






GCAAAGGACATCTCGAACTG

AMVRNIAESALQDAVKKRIA






GAAGAGTCCAACTTTACCAG

RTPSNQDVATYLSGSLEGEF






TCTATGTTGCCGCCACCCCA

GRDGGDFASLWTRARNATRR






TCTCTGCTCCGAAAGCAGAA

LEKRIGCHWTWCEERQELGV






ACACCAC

LVPQVKNTDHTIITPRARTM






(SEQ ID NO: 1196)

LERTLKDAIRCQYVENLKRK








PDQGKAFEVTCKWDASNHFL








PGGSFTRFADWRFIHRARLN








CVPLNGAVRHGNRDKRCRKC








GYANETLPHVLCSCKPHSRA








WQLRHNAIQDRLARAIPPPV








GKVAVNSAIPGTDSQLRPDI








VITNEDRKKIIMVDVTVPFE








NRTPAFHDARARKVEKYAPL








AETLRAKGYQVQTHALIVGA








LGAWDPSNERVLRECGIGQR








YARLMRQLMVSDAIRWSRDI








YIEHITGHRQYQEG








(SEQ ID NO: 1441)





NeS
Utopia
.

Phytophthora

ACCGCCCAAGTCTCCACCGC
TAAGCTGGTCATTTGACCGA
MQDMEEELLLDVEMETETTE


L
2_PCa


capsici

AGCTGCGACTGCTGCTGCTC
CAGGGCACTACCCAGGTAGG
PQTSTAXDATTTTDRPTRWG






ATCGAGCCGCAGTAGTCGCA
GAACCGCCCTTCAGAAACCC
PHPRAVAAAAIAQLVTGEXA






GCTGTACAAGCACCACCTCC
AGGAAGACACAAACACCCTC
XPALPSRQDRRPAPRSHAPT






TACCGGACGCGCCGTCGTGG
CCTTTAGTGACATACATATT
RSRWGPRHQAVGAAAIASLA






AGCACCACGCGCGCGCTGAG
TTAGGCTACATTTCAGTTAC
TGLPASAAPVSRATKHGEGR






CCGCNTCAAGACCAAGAATT
GGAGAGGTTACTAACTGGTA
RRLQTRWGPRVSIPRAARRP






CCAGGCTCGCGCGCGCGTGG
AATAAAAAGCACTTT
GSRWGPPRAAGASGQLPASA






CGCTTCAAGATGGCGACGCG
(SEQ ID NO: 1320)
SGATGOLPEHVEAITTTPRV






ACCAGCAGGAGTGGCCGCTC

ASDADEGPTPPDPWILRFDG






TACTGACGGCGGAGACGAGG

ACRRNPGPGGAGAALFKPSG






CCAAGACCGAGGACAGCGAC

AVVWTCSHYMPSSNETNNTA






GCGGACGCAGCCAAGCGCCA

EYTALLLGVQSAVHHGATRL






AGCCGCTCAAGCAGCACCGA

DIEGDSSLVIAQVKGTFACR






GACCCCATACCCMGGACACC

NAKLRQLRNRVRHALRSVEK






GCGCCGTACGACGGCCACAG

YTLRHIDRKANAHADRLANR






GGACTCGGTGCTGGCTGTGT

ALDRRSSSSECEPHGSCMER






ACGCACACAACGCACCTGCA

CCGTDTTPAVQGPTPQAAAA






TTCACCTGCGCGTTGTGTGT

VPVQVWPQWQRQTMVAWTTS






GTACACAGCACGCAACTTCG

HGGRCRDCSTRCGRSLPSLA






CCGAGCTGACCAAGCATCGC

HRPRLSPRRQPRLRLRQLSD






CATGCAGCGCATCGTCACAC

EERDXAADALQELSDVMASK






CCGCTTCGTGGATCACTTCC

IVDADSWDTGEGYISSIPER






ACAGCGGGTGCACGTGTGGC

IREVLQPYTTRPPRPGHQQQ






ATCGGCTTCCAGTCGCGCGC

QRRRPPRVTRNQREHRLDEA






GGCAGCTACGCGACACGCTC

LDDMQATQQAAPRDQRAIHR






AAGCCTGTGCAGACAGCACA

ARRRVGRVRASMAKQELRQA






CACGCCACCGTAGCTGCCTC

FAKDESKCVSKILAGASAET






GCGCGACCCGGCMNCCASTG

AAEEHVDECPIDAATLHAHF






CSSCCSGNGCCGGMAGCGAS

TGTNAPRTDFDYDAACGQEF






GAGGAGGACCNCGCACCCCC

RGALDSMQPPTVATDAFEEE






CGGTCCTCTSSTCGCGGCAG

LTIDEVEDQLTRAAKTSSPG






CATCTGCAGCTGCAGCAGCC

HDGIGYDIYSRFAAQLVPLL






GCATCAAGCGCCACTCCAGC

HAAYQFCWLHRRVPALWKLG






TGCAGATACTGCCACCACGC

IVRLIHKKGDPMQPTNWRPI






AGAGCGCCGTGCCCATCGCT

CLQPAIYKIYSGLLARRLSR






ACTCCTGGGCCCCAGTACGC

WMEQNQRLPMAQKGFRAFNG






CCCCCACGTGCTGGAGCCAC

CHEHNFVATTLLDQTRRSHR






CTCCAGAGCTCCGAGTTTCC

RLYQVWYDLRNAFGSLPQQL






GGCAAACGCCGMCGCCTCAA

MWSVLRHLGVDASFIARCKN






CACGCCGATCGACCTGCAGC

IYQDSAFVVANAVDGATDPV






CGCTGGACGTGGACGCGCTG

RQEVGVYQGCPLSPLLFISA






(SEQ ID NO: 1197)

LVPLIRRLEKLDGVGVPLAE








GVRPCATAYADDIKVFSDSA








AGIRKCHDAVTRFLEWTGLR








ANPGKCASLAVTTNARGNPV








RDDGVHLELQGEVIAPLSLH








DSYRYLGVGDGFDHVRHRLQ








LEPKLQQIKREAVALMQSGL








AGWQVVKALKTFVYPKVEYA








LRHLRPLQSQLQGFDRAVVR








GLRHLLRLPQSATTEFFYTP








TSGGGLGLQSLVEMHQALQV








AHAWQMLHSKDAAVVAVAKE








QVCQVARKRYRLQEEHWRGR








GDELVRLFLNSELAASPFAD








CLRRNGDIGSLWTDVQRTLR








LHHLSLTAQDDRDGQDPLAL








RVPHHTKWLDHKTVLRHVKL








HMKIRHQTRWKGLVDQGKTV








RVHGGLGAKFVSTGAGLSDD








AYRFGVKARLNQVDTNAVLK








RKRLRSSKTCRDPTCSSAET








LAHALNHCASNMDAIRQRHD








DALEQIGSKIRGALERAKST








TELRLNQTVPEYTGAALRPD








IVLRNVAAKKMVIADLAVTF








EDHAAGARHSSLQLSHDHKT








LKYQPIVAELRVQGWQVQTA








AIVYGSLGSVQPSNFKTYTE








KLKLHKREARQLDLQLSSHC








IQASHRIWGWHCRRHREGQR








SGNTSRASRGSGGTPRRTSQ








VRARR








(SEQ ID NO: 1442)





NeS
Utopia-
.

Phytophthora

GCTCGGCCTCGCGGCTGCCT
TAGGCGGAAACCAGGCCCAA
MLADPAALAAGLARAPPPPS


L
2_PI


infestans

TCCCAGGCGCCGCCGACTTC
GACGGCCGACAGGGCCCCAC
APQDPSPAFPAGPAGQNPRA






GCGCTCTGGCGCGGCCCACA
CCAGGTAGGGAACCGCCCTA
AAPARVEVHTVVAPPGRAGG






CGCCGCCGCCGAGCCTCCAA
GAAACCCATTTCGGTGGTCG
MLPDPGLVDSSPAAATAATP






GCGCGCCCGTTGGCTTCCGC
ACTCGAAGGCCTTACCTATT
APVAATATTARAAARVAVEH






AGACGCAGGGCTCGCGGCGA
TTTTCCTTAGACATTCAATT
HAHAEPNQEHLPMARVLVEP






CGGCCCTCGCCAGCCCCCAA
AGGTAGCGACCAAATTACAA
MQVDECSSCDRSTLTADDGS






GACCCCCCCTACGATGTGGC
ATTTGGTAACGAGTAAGCCA
GDDVAAPSSMLSNDVAAPMD






ACCACCCGGCAGGGCGGCCG
AATGGTAATACACAAAACTT
VDSGTSCPPTLQQPLQRPRA






GCAGGCTGCCCGACTCGGTA
TTCTGTTCTAATCAGTGTGA
LHVGSKRRRLDADDGEEAHQ






TCGCCGGGTGCTACACTCTC
AAACTGGTTTTCGCCTTTTG
LQEEEEAGIHAPALRLSAAS






AGCCGCCACAGCTCGGGCCT
GCGGACTTTTTCACTCGCAT
AQPASVLAVYTHNASRFDCT






TGGCGGTCCGCCATTGGCCC
TTTTGGGCAATCGTCTGCGG
LCAYTAGSFASLLTHRNSRH






TTGGAGCTCGACAGCGACAG
CTAGCTTGCTAGCGGCGGAC
RRTAFLDRFSAGCACGVPFA






CAGCGACGACGAGGACGCTC
GAGCGGTCTCCGGGGGCGTT
SRLAAARHAQACASLSSAPS






AAGACCCCCACGCCGCCGCC
CACCTTTCCCCCGCGAGGCC
AEASSAAGTSSPTADGADST






CCAGGACCCCCCGCAGACGT
AACTACACCGATCTTCTCTA
VSAVAHAEPGLPHHNDTELT






CGCGAGTGTGCTTGCCCCAC
CACTTTTCTAATTCGCCTCC
ASPPLVSSSDVEVQATKTEA






CCGGCAAGGCAGGCAGC
GTCTTCGGTCTTCGGCTGTC
TDNRWGAPLPRVLVASRIAG






(SEQ ID NO: 1198)
GGATTTTTTTCTTTTTGACC
RLAQVPPPRWGPPLPRTTIA







AATCAGAGCGCGCCATGCGA
ARIATRLAATPAPRWDPPLP







CTCTTCTGGCCAATCAGAGA
RSLVVSRIAARLLPALPDAP







CCGGGCCCTGTCCTCGGACA
ACEEEAKDSDTMDWAPTWTN







GCGAGGCCTCCACGGCCAGC
EETKESEPHDEAPGQVDEET







CAATCAAGTCTCGGCAGCGA
IDDADGEWLLRFDGACRANP







CGCGTCTTTCTATAGCGCAG
GPGGAGAALFKPSGPVVWTC







CTGACGAGGCCGATCTGGCG
SHYDPSTTATNNTAEYTALL







GCCCCCGATTGGTCCGACTT
LGARAAADHGVTKLRIEGDS







TCGGCCAATCAGCGACGACG
TLVIQQVRGIFATRSTRLRA







AGGGGGCAGGGGTTTACACT
LRNKVKLELARVGSFSLHHI







TTTGCCCCCGTTTCGACTTC
DRQANGHADRLANAGLDRRR







AACTTCAGGCCAAAATGGCG
TKLECSVHPDGRGCTNTSVA







ATTTCGACCCTCCACGCGCC
TAAPTAPAAPLPSARPPAST







GTGCCACTGCTCGGCACCGG
AAPSPDDDHSDQGDIDDGEV







CGGCGATTCAGCGGGTGCAA
YAAMCISPDAVPHRRPRLRL







CTTCGGGCACGTGTGCAACA
RRLTDEESEEAGNVVERLAA







CATGCAGCGCCCATTGCACG
SLAAKIADAPDWETAEGYIT







CCAAGCGGCATCGCGGGACG
ALPYALYDKLQPYSQSQHQP







ACGCCTCGGCCGCTCAAGCG
PRQQQQQQRQRPRQQQQTRQ







CAGCCCCGCCCTTCCAGCAC
RRQRRCKRGGGSQHRQRKTR







GACCTCGCGCCGTTTGGCGG
RRRPPRVTRHHREHRIDEAL







ATCGCCATCAAGACGTGCGA
DDLHALESRRPQDRTAISKA







GAGCCAGGCGGGGTCGGGCA
RRRVGRIRSALDQHQLRHRF







AAATATACTTACTCTAAGTA
DTDEKACVDGILAAARDKDR







TGCCCGAATCCCTGCCCTCT
AASVTTTAQTAAPPHSAPAS







CAGGCTGAACGCGGCCCCAT
APSSAVDDGICPIPGDLLHA







ACTTGATCTAAGTATGGGAG
FFTDVNTPRTEFDADSPIGA







GGATCCCTGGCCTCTCAGGC
RFREALAQLPAAIAATELLM







TGTACGCGAGACCCGTACGG
EPPSPDEVEDQLQRVRGTSS







CCGAATCCCCTGGCCTCTCA
PGLDGVGYDVYKTFTQQLLP







GCCTGTACGCGGGGC
ALHAAFSRCWTDQRVPQSWK







(SEQ ID NO: 1321)
LGVVRLLFKKGDRQDPANWR








PICLQQAVYKLYAGILAHRF








TRWLDANTRHADAQKGFRAV








NGCGEHNFLAATLTDNARRR








RRELHVVWYDIKNAFGSVPH








ELLWEVLRRMGVPAQFIACC








QGIYDAAAFTVGNAADGTTA








PIQLRLGVFQGCPLSPHLFT








AVISPLLHALKRLPGTGVQL








SAVDRPGASAYADDLKVFSD








TKDGITRQHQLVTDFLRWTG








MVANPSKCSTMSVQRDNRGV








LKTANLTLQLDGAQIPALGM








TEAYAYLGIGDGFDHVRRRV








ELAPKLRELKADTTALMQSG








LAPWQVVKALKVYIYPRVEY








ALRHLRPFQQQLQGFDRHLA








RGLRHLLRLPTSATTEFLYA








PTSRGGLGLLPLTEVHGALQ








IAHAWQTLHSPDPAIRRIAR








VQLRQVADARHRLDAEHWKE








RGEELCERLLNSQLGTSAHA








PPKRRNCDIGSLWVDVQRHL








RSLGLQLQTAPADTHTGAPA








QPLQLRVPHHDKWLTHKDVL








RHVKLHIKNNHWHRWTSMRD








QGKTARAHGGEGSGFLTQPR








GMWEADYRFAVAGRLNQVDT








YSVLKRRRLRSHDRCRQPGC








HRAETLAHVLNHCPGTMDAV








RGRHDGALKRIERELHASAT








DRRDRVELRVNQTVPSLAGP








ALRPDLQLYNHTKKTVAVVD








LAVAFEEQASDDASSSALSL








IASHKRAKYDRIKRHLERQG








WKVHLSALVYGSLGAVASGN








YQVYTTHLGLLKRDAKRLDR








QLSVECIQSSRRIWNLHCSQ








HRTRQHQARPSQGPRGSRAT








ETGGTPSQTSRR








(SEQ ID NO: 1443)





NeS
Utopia-
.

Phytophthora

TCAAGCCCCGCCGCCAAGCC
TGAGCACCTTGGGTTGCTCA
MSGDVVSSDGSSRTTDASGD


L
2_PR


ramorum

CAGCTGCGGCTGTTGCCGCC
AGCGTGATGCGAAGCGGCTG
GDDGAGSSDAAGDVGVVAMD






CCTCCAGCAGCAGCAGTCGC
GACCGGCAGCTCTCGGTGGC
VDQGARRQQPPWQRVGGKRR






AGCTGAACCTACAGCCCCTC
GTGCATCCAGTCCAGCCGCC
RLNDVDDEDTRELAELLLEE






CTGCTGATCGCGCCGCCGTG
GCATCTGGAACCTGCACTGC
EDEAGDHAPAPRLSAASARP






GAGCCCCGCGCGCGCGCCGA
AGCCAGCACCGCGCGCGCCA
ASVLSVYAHNAQRFQCTLCT






GCCGCCCCAAGAACAAGCAC
GCACCAAGCACCAGGGGGAA
YTAASFASLKRHRDSRHRRT






CCCCAGCTAGCGCGCGCGTG
GTCGGGCGGCGGAGACCGGG
AFLDRFSAGCACGAPFASRL






GAGCCC
GGGACTCCGCCGCGCACCGG
AAANHAHACASLNRTLSVAA






(SEQ ID NO: 1199)
CCGCCGCTAGACGGCACACA
TPAAGELSPTAGAANATVKA







GGCCCACAGCGGCCGACAGG
ATVTPDSPRQDPPELAASPP







GCCACACCCAGGTAGGGAAC
LASSPDVAVQAADMQAPTRW







CGCCCTCAAACCCCGCCGGT
DPPLPRTLVATRVASRLTDL







ACATTATGGTCCGACACCTA
TPPRWGPPLPRATVVSRIAA







TGAGGTGCAACCTGTACACA
RLEAAPTPRWGPPLPRVVVA







AGTTACACACCACATAGCGA
SRIAERLAPPELAADDETKD







CTACCAGGTATTTACTACCT
GEEDQSFTEPVAAARSXGGE







GGAAGCCAAGGATTAACCGG
DANGEWLLRFDGACRANPGP







TCGGTAATACACATAACTTT
GGAGAALFKPSGPVVWTCSH







(SEQ ID NO: 1322)
YMPSSSETNNTAEYTALLLG








MRAAADHGATRVHVEGDSTL








VIQQVRGIFATRSTRLRGLR








KSVKAEMARMEHVTLHHIDR








QANGHADRLANAALDRRKTK








LECGLHPDGQGCSSTAATTA








VPSVVPDRPPSSTAAAPTPS








AEPDETEQGDIDDGEVYAAM








CIGPDSIPERRPRLRLRQLS








ETEEEEAGAIVERLAATLAG








KITDASDWATAEGYITALPY








TLYDKLQPFAQHRHQPRPQH








RQQPQRDPPLGTHDGDHGQP








STSRSRRRRRRAKDRLRRRP








PRITRHHREHRLDEALDDLR








AVEHASPHDRPAVARARRRV








GRVNSAIAQQQLRHKFDKDE








KACVDGILAAARASRGLATP








SASASRHPPPVPSTAADDGS








CPIPSDELHAFFTAVNTPAG








TFEPMAPVGAPFRSAVAHLP








AATSQPELLSDAPTTDDIED








QLQRARGSSSPGLDGVGYDI








YKAFAAQLLPALHAAFACCW








RHKQVPQSWKVGVVRLLFKK








GERTEPANWRPICLQQAIYK








LYAGVLARRLTRWLDANGRH








ADTQKGFRAMNGCGEHNFLA








ATLVDQARRKRRELHVVWYD








FANAFGSVPHDLLWEALERQ








GVPSPFIACCRGLYADAVFT








VGNAADGTTAPIALRVGVFQ








GCPLSPHLFTAAIAPLLHAL








KRLPDTGVQLSRVDCLGASA








YADDLKIFSGTEGGTKRQHA








LVADFLRWTGMRANPAKCCT








MSVQRDTRGVLKACNLGLQL








DGAPIPALTMSASYAYLGIG








DGFDHVRRRIELAPKLQELK








HDATALLQSGLAPWQVVKAV








KVYLYPRVEYALRHLRPFHQ








QLEGFDRHLVRGLRHLLRLP








ANATTAFFYAPVSRGGLGLL








PLTELHAALQVAHGWQMLNS








KDPAIRRIARVQLRQIADAR








HRIDAQAWQDREEELAQLLL








NSQLGASTGAPPKRRNGDIG








SLWVDVQRHLRHLSLKLETA








PACAETGTAAAMLQLRVPHH








DKWLDHKTVLRHVKLHYKNK








HWARWAAMXDQGKTARTHGG








AGSGFLTRPRGMWEADYRFA








VAARLNQLDTHSVLKRRRLR








XHDRCRQPGCTQGGDAGARA








QPLRRHHGRGPRPPRRRPQA








HRARAARVVAGRPGPRRAPG








QPDGAVARRPRATARPPAVQ








PHQEDGGGGRPGRGVRGAGE








RRPGELGAGTHRRTQAREVC








RRQATPRAPRVEGPPLGARV








RLARRGAGRQPQGAY








(SEQ ID NO: 1444)





NeS
Utopia
.

Pythium

TGACTGGTGTTTGATCACGA
TAAGCGGGGGGTCCAGACCC
MDYDDSEFFDAICIPDEDAD


L
-2_PU


ultimum

TCAATGAGGTGATTAACATG
CACAAGAGAGAAGCAGGAAT
VLDDGDEGDEGGNDDESSEP






AGCCGGAGCAGGCCCCTTAC
CATGGTCCGCATGGACCAGT
LPLAITNAPSAPLHATMLCG






ACGCTGGTGCTGTAATGGTT
AGGGCACGCTCCACAAAGGT
TVTQPWLLRFDGACRRNPGP






CAGGAATGCTCTTATGAGTA
TATCGCCCTCAAACCCATCA
AGAGATLTRPNGIILWTHYR






ACTCCACAGTATAATTTTTG
CACGAAGGATCTAAAAAGAA
YIPDKTATNNVAEYEALLDG






TTAGCGGGGGTGAGCGCGTG
AGCAACAATCGAAATAGTAA
LRCAAHHGVKHLRIEGDSNL






CGCCGCCCCCACCTTTCTCT
ATAGTAAATAGCTTAGAAAG
VIEQVKGIFACSTSLRPRRD






TTTCGTTTGTATTTGGTTCC
GTCAACATGCGAAATGCATG
QVREILRHFETYSFRHIDRA






TCTGGAGCGACTTCCGTGGC
AGGAACGTAAGATGGTAATA
LNRQADRLANQALDLLKTVS






TTTTGTCGCGCGCTATCGCG
CATTTTTATA
VCALSQTRVQDDTGAAHGCW






CCCGGCATGAGGATCGTCGC
(SEQ ID NO: 1323)
HWTPPDASPTDDASTSILTQ






TCCTGACGCCATGTGCGCCA

DVPVPMDIDDYDPDEPMNAA






GTATATCGGAGCCCCGTGCT

DDPVSINAEREGGTVYPVLR






CCGCTTGGGTGTGTGTTCCG

LGPNVVPERQKRLQIPWLPP






CCACGACGCCACGCGATTTG

REMQKLEKKIEVLGETFASR






CGTGCACGGCATGCGCTGGC

IRDAPDWFSAEGYITALTSE






GACTTCCGCTCAGTGCGCGA

LATLIRQSTAATTGPNAARP






ACTCGCGGTGCATCGTCGAC

CERTISKEKRRARRTTPLQR






GTGCGCACCGGTCTCTGGCC

ALAEAKHELQIIQPDASRKS






TTCCAGGACTGGTATGAGTC

VRKAARRVKRISQAQQRHDL






GTCGTGCGCGTGCAGAGCGC

RRLFSTNERRCVEKILRDPP






TCTTCACCGCCCGTCTCGAC

VGPSSTSSSLPATDDDRCTI






GCGTTCGTTCATTCGAGCCG

DPADLFAYFQTQATAPTNFD






TTGCGACCACAATGCGAACC

FDDEGGELFRSVLDELPRAD






AATCAGCGAACGCCGTCCCC

QEVHLLEDEITRDEIEDQLS






TCGTCCGCCGCCTCGACAAC

RISKSTAPGLDGITNAVYVR






GAGAGCGGACGCGGATGCGA

FKLQLLDALQAAFNACWRYN






CGGCCCCCCCTCCCGCTATC

RVPSMWKAAFVRLIYKKGNR






TCTCTTCCGCGGACGGGCTT

AVPSNWRPICLQQTVYKLYT






CCCCCGCCCCTGTCCTAGTT

AILASRLQRWMDANARFTMS






GCGCCGCTCTCCTTCTGGAT

QKGFRAFNGCHEHNFVATCL






GCGGCAACTTGGACGACCCA

HDQTRRLRKKLAIVWYDLRN






CGTGCACGCG

AFGSLPHEYLWRVLARLGMP






TGCGCTGCCGCCAGTGCGAC

PQFVARVRQLYADASFTVES






GGCGTTGATCCAGCGTCATC

RDGTTDPVQLERGVYQGCPL






TACACGCCGTTCCTCCCGTA

SPYLFIAALIPLVRALHKLK






TCGACAGCAACCGATCTCAA

DQHGIVLAPGVTDCVSAYAD






CGGCGATGATAGCCAACCTC

DIKIFARSGTGAKALHEIVV






CAGACCCGCGTGCCAACATC

RFLSWTNMAANPAKCALMVT






CAACAGCTTCCAGCCATCCA

DGARGGDDTDASMTLSIEGE






TTCGACAACGACAACGACAA

TIPRLTGKEGYVYLGVEDGL






CGACAACGACGACGACCCGC

AHERRATCLRDSLKAASADV






TGCTCGACGACGTGCTC

VRLLRSDLAPWQIVRAIKSH






(SEQ ID NO: 1200)

VLSRFDYVLRHLRPFLSLFD








GFDKMLVRGIKRLCQLPQTA








TSEFLFSPTSAGGLGFLPLK








ELFAALQIVHALQMLHSKDA








NVRAIARHQALQVVRKRYAL








QSDHWSDREEELLEEFFNGT








LERSPFALAKKVSGDIASLW








TDVRVNLTKYGLKFGEAHGR








RLQPLVSHTDKQLAPQQWAS








AIKTHMRLRHLKRWTTLVDQ








GKTARMHERIGSAFLTRPSG








VYDASYAFAVRARLNQVDTR








SALKRKRIVNNSRCRVSGCS








ELETLAHVLNHCRFGSDSIR








ARHAETLLLIKTTMERELTR








PGRQHQRLLVDATVPEARDP








VPSNDAAESNIGAMAPSISH








LRPDIQLYDNKTMEAVIIDL








AVAFEDQSTDDAASSSSFAR








VKGVKTKKYEVIKQFLEYKG








YTVHVAALVYGSLGSVDTGN








FAVYTERLGLRKGAVRRLEC








SLSARHINFAHRMWRRHAIA








HTTGLRLIGTNSVQQQGVQR








APAEKQQHQRPVQRPSRAQA








PRDQPSQQQQSQQSFQQQSQ








QSQQSQQSQQSQQSRQRHAP








TPTPVPVPVPVPVSTPTLTP








TPTPTRRPKPAPSSTQPQQG








APAQRRQQREQKKQPACRRR








HATAVRETPPAAPTAARTAT








PTARPTATTRSTSTTRSTAT








ATSTTRSSAPTSRPSAPRPR








SSAPRPRSSAPTTRSAAPTT








RSAAPTAIATTSVYKSRRTT








NAISGATTRRSASSKRAPMQ








PRTALTPTQQQQRQ








(SEQ ID NO: 1445)





NeS
Utopia
.

Phytophthora

GTACGGCCGAATCCCTGGCC
TAGACGGCAAAGTTCTGGCC
MPRSLASEPVHSSASRLPSQ


L
-3_PI


infesta

TCTCAGCCTGTACGCGGGGC
CGCGTAGGCCGAAAGGGCCC
APPTSRSGAAHTPPPSLQAR






TATACTTGGTCTAAGT
CGCCCAGGTAGGGTAACGCC
PLASADAGLAATALASPQDP






(SEQ ID NO: 1201)
CTCGGGAAAACCATTCTGGT
PYDVAPPGRAAGRLPDSVSP







GTTTGGCTGTTTTTCAACAG
GATLSAATARALAVRHWPLE







TCGAACTTCAACTCGGAGCA
LDSDSSDDEDAQDPHAAAPG







TATCAGATACACTTACTCAC
PPADVASVLAPPGRAGSMLA







ACATTTAGATATCAGATAGG
DPAALAAGLARAPPPPSAPQ







GAACCTTTATTAGGGAGATA
DPSPALPAGPAGQNPRAAAP







ACGGGTACACCGGATGGTAA
ARVEVHTVVAPPGRAGGMLP







ATATACAAAACCTTCTCTGT
DPGLVDSSPAAATAATPAPV







TCTAATCAGTGTGAAAACTG
AATATTARVAVEHHAHAEPN







GTTTTCGCCTTTTGGCGGAC
QEHLPMARVLVEPMQVDECS







TTTTTCACTCGCATTTTTGG
SCDRSTLTADDGSGDDVAAP







GCAATCGTCTGCGGCTAGCT
SSMLSNDVAAPMDVDSGTSC







TGCTAGCGGCGGACGAGCGG
PPTLQQPLQRPRALHVGSKR







TCTCCGGGGGCGTTCACCTT
RRLNADDGEEAHQLQEEEEA







TCCCCCGCGAGGCCAACTAC
GIHAPALRLSAASAQPASVL







ACCGATCTTCTCTACACTTT
AVYTHNASRFDCTLCAYTAG







TCTAATTCGCCTCCGTCTTC
SFASLKTHRNSRHRRTAFLD







GGTCTTCGGTTGTCGGGCTT
RFSAGCACGVPFASRLAAAR







TTTTCTTTTTGACCAATCAG
HAQACASLSSAPLAEASSAA







AGCGCGCCATGCGACTCTTC
GASSHTVDGADSTVSAAGHA







TGGCCAATCAGAGACCGGGC
EPDLPRHNATELTASPPLVS







CCTGTCCTCGGACAGCGAGG
STDVEVQATETEATENRWGT







CCTCCACGGCCAGCCAATCA
PLPRVLVASRIAGRLAQVPP







AGTCTCGGCAGCGACGCGTC
PRWGPPLPRTTIAGRIATRL







TTTCTATAGCGCAGCTGACG
AATPAPRWSPPLPRSLVASR







AGGCCGATCTGGCGGCCCCC
IAGRLLPALPDAPACEDEAK







GATTGGTCCGACTTTCGGCC
DSDEMDWEASEPHVEAPGPV







AATCAGCGACGACGAGGGGG
DEETIDDADGEWLLRFDGAC







CAGGGGTTTACACTTTTGCC
RANPGPGGAGAALFKPSGPV







CCCGTTTCGGCTTCAACTTC
VWTCSHYDPSTTATNNTAEY







AGGCCAAAATGGCGATTTGG
TALLLGARAAADHGVTKLRV







ACCCTCCACGCGCCGTGCCA
EGDSTLVIQQVRGIFATRST







CTGCTCGGCACCGGCGGCGA
RLRALRNKVKLELARVGSFS







TTCAGCGGGTGCAACTTCGG
LHHIDRQANGHADRLANAGL







GCACGTGTGCAACACATGCA
DRRRTQLECSVHPDGRGCTN







GCGCCCATTGCACGCCAAGC
TSVATAAPTASAAPSTPTRP







GGCATCGCGGGACGACGCCT
PATTAAPFHSDQGHIDEDDE







CGGCCGCTCAAGCGCAGCCC
RRADIDDGEIYAPMTLGPDE







CGCCCTTCCAGCACGACCTC
VPARRPRLRLRQLSDEELEA







GCGCCGTTTGGCGGATCGCC
AGAIVERLSASLSAKITDAE







ATCAAGACGTGCGAGAGCCA
DWGTAEGYITALPHLLYDKL







GGCGGGGTCGGGCAAAATAT
LPYSRTAPRHQRPPRPSRNQ







ACTTACTCTAAGTATGCCCG
QDHPQPRRDQPQRNVDEQQH







AATCCCTGCCCTCTCAGGCT
AESQQGEDQRQQQPPTRRRR







GTACGCGGCCCCATACTTGA
RRGKRRGRRQRRHPRQPGQS







CCTAAGTATGGGAGGATCCC
TRASQQSRQRRPRPPRVTRH







TGGCCTCTCAGGCTGTACGC
HREHRIDEALDELHTLERAR







GRGGACCAAGTACAGCCGAA
PQDRSAIDKARRRVGRVRGA







TCCCTGGCCTCTCAGCCTGT
INQHLLRHRFDTDEKACVAD







ACGCGGGGCTATACTTGGTC
ILEKAHAARAARTAQAAGAA







TAAGTATGCCCCGGTCGCTG
TSTGGAATAPTQQAATSALG







GCCTCTGAGCCCGTACGC
DADDGTCPILADELWQYFTG







(SEQ ID NO: 1324)
TNTPRWEFNPATPVGEAFRT








AMARLPPATRLRELLTEAPT








ADEIETQLQHVRGSSSPGLD








GIGYDVYQRFAQQLLPVLTA








SFKRCWTAKMVPQSWQVGVV








RLLYKKGAHDDPANWRPICL








QQAIYKLYTGVLARRLVRWL








DVNDRHAPGQKGFRAVNGCG








EHNFLAATLIDQARRKRRSL








YEVWYDFRNAFGSVPFQLLW








DSLQRLGAPADFIDMCKGLY








HQAAFVIGNAADGPTAAIRQ








QVGVFQGCPLSPQLFNVAIS








PLLFALRRLPETGVQLSGDD








RVGVSAYADDLKTFSSTKAG








ATKQHELVAAFLAWTGMKAN








AAKCSSMGVRRNSNGATEAD








NLDLALDGTPIPSMTHMQSY








TYLGIGDGFDHVHRRIELAP








KLKTLKQDTTALLESGLAPW








QVVKAVKVYLYPRVEYALRH








LRPEDQLLESFDLHLRAGLR








HLLRLPKNANNDFFYSPVSR








GGLGLLPLVELHAALQIAHG








WQMLNSTDPATRRIAREQLH








QIADARHRLDKAHWKERGDE








LCQLFLNLDLGTSAHAPPKR








RNCDIGSLWVDVRKNLQAFG








LKLETAPADAESGTPALPLQ








LRVPHHEKWLTHRDVLRHVK








QHLKNKHWRAWCAFQDQGRT








ARAHGGVGSSFITRPRGMWE








SDYRFAVAARLNMVDTSATL








ARRRLRAHDRCRYPGCRWKE








SLEHVLNHCPGTMDAVRGRH








DGVLREIEHALRAPSGARRE








LRVNQTVPGLPGPALRPDIQ








VYNHDQRTVAVVDLAVAFDR








QDRDDPETSGLAKAAAEKKA








KYTGIQRHLERQGWKVHLSA








LVYGSLGSVAPNNYKVYTEH








LGLLKRDAKRLDRTLSVACI








QSSRRIWNLHCAKHRARQHQ








TPSQSRGRRVTETGGAPSRT








DRR








(SEQ ID NO: 1446)





NeS
Utopia
.

Phytophthora

TGCGCGGCAGACCAAGACGC
TAGACGGCAACACTCTGGCC
MQDQVDAEQQARNRWGPPLP


L
-3_PR


ramorum

GCAGCCAACAACAGACCGTG
CGCGTCGGCCGAAAGGGCCC
RPLVASRVAARLGEVPPPRW






CAGCGTGCGGGTGGAAAGCG
CACCCACGTAGGGAACCGCC
GPPLPRGVVVSRIAARLEAV






CCGCCGCCTGAACGCTGGTG
CTCGGGAAACCCAGTCTGGT
PVPRGGPPLPRSFVATRIAD






ACGATGAAGACCAGCGAGAG
GTTCGGCCAAGAAATGCACC
RLAPPSPDLSLLDEEMKESE






CTGGCCGAGCTCCTGCTCGT
ACCACCACGGCGGAGGTGCA
PPDPTHHSADEDSTDAETAD






GGACGAGGACAAGGCTGGCG
TTTCGACAGTCGAACTTCAA
AVMEPAFVSDPPTATPREWR






CCGAACACCCCGCGCTCAGG
CCCGCCACATATCGGATATA
LQFDGACRGGPNPGGAGALL






CTGCCCACGGCCAGCGCTCA
GTTACAGCTCTAGTTAGACA
YNPEGAVVWTGSHYMPGAKE






TCCGGCCTCCGTCCTCTCCG
TCGGATAGGAACTTCTTAGA
TNNSAEYTALLIGARAAADH






TGTACGCGCACGCTGCAACT
AAATTAACGGGTATACCGGA
GARQLRIEGDSLLVIRQVKG






CGCTTCGACTGCACGCTGTG
TGGTAAATAAAATAAAAACT
LYATKSTRLRQLRNAVRHEL






CACGTACACGGCTGCCAGCC
TC
ARVGQHSLHHIDRQGNAFAD






TCGCTTCGCTCAAGCGCCAC
(SEQ ID NO: 1325)
RLANRALDLKSDKVECKEHP






CGCTCGTCTCGGCACCGACG

VAGACTTCMGSPSAGPPATP






CACGGCCTTCCTCGACAAGT

PPTTADIEMADAGSDDELRA






TCTTGGCGGGCTGCGCGTGC

DIDDGEVYAPMRLEPGVIPT






GGCACGCCCTTCGCATCGAG

RRSRLRLRQLTDDEMEAAGE






GTTGGCCGCAGCCAGACACG

VVERLSAGLSAKIADADDWE






CGCAAGCGTGCGCCAACCTC

TAEGYITALPYMLYDKLQQY






TGCACCACCTCGGCGACGAC

TQVRHGTARSPAPHPQRRDV






TTCGACGGCAGCAAAGGCAT

QGQVETHREPRHETIGQPDQ






CAAGCCCCACTGCTGCCGGA

PGEPSPTRRRRRGKRKGRRQ






GGCAGACCCACCGTCCGTGC

RRHPRRTNCGGGGRQQRKQR






AGTGGTCACCGCCGCGCCCG

HPRPPRGTRHHREHRIDEAI






ACCTGCCCCGCCAGTATCCC

DELHALERARPQARPAIAKA






TCGGAGCTCGTTGCGTCCCC

RRRVGRIRSAIDQQLLRHRF






CCCGCAGCCGAGCTCCACCA

DTAEKECVDGILAAARTARD






ACGTTGCA

ARTTVRAAAATGTTATPETA






(SEQ ID NO: 1202)

VTSGTEQQDDNGTCPIPSEV








LWRHFDSVNTPQRDFDPEAP








EGAAFRSAMARLPAATRFME








LLKEEPSTDGIEVQLQHASS








TSSPGLDGVGYDVYKRFASQ








LLPVLKAAFKCCWTHKQVPQ








SWKLGVVRLLYKKGDREDPA








NWRPICLQQAIYKIYTGVLA








RRLTRWQDANDRHAPGQKGF








RPVNGCGEHNFLAAMLIDHA








RRKHRPLYEVWYDFRNAFGS








VPLGLLWDALERTGVPAEYI








AAVQGLYDHAAFMVGNAVDG








STAPILQRVGVFQGCPLSPP








LFSAAISPLLHALQLLPSSG








VQLSGDDRPGVSAYADDLKT








FSGTKAGVTEQHELVAMFLR








WTGMADGFDHVRRRVALAPK








LKLLKQDATALMESGLAPWQ








VVKAVKGYLYPRVEYALRHL








RPDDQLLESFDLHLRRGLRH








LLRLPKSANNDFVYAPVSRG








GLGFLPLVELHAALQIAHGW








QMINSPDPAIRRIAREQLHQ








VADARHRLDKDHWKQRGDEL








CELLLNGELGTSAHAPPKRR








NGDIGSLWVDVRKNLKAFGL








KLATAPADPESGAPAKPLQL








CVPHHAEWLDHRNVLRHVKQ








HMKNKRWRAWCSHVDQGRTA








RAHGGVGSGFLTRPRGMWES








DYRFAVAARLNMLDTVNVLA








RRRLRAHDRCRHPGCRWKET








LAHVLNHCPGTMDSIRGRHD








DALKEIERTLHASSGDRQGR








TELRTNQTVPGLAGPALRPD








LQVYNHDQRTVAVVDLAIAF








DEQPRDDPESSGLAKAAAEK








KAKYAGIKRHLERQGWKVHL








SALVYGSLGSVAPSNYKVYT








EHLGLLKRDAKRLDRQLSVA








CIQSSRRIWNLHCAQHRARQ








HQDQPAPRGRRVTETGGTPS








RTDRR








(SEQ ID NO: 1447)





Nes
Utopia
AATU0

Phytophthora

AGCTCGGCCTCGCGGCTGCC
TAAACGGGTCACTTGACCGA
MLADPAALAAGLARAPPPPS


L
-4_PI
10012

infestans

TTCCCAGGCGCCGCCGACTT
CAGGGCACCACCCAGGTAGG
APQDPSPAFPAGPAGQNPRA




81.1

CGCGCTCTGGCGCGGCCCAC
GAACCGCCCTTTAAAACCCA
AAPARVEVHTVVAPPGRAGG






ACGCCGCCGCCGAGCCTCCA
GGAAGACACAAACACCCTCC
MLPDPGLVEEPIQATYAHDA






AGCGCGCCCGTTGGCTTTCG
ACATAGTGACATACATATTT
AQFECALCPYVAESMAVLVQ






CAGACGCAGGGCTCGCGGCG
TAGCCTAGATTTCAGTTACG
HRRSAHRGTRFKDIFTSGCQ






ACGGCCCTCGCCAGCCCCCA
GAGAGGTTACTAACTGGTAC
CSLVFYARIVAASHAVACAR






AGACCCCCCCTACGATGTGG
ATAAAATTACACATTCTGTT
RNQRAVPPAPTPVAPTRPEA






CACCACCCGGCAGGGCGGCC
CTAATCAGTGTGAAAACTGG
TPQPTGYLAAAMTAAAAAAS






GGCAGGCTGCCCGACTCGGT
TTTTCGCCTTTTGGCGGACT
SDTVVAAATNMQSAVPAAAK






ATCGCCGGGTGCTACACTCT
TTTTCACTCGCATTTTTGGG
TTGLQLVPPELEPALPQRAS






CAGCCGCTACAGCTCGGGCC
CAATCGTCTGCGGCTAGCTT
CHAGKRRRLNADEAVTPCTP






TTGGCGGTCCGCTATTGGCC
GCTAGCGGCGGACGAGCGGT
TARVSPQTEVAMAPHDAPQD






CTTGGAGCTCGACGGCGACA
CTCCGGGGGCGTACACCTTT
DTVLQREAAEPQPDPAATQG






GCAGCGACGACGAGGACGCT
CCCCCGCGAGGCCAACTACA
AQVQRVEDTTAAQDDTVQQD






CAAGACCCCCACGCCGCCGC
CCGATCTTCTCTACACTTTT
HDADTAQVSPPRRTPTRWGP






CCCAGAACCCCCAGAAGACG
CTAATTCGCCTTCGTCTTCG
RPSSTQEPSPMTGEPAATLA






TCGCGAGTGTGCTTGCCCCA
GTCTTCGGCTGTCGGATTTT
ARRPLTPAATGTRATRWGPC






CCCGGCAGGGCAGGCAGC
TTTCTTTTTGACCAATCAGA
HRAIGAAAIARLVTGLPTEP






(SEQ ID NO: 1203)
GTGCGCCATGCGACTCTTCT
AQPQRRQPPPPQEPPLQPEP







GGCCAATCAGAGACCGGGCC
QAAAATVAADIAATVAADIA







CTGTCCTCGGACAGCGAGGC
AAAANAAMDVDGGPAADETW







CTCCACGGCCAGCCAATCAA
LLRFDGACRRNPGPGGAGAA







GTCTCGGCAGCGACGCGTCT
LFAPSGAVVWTCSHFMPSRS







TTCTATAGCGCAGCTGACGA
ETNNTAEYTALLLGAQSAVH







GGCCGATCTGGCGGCCCCCG
HGAKRLNIEGDSHLILSQVR







ATTGGTCCGACTTTCGGCCA
GAFACNNKRLRSLRNRVQAS







ATCAGCGACGACGAGGGGGC
LRQLDWYRLQHIDRKANQHA







AGGAGTTTACACTTTTGCCC
DRLANRALDLRRTVTECGPH







CCGTTTCGACATCAACTTCA
AETRNRCFQTPQPLVEPGET







GGCCAAAATGGCGATTTCGA
HCVPGSDEVLAANTAMEDAT







CCCTCCACGCGCCGTGCCAC
AVPTEDDEAEVAARDGGEVF







TGCTCGGCACCGGCGGCGAT
PTIAIGPDSAPAKQPRLRLK







TCAGCGGGTGCAACTTCGGG
KLDEDDFDAAAAAVTRVSEE







CACGTGTGCAACACATGCAG
LASKIVDAGDWTSGEGYISA







CGCCCATTGCACGCCAAGCG
IPERLRAALRPFALPTQPAR







GCATCGCGGGACGACGCCTC
PQPREPRMQQPPRRPPRVTR







GGCCGCTCAAGCGCAGCCCC
DHLEHRLDEALDTMENVQRS







GCCCTTCCAGCACGACCTCG
TPQNQKAVRRARRRVGRLRS







CGCCGTTTGGCGGATCGCCA
AMDRTRLRKKFATHERECVA







TCAAGACGTGCGAGAGCCAG
EILRRASTEEAANPSQEKCP







GCGGGGTCGGGCAAAATATA
IDRATLHEYFTATSTORTPF







CTTACTCTAAGTATGCCCGA
DYDSAKGTEFRTFLEVMSTP







ATCCCTGCCCTCTCAGGCTG
SHETSALTAEPTLDEIEDQL







TACGCGGCCCCATACTTGAT
AHVKAGSSPGHDGVGYDVYR







CTAAGTATGGGAGGATCCCT
RFQVQLLPLLHAAFRFCWRH







GGCCTCTCAGGCTGTACGCG
RRVPALWKVGFVRLLHKKGD







AGACCCGTACGGCCGAATCC
PQQPNNWRPICLQTAIYKLY







CTGGCTTCTCAGCCTGTACG
SGLLARRLSKFLEANELLPM







CGGGGCTATACTTGGTCTAA
AQKGFRAFNGCHEHNFVATT







GTATGCCCCGGTCGCTGGCC
LLDQTRRMHRRLYQVWYDLR







TCTGAGCCCGTACACA
NAFGSLPQQLMWGVLRQLGV







(SEQ ID NO: 1326)
TEEFVARCSGIYEDSYFVVG








NASDGATEPVRQEVGVYQGC








PLSPLLFITALVPLLRALEN








QDGVGVPLADGVRPCATAYA








DDIKVFCDSATGIQRCHALV








TRFLEWTGLQANPAKCAFLP








VTRSQHSNPTRDRDIELRIH








GEAIATLGLQESYRYLGVGD








GFDHVRHRLQLEPKLKQIKR








EAVALLHSELVPWQILKALK








VYIYPKVEYALRHLRPLKSQ








LQGFDSAIVRGLRHLLRLPE








NSHDGLFFSPTSAGGLGLLS








LVELHEALQVAHAWQMLHSK








DPAIRAIARTQVGQVARKRF








KLVEEHWRGREDDLAQRFLN








TELAASPHATETRRNGDIGS








LWNDVRDTLQTLGLKFAAGD








EEEAPGLLQLRVPHHTKWLS








HSTVLRHVKLHMKLRRMDTW








KSKVSQGTTVREHGGVGSRF








ITAGAGLSDAEYRFAIAPRA








HLIDTNSTLKRRRLRANDTC








RAPGCSYTEPPAHILNKCSP








NMDAIRKRHDDALERIADAL








RRKVEKSGGRLEVAINKTVP








EYDGAALRPDIVLRNTETKR








AIIADLAITHENQPTDATTS








SALQQSRDNKITKYQTVAAA








MMRAGWRVRVTGIVYGSLGS








VLPSNFKVYTELLALLKRDA








RRLNRQLSSHCIRASARIWS








AHCRRHRERQRSGNASRASR








GSGGAPRRTSQASARR








(SEQ ID NO: 1448)





NeS
Utopia
.

Phytophthora

CCAGGAATCACCCCCGCCGC
TAGACGGCACACAGGCCCAC
MSGDVVSSDGSSRTTDASGD


L
-4_PR


ramorum

CCCCAAGCGCTCCGCCGCCG
AGCGGCCGACAGGGCCACAC
GDDGAGSSDAAGDVGVVAMD






AGCCCAGCTGCGGCTGCTGC
CCAGGTAGGGAACCGCCTTC
VDQGARRQQPPWQRVGGKRR






CGCCCCACTGGTGACGGCAG
AAACCCGGCCGGTACATTAT
RLNDVDDEDTRELAELLLEE






TCGCAGCTGCCCCTGCCACC
GGTCCGACACCTATGAGGTG
EDEAGDHAPAPRLSAASARP






TCTCCTGTTGGCCGCGCCGC
CAACCGGTACACAAGTTACA
ASVLSVYAHNAQRFQCTLCT






CGTGGAGCCCCGCGCGCGCG
CACCACATAGCGACTACCAG
YTAASFASLKRHRDSRHRRT






CCGAGCCGCCCCAAGAACAA
GTATTTACTACCTGGAAGCC
AFLDRFSAGCACGVPFASRL






GCACCCCCAGCTAGCGCGCG
AAGGATTAACCGGTCGGTAA
AAANHAHACDSLNRTFSVAA






CGTGGAGCCC
TACACATAACTTT
APAAGELSPTAGAANATVKA






(SEQ ID NO: 1204)
(SEQ ID NO: 1327)
ATVTPDSPRQDPPKLAATPP








LASSALVVDPDHAEQQARER








WGPPLPRTLVAGRVAARLSE








VPAPRWGPPLPRGVVAFRIG








HRVLPPEMTSDEETKDDSSV








QDGDRQDYPVAAMDVDSGMS








GEWLLRFDGACRANPGPGGA








GAALSQPDGSVVWTCSHYMP








SSSETNNTAEYTALLLGTRA








AADHGTTTLRVEGDSTLVIQ








QVRGIFATRSVTLRHLRDQV








KLELARVGRFSLHHIDRQAN








AHADRLANRALDLRRTVSEC








GVHPDGNGCTPTAIDDRPLA








PTQQPPDAPPPPPAADIEME








DPDDEDLADIDDGEVYAAMR








VGPNATPQRRRRGRSGTAKK








HRRQRPPRVTRHHREHRLDE








ALDDLHAVERSTPSDRTTVR








RARRRVGRVNSAIEQQRLRH








RFDTDEKACVTDILAKACAT








REAARTTASGGDPPAGPATP








AAGSADDGTCPILGEELWRF








FDSVNTPRQEFAPDAPVGAA








FRSALARLPAATSCKELLTA








APSAGEVEDQLQHVRGASSP








GLDGVGYDVYQHFAAQLLPA








LTAAFKACWTAKRVPQSWKL








GVVRLLHKKGAREDPANWRP








ICLQQAIYKLYTGLLARRLV








RWLDANDRHAPGQKGFRAVN








GCGEHNFLAATLVDQARRKR








RTLFEVWYDFRNAFGSVPFA








LLWDALARLGVPDDYVTMCK








GLYESAAFVVGNAIDGTTDP








IALRVGVFQGCPLSPQLFNA








AISPLLFALQRLPATGVQLS








GDDCPGASAYADDLKIFSGT








EDGIKRQHALVADFLRWTGM








AANPNKCCTMSVQRDGRGVL








KTDDLQLDLAGTPIPALSMS








ASYTYLGIGDGFDHVRRRVE








LAPALKQLKDDATTLLQSGL








APWQVVKAVKTYLYPRVEYA








LRHLRPFQQQLEGFDRHLAR








GLRHLLRLPGNATAECFYAP








VSRGGLGLLPLTELHAALQV








AHGWQLLNSKDPAIRRIARV








QLRQIADARHRIDSRAWEGR








DEELCELLLNSQLGTSPDAP








PKRRNGDIGSLWVDVQRHLR








TLGLKFATAPACADAGSAAT








TLQLRVPHHDKWLDHRTVLR








HVKLHVKHRHWSKWAAMRDQ








GKTARAHGGAGSGFLTRPRG








MWEADYRFAVAARLNQLDTH








SVLKRRRLRAHDHCRQPGCS








RAETLAHVLNHCAGTMDAVR








GRHDDALKHIERALHASSPG








GQDRVELRVNQTVPSLAGPA








LRPDLQLYNHTKKMVAVVDL








AVAFEEQASDDPESSALARI








AAHKRAKYAGVKRHLERQGW








KVHLSALVYGSLGAVPAGNH








KVLTEHLGLLKRDAKRLDRQ








LSVACIQSSRRIWNLHCSQH








RARQHQAPGGSRAAETGGTP








PRTGRR








(SEQ ID NO: 1449)





NeS
Utopia
.

Phytophthora

CTCAAGCCTAGCAGCGGCTA
TGACGCACCGTGACATAGTG
MARVLVEPMQVDECSSCDRS


L
-5 PI


infestans

CCGCAGCTACTCCAGCTCCG
CGGCACGTGAAGATGCACAT
TLTADDGSGDDVAAPSSLNS






GTAGCTGCTACTGCTACAAC
GAAGCTCCGACACTGGGCCA
NDVAAPMDVDSGTDCPPALQ






TGCTCGCGCTGCTGCTCGCG
AGTGGGCGGCCATGCGCGAC
QPPQRPRALHVGSKRRRLDA






TCGCCGTGGAGCACCACGCG
CAAGGCAAGACAGCTCGTGC
DDEEEARQLQEEEEAGIHAP






CACGCTGAACCGAACCAAGA
ACATGGTGGGGTTGGTAGTG
ALRLSAASAQPASVLAVYTH






ACATCTACCG
GCTTCCTCACACGGCCGCGA
NASRFDCTLYAYTAGSFASL






(SEQ ID NO: 1205)
GGCCTGTGGGAAGCCGACTA
KTHRNSRHRRTAFLDRFSAG







CCGGTTCGCGGTGGCCGGCC
CACGVPFASRLAAARHAQAC







GCTTAAACCAGGTAGACACG
ASLSSAPLAEASSAAGASSH







CACAGTGTCCTCAAGCGCCG
TVDEADSTVSAAGHTEPDLP







GCGCCTCCGAGCACATGACA
RHNATELTASPPLVSSPDVE







GGTGCAGACACCCAGGATGC
VQAPETEATENRWGTPLPRV







ACGCGCTCCGAGACGCTGGC
LVASRIAGRLAQVPPPRWGP







GCATGTGCTTAACCACTGCG
PLPRTTIAGRIATRLAATPA







ACGGAACCATGGACGCAGTC
PRWDPPLPRSLVVSRIAARL







CGTGGCCGGCCATGACGCCG
LPALPDAPACEEEAKDSDTM







CACTCAAGATTATTGAGCGT
DWAPTWTNEETKDSEPHDEA







GCGCTCCTCGCATCGTCGGC
PGQVDEETIDDADGEWLLRF







CGACCAGCAGGACCGTGCTG
DGACRANPGPGGAGAALFKP







AGCTCCGCGTGAACCAGACC
SGPVVWTCSHYDPSTTATNN







GTGCCGTCACTCGCCGGCCC
TAEYTALLLGARAAADHGVT







CGCGCTACGGCCCGACCTTC
KLRVEGDSTLVIQQVRGIFA







AGCTCTACAACCACACCAAG
TRSTRLRALRNKVKLELARV







AAGACGGTGGCGGTGGTCGA
GSFSLHHIDRQANGHADRLA







CCTGGCCGTGGCGTTGAGGA
NAGLDRRRTKLECSVHPDGR







GCAGGCGAGTGACGACGCGA
GCTNTSVATAAPTAPAAPLP







GTAGCTCGGCACTGTCCCGG
PARPPATTAAPSHDDDHSVQ







ATCGCCAACCACAAGCGAGC
GDIDDGEVYAAMCIGPDAVP







CAAGTACGACCACATCAAGC
HRRPRLRLRHLTDEESEEAG







TACACCTCGAGCGCCAAGGA
DVVERLAASLAAKIADAPDW







TGGAAGGTACACCTCTCGGC
ETAEGYITALPYALYDKLQP







ACTCGTGTACGGGTCGCTTG
YSQAQPQPPSQQQQQQQQRP







GGGCGGTCGCTAGTGGCAAC
RQQQQTRQRRQRRGKRGGGS







TACCAGGTGTACACCACACA
QRRQRKTRRRRPPRVTRHHR







CCTGGGGCTACTCAAGCGCG
EHRIDEALDDLHAIESRRPQ







ATGCAAAGCGGCTGGACCGG
DRTAISKARRRVGRIRSALD







CAGCTGTCTGCCTAATGCAT
QHQLRHRFDTDEKVCVDAIL







CCAGTCCAGCCGCCGCATCT
AGARASQGATTAPPSATTDP







AGAATCTACACTGCAGCCAG
PAPMDDSRCPIPGDDLWRFF







CACCGGACTCGCCAACACCA
DSVNTPRRSFDAEAPDGAAF







GGCGAGGCCCAGCCAAGGAC
REAMACLPAATRAQELLTEA







CAAGAGGCAGCCGGGCGACG
PTVDEVEDQIQHARASSSPG







GAGACCGGGGGGACTCCGTC
LDGVGYDIYKQFAAHLLPAL







CCAGACAAGCCGCCGCTAGG
HTAFVCCWNHKRVPQSWKLG







CGGAAACCAGGCCCAAGACG
VVRLLHKKGDRQDPANWRPI







GCCGACAGGGCCCCACCCAG
CLQQTIYKLYAGILSRRFVR







GTAGGGAACCGCCCTAGAAA
WLDANARHAEAQKGFRAMNG







CCCATTTCGGTGGTCGACTC
CGEHNFLAATLVDHARRKRK







GCAAGCCTTACCTATATTTT
ELHVVWYDLANAFGSVPHDL







AGACGTAGCGACCAAATTAC
LWETLARQGVPPTFVDCCRG







AAATTTGGTAACGAGTAAGC
IYSDAAFTIGNAADGTTAPI







CAAATGGTAATACACAAAAC
RLRVGVFQGCPLSPHLFTAA







TTT
IAPLLHALKRLPVTGVQLTG







(SEQ ID NO: 1328)
VDRPGAAAYADDLKTFSSSV








DGIKRQHELVATFLRWTGMA








ANLSKCSAMSVQRDSRGVLK








TGDLCLKLNDAEIPALSMTA








SYAYLGIGDGFDHVRRRLEL








APMMKQLKHDATALMQSGLA








SWQVVKAVKVYLYPRIEYAL








RHLRPFKQQLEAFDEHLRRG








LRHLLRLPTNATSAFFSAPT








SRGGLGLLPLTELHAALQIA








HGWQILNSPDGATQRIAREQ








LREIPDARHRLDTAHWRNRD








AELCELLLNSQLGQSSHAPP








KRRNCDIGSLWIDIRRQLGT








LGLKFETAPGRRSHQPARPA








IAAFACRTTTSG








(SEQ ID NO: 1450)





NeS
Utopia
.

Phytophthora

CCAGGCATCACCCCCGCCGC
TGATGCCCGCCAACTACAAG
MSGDWSSDGSSRTTDVSGDG


L
-5_PR


ramorum

CCCCAAGCGCTCCGCCGCCG
GTGCTTACTGAGCACCTTGG
DDGADGAGSSDAAGDVGVVA






AGCGCAGCTGCGGCTGCTGC
GCTGCTCAAGCGTGATGCGA
MDVDQGARRQRPPWQRVGGK






CGCCCCACTGGTGACGGCAG
AGCGGCTGGACCGGCAGCTG
RRRLNDVDDEDTRELAELLL






TCGCAGCTGCCCCTGCCACC
TCGGTGGCGTGCATCCAGTC
EEEDEVGAQAPALRLFAASA






TCTCCTGTTGGCCGCGCCGC
CAGCCGCCGCATCTGGAACC
HPASVLSVYAHNAQRFVCTL






CGTGGAGCCCCGCGCGCGCG
TGCACTGCGCGCAGCATCGA
CAYTAASFASLKRHRDSRHR






CCGAGCCGCCCCAAGAACAA
GCACGGCAGCACCAGGGCCA
RVSFVDKFSAGCACGTPFGS






GCACCCCCAGCTAGCGCGCG
AGCGCCAAGGGGCAGTCGGG
RLAAARHAQACASLSIPRTV






CGCGGAGCCC
CGGCGGAGACCGGGGGGACT
TAPAAAGDLSPTATGANATA






(SEQ ID NO: 1206)
CCGCCACAGAGCGGCCGCCG
SAAATSPDLPRPASPELAAS







CTAAGCGGACATCGGGCCCG
PPQTSPFDVAIQADAAEQTA







TAGCGGCCGACAGGGCCACA
WTRWDPPLTRAAVAARVASR







CCCATGTAGGGAACCGCCCT
LAWPAPRWGPPLSRTLVASR







CTAAACCCGCCCGGTACATT
IAARLDAQTSRWGPPLPPAM







ATGGTCCGACACCTATGAGG
VASRIASRLAAMPAPRWGPP







TGCAACCGGTACACAAGTTA
LLRTVIASRIADRLLPPELA







CACACCACATAGCGACTACC
ADEETKDDDVHMDNAASVDV







AGGTATTTACTACCTGGAAG
DEESEVADVVMTDHDGEWLL







CCAAGGATTAACCGGTCGGT
RFDGACRANPGPGGAGAALF







AATACACATAACTTT
KPSGPWWACSRYMPSSSATN







(SEQ ID NO: 1329)
NTAEYTALLLGARAAADHGA








THLRVEGDSTLVIQQVRGIF








AARSTRMRALRNQVQSELAR








VGSFSLHHIDRQDNAHADRL








ANRALDLRRTVIECGIHCDG








VGCTATTTEVQSSSAPEIPT








RPVADDHDEHEWDVVDVCGV








CGVCGDRGTCGVCDVSGDI








DDGEVYAAMRTGPDAVPARR








PRLRLRKLTDEEQEEAGTLA








ERLGATLAAKIADARDWESA








EGYITALPYLLYDKLLPYSQ








GPARSLPVRQHQRQQQQPDG








QFQRPTQSRSAARRQRRQRH








RARRRPPRVTRHHREHRLDE








ALDDLHAVERATPSDRRSIR








RARRRVGRVNSAVEQQRLRH








HFDTNEKGCVEILLAKARAQ








RSTTVARTAVGEPNSGAAED








DGTCPIPSERLHRHFTEVNT








PGSSFDAMAPVGAPFRAALA








HLPAATEASELLTEAPTPDE








IEDQLQRAKGTTSPGLDGVG








YDVYKAFSTQLLPVLHAAFQ








CCWQHHRVPQSRKQGIVRLL








YKKGPREDPANWRPICLQQV








IYKTYAGVLARRFTRWLAAN








GRHADAQKGFRTVNGCGEHN








FLASTLIDHARRSRRELHMV








WYDLKNAFGSVPQELLWEVL








QRMGVPPAFVEVCQGLYQDA








AFTVGNAADGPTDLVRQLVG








VFQGCPLSPHLFTAAISPLL








HALDRLKDTGVRLSADDRPG








ASAYADDLKIFSGTADGVKR








QHALVADFLRWTGMVANPNK








CCTMSVQRDGRGVLKACDLE








LQLDGARIPSLIMNASYAYL








GTGDGFDHVRRRIELVPALM








QLKDDATALLQSGLAPCQVV








KAVKTYLFPRVEYALRHLRP








FQQQLEGFNRHLVCGLRHLL








RLPVSATTSFFFVPVSRGGL








GLLPLTELHAAPADRRPAPP








PRPRPLEGAGGENMRAADQL








AARDVGPRPTQAPQRRHRLV








VGRRPAPPPRTRPQARDRAG








VRGDRHRGGDAAASRAAPRE








VAGPPHGPAAGEAAHEEQAL








AAVGRDEGPRQDRPHPWWCR








ERLPHAASRPMGDRLPLCSG








GSAQPAGHAQRAEAPAPPRA








RPVSTAGLLPCRDTGTRAES








LRRHHGRGPRPPRRCAQDHR








ARAHRVVRAARTAPSSGSTR








PCPRSPAPRCGPTSSSSTTP








RRRWRWSTWPWRSRSRRATT








PRALRWRASPRTSEPSTPAS








SGTSSAKGGRSTSRRSCTAR








WAR








(SEQ ID NO: 1451)





NeS
YUREC
.

Ciona

ATCAACCCACTACTACACCC
TAGCCAAAAGATTTGGTGTT
MATSSSSVSSGNVQTEVRCV


L
i


intestinalis

TCTACAAAGAACCCACTACA
CTTGGCGAGCTGCAGTCCCG
YHGKGDLLLECPVAHCPSIH






GCAAGATCTTGGCGACCAAC
GCAGAGTAGGCGAGATCGTG
PTVATITKHLKKHHTPQFEQ






TACACCTGCAGCCTCCCGTC
AGCTCTACACCGCTTCACGA
ITTKNLTITYTCSQCSFSTT






GACTCTTCACCACCTGCGCA
TTTGACGAGTTCTTCTTGAG
GLTQHHISKHYKTCKGVGAV






CCTCCCTCCGACTCCAAGCG
TCGATCCCCAACGGCAACGC
QEGNKGRFCCPACGTRWALL






GCAGACAGTGGCACCACCAG
ACTCCAAATTTAACGACCGC
CKARHHFNNVHFEYDTPPIA






CACTCCAAGGGTCCACAGCC
ATGGCGACGCCACTGATCTC
AFSGTPYKLKKRKFTIINKA






AGAAGAGTGCACCCCCCACT
CCAGTCATCCGTGCTCCGAG
LTYSCPLPLNQLLCPLWSCS






GCTAGGAAGAGCTAGGCCAT
GGCACTTAAGGGGAACCGAT
LTILNKPLSSVQQETAHGDG






TGCCCGCTGTGGGCCTTGTG
TCACTGATGCTGCAAGTTGG
SQGQSYVPTQLRQVLRARCH






CTGGCTGTCTGCCGCTGATT
CGGGGGCGCACTCACCAATG
CGNPPIGKGHWASCQGKRPL






GGCACGTTAGGGAGTGTGCG
GAAGATGAGGGAGCCCAACG
SSPKGGRSSPTPPANLTLHF






CTGAGCACGATAGGGAGGCT
ATGCCCCGAACTACCGCACC
LNYLPFQLPSQSSSPQSSTL






GCTGAAGCCGAAGAACCCGC
GTCCCCAGCACCGTGTCCTT
DPTACKARVPIPSFLRGDCE






GCCGGGGACTTTAGGACTTT
GTGAGTAATTATTTTCACTA
VTFFIIPSVNFYRPYLSYPL






AACTAAAATCTGTAACACTA
ATCCAGGGACGGGGCCATGA
RMFWRNRTSSGHCSLHRWRG






AAATTGGAATACTGGATTAC
ACTGAACTATGTCTGCACGC
FVRERLVPHPRSKSPARTPL






TATTGGATTACTACTGGACA
TGCCCCGCCTAGCCTCGGCC
EFLCEFRLAGVFPDPGKVAS






ATAACCTGATCTACAACAAC
ACAAATAAATAGTCAAGCCG
LRPVPAPLTLCLSPPVAGPM






CAATTGGATTACAGAACCAA
GGGCGTACTACCAATTGATT
ISCEDHSAPPSVRSSSPIPN






ATTACAATCTCAACTACAAA
CGGATTTGGCGGGGCCCATC
SPASVSSVEAHLSDLLDKVS






CAAAAAGTGAGTCCCCGGCC
ACGCGCCTCCGCCCCCACAC
SGELRPLSPTLPSSGFFGPL






GGGTTTCTATATTACCCAAT
ACAAAAACACTTTTAAACTC
LPPTPPPRPTPSAEKASPSG






TACTGTTTTTACAATTTTAA
TTCGGTTCCCCAACCACTAC
LSYLPCREVKIASIARPSPA






AATTTTATAGTATTTTACTA
AACAAAGCGAGCGGCCCCTC
SQRVGCDADRTGPSLNPNYQ






ATTTTTCCGCCCCGCTAGCA
TTAGATCCAATTTTAAAATT
QTSPPSTPSFSPIVRPPKFP






CTTAAATTGGCACCCCCCCC
TTAAATCAGTGCACTCAACT
RSGAKVNSKSKPPGVRPRRA






CCCCSTCCAAAAAAAAATAG
TTTTTACGTGTTGTGTTTTI
KPIEPGTESASPVDVDTISS






ATAACCCTCCACCACCTAAA
IGTATTTTTTCCCACAGATT
SVQEPCTPENRTPEFFYERK






CCCCGGTCACCCCCTCAGAA
TTGTATTTTTATATTATATT
WLVSILNIHEREGSNFFQFN






CTAATTGGAACCCGGATCTT
TTATATACACAACACTAITT
RDLEYWTQLLSGSQKGGRAK






TTCCCTCTATCAAATTTCCG
TIATACACTACCTTGCACTG
RASYNRGAANQAMKNRDSGR






GTTCAGTTTGGCTTAATTAC
TCCCACITTTTGTAATTATC
KDFDPRPVAGHSSGGGTELG






CTAAAACCCGTCTTTATTTT
ACCTTTTACCTTTTATGCCG
SRPRYPKGARLRADFWRDMK






ATCCCTTTATTGCCCCTTAA
CTCGCTGAGCTCCTTGCACG
GTVRKLLDGSNGERRCGIPL






ACCAAATTATTCAAATTGAA
GATGACCAGACAAACTTTTA
DIIERKFRQVSMPGWIDHRR






ATCCGGCCAAATTGCCTTGC
TAAAATTATAACATTGTTTT
YAAGASPSLVTQAETDVAIT






TGAACCACCATTTTTGTTTT
AATTGTCGCGGGGTATCAGT
SEEVEAVLSGLNVQSAPGSD






TCTTATAAAAGTAAATTTTT
GGCGCCCCCTTGCGGCCGTG
GLSYRFWKGLDPSGRLLSCL






CAACAACCCAAAATCATAAA
AAAGCCCTTTTCACCTTAAT
FEIVRRHGRIPGAWPTCSVI






ATTAAAACTGTTATCTGATT
TCGCCCCTTAGTCCAAATTT
LLCKDAQGDVQDVGNWRPIT






AGTCTAAATTCTGTTATTGA
TTCCCCATTGCCTGTAAAAG
ICRTLYKLYAAVIARRIQTW






CAAAAACCTCCATACCAACC
TGCGTTGCCGTCGACTCGAA
AKQGGVLSRLQKGFMPVEGV






TTAATCTTATCATTATCTTC
CTCGTCCTTTTTACCGCCTC
FEHVFMLDTVLSDAKLRRKN






ACCCAGACTGCTCAATCTAG
TCTGTTACTGAACTATGACC
LLAVFLDVRNAFGSVRHECL






TTCTTTTTTCAATCAATAAG
AGCTTGGCTGTTGAAGTCGG
LKVLRHFDAPHYLVELVRDI






GAGGTGTTTTCTTTGGTCTG
CTTTAATTCGCCGGCTTCAC
YTGATCRVRSSVGETGDIPC






TTGTTTTATAGTTTACCGTT
ATTATATTTTTTTGTTGGGT
DRGVRQGYPLSGILFNLVTE






ATAATAGTCTGTTGTCCTAT
AATCTGTTTTTTTTCATATA
VLIPGLSAGNDGYRMACLGG






AGTTTTCCGTGGTGTATTTC
TAAAACCATTCGGCTTAAAA
KLTQVLAYADDLVWTENRDQ






TGGTCTACTATAGTTGGTGC
TCACCAATCCCCATCATCCA
MLRQLGVCEEFGRWAGLAFN






CTCTAAAACATTATTATACC
CCCTGGTCACCCATTGAAAC
QRKCGLIGWRTLRGGRRVAL






GAAGAGTGCTAGCGATTTCT
ATCTCTTTTTAGTCAATTAT
EDPLLLNGVEIPLLRPGEHY






ATTATTAACTCACCCACCAG
TTTTTCAGATTACCCCTCCC
KYLGAMTGVMSVPRTGSQLI






TACTGCTGTGCATCCACGCA
AAATTGTCATATAGTTTAAC
KDFRARLQRLFTSFLTPHQK






GCACCACGCCTGCCATCACG
ACCCCGTTTGCCAAGTTGGT
LIALKRFLLPSLSFHLRVRP






TACAGTGCGCTTGCTGCCTT
CTTTTCCCCATCCCCTTCTG
IARSELIALDRRVRECLRVA






GTGTTTTGTTTTGCAGACAC
TCCTTCCGGTAATCCCATAA
FRLTKPSCQAVFHTPTDMDG






CACTGGACGATAG
CATTTTATTCTTTAATTCGG
LGVPSVCSESSILTIAQGFK






(SEQ ID NO: 1207)
CCCCATAAATAGTGTAATCT
VLTSPDGTVSATASARVKLY







TAAGACGAAGTCCCCAGAGG
AAKFGGLTEAGPSDWARYLS







CCCGGTCCCCCCCTTCTTTG
GDDVNGNSTRKPGANLPSGL







TCATGGACCGGGCCAGCCCC
WTRVRCASRQLGAVWRVCPE







CCTGTTACCACAACAACCCA
NGITVRVRNSVITSRDRRKL







CCATTTTATTCTTTCTTTCT
IRSFHDCSNQQWKEQWMQHP







TTTTATTAGTATTTATTTAT
NQEKTAAAHMAYADANRWVK







ATTGCCGCCCAACTGTCGTG
QPSVMEPHTYFFALRARLNL







TCGTGGCGCAGGGGGGTTCC
LPTRVSRAIYSRDQHPDILC







CTCTGTCGGCCCAGTGGACG
RRCGASVESLPHVLNHCPPN







ACCGTCTAAAAAACAGGCAC
MSIILGRHNLVLQEVLNAVD







AGGCAGCAGGCACTTATCAC
KTQFKEISVDRTVPEHMSET







CCGGTACCTCCGGGTACCGG
GEALRPDIVARRNDGSWVVD







AGGCTGGTTTTCAGCGCACT
VACPFDQKANFDEAAKRKLL







GTACGTGATGGCCAATTTTA
KYDKLCCNIAASTGKPVECH







TTCATTGCATTTTATCCGCG
SIWGSLGSLAEGLSTSLRAL







TCGTGGTGTTTGCGTGGATG
GITDFARSKLVACHQG







CATTAATAAAAGATGAAATT
(SEQ ID NO: 1452)







CC








(SEQ ID NO: 1330)






R2
PERER
BN000

Schistosoma

ATCTCACGTTTTAATTTATT
TAACGGCTGAACGAATAGCC
MPVSTGAETDITSSLPIPAS



E-9
800

mansoni

TTTGAACTACTGCAGTCTGA
CCCTTCACTCTTAGACATTC
SIVSPNYTLPDSSSTCLICF






GTGCTTCTAACGACCCGAAG
CCCCACTGTTGTTGCTTATC
AIFPTHNILLSHATAIHHIS






GCTCAGAAACTACCCACTTC
TTCATGCTCTTGTGTTAATT
CPPTPVQDGSQQMSCVLCAA






TTGAACTGCTACTTTTTGCT
GACTGCTCTCTTCTGGGTTG
AFSSNRGLTQHIRHRHISEY






GTTTATCCACAACAACAGTT
ACGTCTGATTGTCTCTCTCT
NELIRQRIAVQPTSRIWSPF






GTGATTCTATTCTCCANATA
CTTTCCATATTGCTTGCTCT
DDASLLSIANHEAHRFPTKN






TTCCTTGTGCTTTTGTCAAC
GCCCGCTTACTTCCAATAGT
DLCQHISTILTRRTAEAVKR






ATTATTCTATACCAACTGTA
TGTCATATTATGTCTTTGTT
RLLHLQWSRSPTAITTSSNN






CCACCTACTTCTTCATCTCA
TACTTGCCATGTCTAACGAC
HTITDIPNTEARYIFPVDLD






CGTTTTAATTCTGGTCTTAT
AATTACTTTATCTACCTTAG
EHPPLSDATTPNASTHPLPE






TTTCTCATCATTAGTCACGG
TTTGTCCTCTTGGTTTCGAT
LLVILTPLPSPTRLQNISES






AGAGGGCCTATGAACGGTCC
TGCCTTCATATGTTCATGGC
QTSHESNKNSMHTPPTYACD






GTGACGCGAAATTCTATCCG
GGAATCTGATGTTTATAATG
PDETLGATPSSTIPSCFHSY






CGATTTCGACCTCTCCTGCT
ACTATTCCTATTACCACCAC
QDPLAEQRGKLLRASASLLQ






AGTGGTCCCCGAAGTACGGT
TACAACTACTATTATTATTT
SSCTRIRSSSLLAFLQNEST






TCCTCTGGCCTGTCAGTTGT
TCATTACTATTAACATTATT
LMDEEHVSTFLNSHAEFVFP






GTTAAAACTATATAATAACG
ATAAACATTATTACTATTAT
RTWTPSRPKHPSHAPANVSR






(SEQ ID NO: 1208)
TATTATTACTATTATTACTT
KKRRKIEYAHIQRLFHHRPK







CTACAATTAATATTATGGCT
DASNTVLDGRWRNPYVANHS







ACTCCTCTCAGCACACCAAT
MIPDFDCFWTTVFTKTNSPD







AAAATATCAATCAAACATCT
SREITPIIPMTPSLIDPILP







CAATTATATCCACCTATTAA
SDVTWALKEMHGTAGGIDRL







ACTCTCTCTATTTCCCCTGA
TSYDLMRFGKNGLAGYLNML







GTTATAAACTTACAATTCAG
LALAYLPTNLSTARVTFVPK







TCTAACCGAATATCTCTCTT
SSSPVSPEDFRPISVAPVAT







TTACAAATCTTAAGTATGTA
RCLHKILAKRWMPLFPQERL







ATTTTGTGCCAAACCCATTT
QFAFLNRDGCFEAVNLLHSV







GGGTCTGTACAATTTGATAC
IRHVHTRHTGASFALLDISR







TTAAAAATAAATGTTATTAG
AFDTVSHDSIIRAAKRYGAP







CC
ELLCRYLNNYYRRSTSCVNR







(SEQ ID NO: 1331)
TELHPTCGVKQGDPLSPLLF








IMVLDEVLEGLDPMTHLTVD








GESLNYIAYADDLVVFAPNA








ELLQRKLDRISILLHEAGWS








VNPEKSRTLDLISGGHSKIT








ALSQTEFTIAGMRIPPLSAA








DTFDYLGIKFNFKGRCPVAH








IDLLNNYLTEISCAPLKPQQ








RMKILKDNLLPRLLYPLTLG








IVHLKTLKSMDRNIHTAIRK








WLRLPSDTPLAYFHSPVAAG








GLGILHLSSSVPFHRRKRLE








TLLSSPNRLLHKLPTSPTLA








SYSHLSQLPVRIGHETVTSR








EEASNSWVRRLHSSCDGKGL








LLAPLSTESHAWLRYPQSIF








PSVYINAVKLRGGLLSTKVR








RSRGGRVTNGLNCRGGCAHH








ETIHHILQHCALTHDIRCKR








HNELCNLVAKKLRRQKIHFL








QEPCIPLEKTYCKPDFIIIR








DSIAYVLDVTVSDDGNTHAS








RLLKISKYGNERTVASIKRF








LTSSGYIITSVRQTPVVLTF








RGILDRASSQSLRRLCFSSR








DLGDLCLSAIQGSIKIYNTY








MRGT








(SEQ ID NO: 1453)





R2
R2-
.

Bombus

TCAATAGTTACTCGGGGGCA
TGAAAACACGATAACGATTA
IAKFDNNTNSASDAAPLSPG



1_BTe


terrestris

GGCGGGATATTGGTCTTGCC
TGAATCAAAATAAGAAAGTA
GAVADLSASEGTTDNDQAMS






TTGCCCAAGTCACACTCCTA
AACATCCCAGAAATTGTCTA
PAMSLXTVPLVGNRVACPXC






CCTCCTCGTGGTACCGCCGG
CGTCTTATTTGTTATCTATT
EKREANLFFLNLSDLDRHLT






TAACACGCGCACGTCCACAT
TATTTGTTTA
QHHPDAPIXWSCIDCAKCFP






CAGCGAGGGGCGTACTCCCC
(SEQ ID NO: 1332)
KLHGARCHIPKCGGASSQAR






CGGATGTGGCGGCGCGTGGC

TGEFQCEACPMSFGSRRGLS






TAAACGGAGTGTGGCGACGA

THERHAHPAVRNIKRRGADP






AGGAGCGAAAGACTAACAAC

PEENTKSWKVEXVARLKGLW






TATAACGGTCTTCCGTAACG

EIFKDHKYPNKEISKFLTTK






GCTACTTGGAGCCGTGAATA

TVDXXKYQRKKLNLIGXESP






ATGGAGCCTATATTAAACCC

QEATSLATEGGCDLVSSGNA






TGGAACTCGTTCCTTCGTTC

SFGSPVGRNENEEELIHEWK






TGTTGACGACTGGAACGGCA

LSLKNEINKPTEVPPILKEV






AAGGACATGATTTGGATAAC

YNRLMLIWEEHQDDRDSLTE






AATTGGAAACTTAATATCGA

SLDHFIRTALYELINKINKN






ATTCACAATTAA

QTDLKTKRAAKTKSPKNNRN






(SEQ ID NO: 1209)

SRKRFSYARCQELFHECPRR








LADAVVNNDQAYLEPARQPP








GSEEVRGLYEKLWGQVGSTY








VPAPVTRVPKLSLSEIFPPI








AAEDVGERIGKIRKKAAAGP








DGLQRDHLTIPGLPIIMAKI








YNILVYCSYFPSAWKENRTT








LIPKINKPCSLVENWRPITI








SPILGRIFSSIIDGRIRRGT








VLNMRQKGFTSENGCKINIE








LLNSALNYSKRNSGGIFTIV








DISKVFDTVPHAALKPCLAK








KGVPALIVDLIDEMYKNVKT








TIKTKDGGVEIMIRRGVKQG








DPLSPLLFNLCLEPLLEEIE








EQASGINVSEHRKVSVLAFA








DDIVLLGADAREAQHQINVL








TDYLQSLMMNLSIEKCQTFE








VVAKKDTWFIKEPGLKIGNQ








IMPTVDPDEAFKYLGAKIGP








WKGVHCGVIVPELLSVVKRV








RKLSLKPGQKVELLTKYIFP








RYIYHLLVSPPSDTVLKLLD








SEVRQEVKTILHLVPSTATG








FFYTPKACGGIGIPRFEHII








KLGTLKSAIKIANSIDPAVA








GLIDDAAIKKLKQTANSLRI








NWPASLEDIEKARKRLRKEH








ISQWADLKCQGQGVPDFIKN








KTGNLWLEDHSLLKPSRLID








ALRLRTNTFGTRSVLARADK








NIDVTCRRCRAQPETLGHIL








GLCQHTKGLRIKRHDEVKSL








LEGRLKSKKNNEVFVEPTIK








AGGSLFKPDLVIKNGERVLV








VDVTVRYENKNYLALAEKEK








IEKYRPCLRALKEIFNAKGG








EILPVVLGSRGTITPNTEKV








LKRLGIANNDIKTILLNVLR








SSIELCNIFIDD








(SEQ ID NO: 1454)





R2
R2-
.

Crocodylus

AGGCGTCTCCTTTAAGGGCA
TGAATCCCACTCTGGGGACC
VPPGAEARGRYHHPRXEXAR



1_Crp


porosus

ACGGTCTGGTTACGCGGTCG
CCCAAAAATTAGAAAAACCC
QGEPPSXRVFLVXLPDSNPP






CAGCAGKCTTGCKCCAGGTA
AAAACAGTTGTGTTTAAGTG
CPICGDHVXXXSVLALHCVE






CCTCCWCGTGGTTCCCGCCG
TGTTCTTGTTTGTCCCTTTG
GHXWAXVQYQCTHCGILCHI






GGTGCCGMAGMCCCAGGSCT
GCTTCACCTCCAAGTTGCGA
PRCQGRVXEXTGKDXXCPEC






GTCGGTAGCTCGATCCTGGC
TCCCCCATCTCCCCTGCGCT
PASFDEKAGLSQHKRHTVTX






ACAGTAMGGCCAGGGGAGTK
GTCTTTCTGAATGACCAGTG
SXERVAGXLLRAXLRHGCWS






CTTCCTTGCTGCWGGTGCCC
GTGTTGAGGCTGGTGTGACC
VEEEETLTRLDAMFXGARNI






CACAAGCGTKTGGCAGSMCM
TCGGTCACCTCCAAGGCCAA
NQLIAAEXVSKMXKQISDKW






CCTGCTTCWTCGCAAMAATM
GTGCCCTGGCCCCGAGTAGG
RXLXLXPEQTTXGGXAESAS






ASAGKGTSMTCAGTAGTCGG
ACCAGGTGGCCCAGCTTGCT
VVXXESMTPEMEAQSPAXPP






CCCCGCCGCTAGCCAAAACT
GGGCACCCGTCACCGCCCAG
GKIRKIFTGQDGHAGGXAWE






GTTCGCCACSCAGTTACAGA
GGCAAATGGAAGGGATCTAT
NQEDFHWTRWARRWLKRGQX






TGGTGCCTGTGCTGACCKGK
CCTGACCACTACCAGGCTAA
LSDKVQEVLGXWVEGQPRIX






KGCCCCGTGGGCTCGGWGGT
GTGTGGTGCTGCCTAGCCTG
AWVDXVSLDVLTLFLGVPPG






GCAGGGGWGGCCGGCCCATG
CCGTAAGGTCAAGCGCCCTG
PQRAPSKKGPXEGGKPTSWM






GCTGGGCCAGACSTGGGCCK
CTGCCGCTCGGGTAKCAGTC
NKRAIKWGTFLRYQHLFGAN






TGGAGCCCGCTCCCAMCCCA
CTCGTTCACTCGTCCCTCCT
RKLLAAXILDGAXRNQXTLL






GAGTTCCCCCTCAAMGCCGC
AGTACCCTCCGCMTCTGCGC
LEEVXQXYXGKWEAEPPFEG






CAGGKCAGMAMCAKCCGGGG
TTCTGCTATCCACTGTGGCC
LGRFGXXRDVDSFAFEALIT






AGKGMCACGGCCCCGGCCGT
GGWGATGCCGAGGWTGSWMC
XEEAVKHMMXMAXXSAPGPD






GA
AWCCTCGACACCTCCAGGGC
KLTLRDLRRADPEGDALAEL






(SEQ ID NO: 1210)
CAAGCGCCTTGGCCMCAGGT
FSLWXITGVVPDRLKEXQXV







AGGACCTGGCACCTGCCCAG
LIPKAVDFEKLRQLGNWRPI







GGGGCCAGACACTGCCTGCG
TIXSIVLQLXSRVLTARLTA







GCAAGGGAAAGGGAGCCGTC
ACPIXPHQQGFISAPXCAEN







CCTGACCGTTACCGGGCTTG
LKXPELIXRKVKXDRRPLGV







ATGGTGCTGGCTAGCCCGCC
AFVDXARAFDSVSHDXISWV







GTATGTCAAGCACTCCACAG
LKAKGVDQHIVNLIEDSYQK







CTGCTCGAGTTCGCTGGCTT
VTMRVQVFSGSTPPISIKXG







CACCTTCATCCCWCCTAGTG
VKQGDPMSPLLFNIAMDPLI







CCTTCCGCCTCTGCGCTATT
XKLKTVRQGVKVGSASLTTL







TTCGTCCCGACTCGTACCTC
AFADGLXLLXDSWEGMQHNI







CCCACCTCTGCGCTACMGCT
TTSXTPGRACNTTSRHPRGL







ATCCCTGMAAGGACCAAGTG
LQPHGPTSATXKMXGVLLES







GGCAGGGGSGTTCGCCCCCC
XMRLLYGEQLRGLEDXRPXX







GCCCGCAGGAGGCTCGGCGT
HDAXARRADTISGLEXRSLG







ATCCGTGGCKTCWTGCCTCC
WDXQTRFGYATXLLAREDGD







ACCGTCTTGTGCCGCTAGAG
CXAQTNAEALCWXSXPFPGC







GGGTACCTCMGAGACCGGCG
AXRPXYANXGWVASEALDSM







CAACACGACCTTGACGSTTA
SRRXVKEWFHLPACTDXLLX







GACAGTAGGGTGMAACAKCC
SRHRDGGLGLLRLARXXLAA







CTGCTGCAGGCSTGAAGGGC
XVRRPIRVATSSDEVTRKVS







CAAACGGCTGTGCCATGAGA
YACGISDEVERLXLAXGGDX







GGGGAACCTTGAAGACCGGG
SNVPRFEDPXAPKSXXVQGP







SCAGTCAGCCAGTTAGTCAG
HEAAQETPRVVRTQAIPWPS







TTGGGCGAAACAATCCCAGC
NWRAEEHSKWAQLSCQGERV







TGCAGGCCCAMMAGGGCTGT
ELFCNDPVSNGWINSRGQLA







CAGGTGAGGGGGTATCCCCA
ERLWIMALKLRSNIYPTREF







WCCACCCCCCGCCGCCGACT
LGRGQAGTNIGCRHCTHPRE







ACGGAGGCAKGAAGTCCCTA
TLGHILGICPAMQEARILRH







GTGACTTCKGACCCCCACGT
NKLCKILAAEGKNCEWTVYY







CTTGTGCCGGGAGAGGGGAA
EPHLHNAAGELRKPDLIFVR







CCTTGAAGATCGGGGCAAGC
DGTALVVDITVWYEGGPATL







CGCACTTGATAGTTAGCCAG
LSTTAEKATKYLDLNTQIQE







TCKGGTGAAACAATCCCAGC
LTGAEQVTYFGFPIGARGKW







TGCGGGTCCGAAAGGGCCGA
HADNWRVLSELGLSNSRKER







CTWCCAGGCGAGGGGGGCCT
VTRLLSWRALLGSVDMVNIF







GCGGAAAMCCCCCTCCATGG
VSKHRQENLLDEHCTPAEQV







TACGGAGKTCTGGCGTCCTM
VSSYAS







ACCGACWCCTTGCCACCAAC
(SEQ ID NO: 1455)







GTCTTGTGCCGGGAGAGGGG








AACCTTGAAGATCGGGSCAA








GCTGCACTTGATGGTTAGTC








AGTCGGGTGAAATAATCCCA








GCWGCCCCCGCTGTGACTGC








TAAGMCWGGTCCCCAAGGGG








CATGAGGCATSTGCGCTGAG








CCGGSAGGGGTGACACMCGG








CGATCGGCGCAGCACAKAST








GAAGGGAGGCACTTGCTGAG








ACTGCTTCTGAGGCCCCAGA








CTTGGGGTGGTGCAGCCTTG








TCTGGGGTATGGTACAGCAC








CCTACTGCTCCCTTTGGKCA








GCAGAATTCGTCCCGACCTC








TTACCCACCCGAGTCTGCGC








CTTGTTCCGCTATCCTGCAT








CTCCGATCCACCTCGCTGTC








TCCCCGCTGCGCTGCTTTTC








TCTCAAGTGGGTTAAATCTT








GTCATGATTACCTCCCACGT








TTCCGCTCAAGGGCAATGCC








CAAMATGACGGGGATCGCTG








GTGCATGGCAGTCATGAGAC








CATCCGGACCCTCCGGTGGT








CGCTATAGTCATTTTKTGTT








GCATGGGGCATSCTGAGTCA








CTTAACCGAAAGACTCWAAA








TAACTCAAAAGAGGKAMCCT








CTGSGGTTCGGTAAA








(SEQ ID NO: 1333)






R2
R2-
.

Drosophila

GAAGCTGGGTCGGATGAGCG
TAGATGTACTAACCTCTAGC
FERRSNSWGYRPLEPRSVGT



1_DWi


willistoni

CAGAAGGGGTGTTCTTTGGA
TTTTCTTATACTTTTGCCTG
ESNNNSPRSNITITSATSRP






ACACTGTAATTCATAAGTCG
CTACCTTGGCATTACATCTA
GDQPREAIAVVNLAGEIPCA






TAAGTCTGATCAAGTCGACT
AAAAGGTACAAACATCGCAT
VCGRLFNTRRGLGVHMSHQH






CGAAACCTCCTCGTGGTGTT
TGTCATAAAGAGGTGGTTTT
KDELDTQRQREDVKLRWSEE






TCCTGGGTGCTGTTGAGTTC
AGTACGTAGGCGCTGTGGGA
EAWMMARKEVELEASGNLRF






CTAGTCTCTAGGTTCTTTTC
CTTCATTGTCCCGGTGATGC
PNKKLAEVFTHRSSEAIKCF






AGTAGCTAA
AGTGAATCGTGCATACGAGA
RKRGEYKAKLEQIRGQSTPT






(SEQ ID NO: 1211)
TTGTCCAGTAGTTGGTTGCT
PEALDSITSQPRPSLLERNH







CGTATCTTTAGAAGATTTCC
QVSSSEAQPINPSEEQSNWE







TTCCTCGGCGATCAAAAAAA
IMRILQGYRPVECSPRWRAQ







AAAAAAAAAAAAAAAA
VLQTIVDRAQAVGKETTLQC







(SEQ ID NO: 1334)
LSNYLLEVFPLPNEPHTIGR








SNLRRPRTRRQLRQQEYAQV








QRRWDKNTGRCIKSLLDGTD








ESVMPNQEIMEPYWKQVMTN








PSTCSCDNTRFRMEHSLETV








WSAITPRDLRENKLKLSSAP








GPDGITPRTARSVPLGIMLR








IMNLILWCGKIPFSTRLART








IFIPKTVTANRPQDFRPITV








PSVLVRQLNAVLASRLASKV








NWDPRQRGFLPTDGCADNAT








LVDLILREHHKRWKSCYLAT








VDVSKAFDSVSHQAIIKTLQ








AYGAPTNFVSFIEEQYKGGG








TSLNGAGWSSEVFIPARGVK








QGDPLSPLLFNLIIDRLLRS








YPREIGAKVGNTMTSAAAFA








DDLVLFAETPMGLQTLLDTT








VGFLASVGLSLNADKCFTVS








IKGQAKQKCTVVERRSFCVG








ERECPSLKRTEEWKYLGIRF








TADGRARYSPADDLGPKLLR








LTRAPLKPQQKLFALRTVLI








PQLYHQLTLGSVMIGVLRKC








DRLVRQFVRRWLDLPLDVPV








AYFHAPHTCGGLGIPSIRWI








APMLRLKRLSNIKWPHLEQS








EVASSFIDDELQRARDRLKA








ENVQLCSRPEIDSYFANRLY








MSVDGCGLREAGHYGPQHGW








VSQPTRLLTGKEYLHGVKLR








INALPSKSRTTRGRHELERR








CRAGCDAPETTNHILQKCYR








THGRRVARHNSVVNAVKRGL








ERKGCVVHVEPSLQCDSGLN








KPDLVGIRQNHIYVIDVQVV








TDGHSLDQAHQRKVERYDRA








DIRSQMRRFFGATGEIEFHS








VTLNWRGIWSGQSVKRLIAK








DLLIAEDTKLISVRAVNGGV








TSFKYFMYCAGYTRS








(SEQ ID NO: 1456)





R2
R2-
.

Gavialis

AGGCATCTCCTTKAAGGGTA
TGAGAAGTTGCGAGTTCTTA
PAAPRAWGAVEAGPWPGRTR



1_Gav


gangeticus

ATGGTCTGGTTACATGGTCA
TGCAAGTTGAATACCACTCT
AVEPAPSPESSPSEAARAAP






TAGCAGGTTTGTGTCAGGTA
KGKGACCCCAAAAAAWWAAA
AGEGHGPGHESPSVQRPEAD






CCTCCCAGTGGTTCCCGCCG
CCCCAAAACAGTTGTGTTTA
TTAPGVSAPTREGEPPSTRV






GGTGSCAMAGCCCCAGGGCT
AGTGTGTTCTTGTTCGTCCC
FLVRLPDSNPPCPICRDHVG






GTCGGTAGCTCGATCCTGGT
TTTGGCTTCACCTCSAAGTT
KPSALALHCVESHAWADVQY






ACAGTACGGCCAAGGGAGTT
GCGATCCCCCCATCTCCCCT
QCTHCKKVSANKHSILCHIP






CTTCCTTGCTGTCGGGTGCC
GCGCTGCCTTTCAGAACGGC
CCQGRVPEWTGKDWACPECP






TCGCAAGCACKTGGCAGCCC
CGGTGGTGTCGAGGCTGGCG
ASFNKKVGLSQHKRHVHPVT






CAATCGCTTCATTGCGAAAA
CGACCTCGGTCACCTCCAAG
RNVERVAGSLSRAGLRPQTR






ACACAAACGTCCTAAGGGGA
GCCAAGTGCCCTGGCCCCGA
RGCWSVEEEETLTCLDAMFR






TGATCAGCTAGTCAGTTCTG
GTAGGACTGAGTGGCCCAGC
GARNINQLIAAEMVTKMPKQ






CCGCTAGCCAAAACTGTTTG
TCGCTGGGCACCCGTCACCA
ISDKRRQLGLCPEQTTLGGD






CCACCCAGTTACAGATAGCG
TCTGGGGCAAATGGAAGGGA
AESTSVVEEESMTPEMETQS






TCTGTGCTGA
TCTGTCCTGACCACTACCAG
PINPPGKIRKILAQRARRWL






(SEQ ID NO: 1212)
GCTAAGTGTGGTGCGGCCTA
KKGQGLSDKVREVLGAWVEG







GCCTGCCGTAAGGTCAAGCG
QPRIHAWVDSVSLDVLTLFL







CCCTGCTGCCACTCAGGTAT
GVPSGPQRAPNKKRPKEGGK







CAGTCCTCGTTCACTTGTCC
PTSWMNKCAVKWGTFLRYQH







CTCCTAGTACCCTCTGCCTC
LFGANRKLLVAIVLDGADRN







TGCTCTTTTGCTATCCACTA
QCTLLLEEVFQAYREKWGLE







TGGCCAGTGATGTTGAGGTT
EVLRAYRGKWEVESSFEGLG







GGTGCATCCTTGGTCACCTC
RFGVRRDADNFAFKALITPE







CAGGGCCAAGCGCCTTGGCC
EVVKHMMAMASKSAPGPDKL







ACAGGTAGGACCTGGCACCT
TLRDLRRADPEGDALAELFS







GCCCAGGGGGCCAGACACTG
LWLITGTVPDGLKECRSVLI







CCTGTGGCAAGGGAAAGGGA
PKTVDREKLGQLGNWRPITI







GCCGTCCCTGACCGTTACCA
GSIVLRLFSRVLTARLAAAC







GGCTTGAGATGGTGCTGGCT
PINPRQRGFIAAPGCAENLK







AGCCCACCATATGTCAAGCA
VLELLLRKRKRDRQPLGVVF







CTCCACAGCTGCTTGAGTTT
VDLARAFDSVSHDHISWVLK







GTTGGCTTCACCTTCATCCC
AKGVDEHIVNLIEDSYQKVT







ACCTAGTGTCTTCTGCCTCT
TRVQVFNGVTPPISIKTGVK







GCACTATTTTCATCCCAACT
QGDPMSPLLFNIAMDPLIAK







CGTACCTCCCCATCTCTGCG
LETDGQGVKVGSASLTTLAF







CTCCTGCTATCCCTGCAAGG
ADDLVLLSDSWEGMLKNISI







ACCAAGTAGGCAGGGGGGTT
LEDFCNLTGLRVQPKKCQGF







CATCCCCCTACCTGCAGGAG
FLNPTCDSFTVNNCEAWKIA







ACTCAGCATATCCATGACTT
GREITMLGPGESTRYLGLNV







CTTGCCTCCACCGTCTTGTG
GPWVGIDKPDLGTQLSSWLE







GCGCTAGAGGGGTACCTCAG
RIGTAPLKPMQKLSLLVQYA







AGACCGGCACAACATGACCT
IPRLNYQADYAGIGRVALEA







TGACGGTTAGACAGTAGGGT
LDSMNRRKVKEWFHLPACTS







CAAACAACCCTGCTGCAGGC
DGLLHSRHRDGGLGLPRLAK







CCAAAGGGCCAACAGCTGTG
AIPEAQVRRLIRVATSSDEV







CCACGAGAGGGGAACCTTGA
TRKVSYACGISDEVERLWLA







AGACTGGGGCAGTCTGACCA
RGGDMSSVPRFEDPEAPRSP







TGCTGGTTAGTCAGTTGGGT
GVQGPCEAAQEIPSVVRKLA







GAAATAATCCCAGCTGCAGG
IPRPSNWRSKKHSKWAQLSC







CCCAAAAGGGCTGACAGTCA
QGEGMELFCNDPVSNGWNNS







GGTGAGGGGGTATCTCCATC
RGQLAEHLQIVALKLRSNIY







TGCTCCCCACTGCCAACTAC
PTREFLGRSQASTNVGCRHC







GGAGGCATGAAGTCCGTAGT
THPHETLGHILGICPAVQEA







GACTTCTGACCCCCACGTCT
RIIRHNKLCKILAAEGKKCE







TGTGCCATGAGAAGGGAACC
WTVYYELQLLNAAGELCKPD







TTGAAGATTGGGACAAACCG
LIFVRDGTXLVVNVTVGYEG







CACTTGAAAGTTACTCAGCC
GPAXLLSTAAEKATKYLDXN







GGGTGAAAATAAGTCCCAGT
AQIQELTGAEQVTYFGFPIG







TGCGGGCCCCTCGGGGCTGA
ARGKWHADNXRVLSELGLSN







CAGTCAGGTGAGGAGGGCTG
SRKERVARLLXWRALLGSVD







CAAAGCCCATCTCCTGACTC
MVNIFASKHRQENLSDXALS







CAGAGGCCTGGCGTCCTAAC
PS







CGACTTCTTGCCACCAATGT
(SEQ ID NO: 1457)







CTTGCGCCAGGAGAGGGCAA








CCTTGAAGATCGGGGCAAGC








CGCACTTGATAGTTAGCCAG








TCGAGTGAAACAATCTCAGC








TGCGGGTCCGAAAGGACTGA








CTTCCAGGCGAGGGGGGGGC








CTGCGGAAAACCCCCTCCAT








GGTACGGAGGTCTGGCATCC








TAACCGACACCTTGCCACCA








ATGTCTTGTGCCAGGAGAGG








GGAACCTTGAAGACTGGGGC








AAGCCGCAGTTGATGGTTAG








TCAGTCGGGTGAAATAATCC








CGGCTGCACCCTGCTGTGAC








TGCTAAGCCCGGTCCCCAAG








GGGCATGAGGCATGTGCGCT








GAGACGGGAGGGGTGACATC








TGGCGATCAGCACAGCACAG








ACTGAAGGGAGGCACTTGCC








GAGAATGCTTCTGAGGCCCC








AGACTTGGGGTGGTGCAGCT








TTGTCTCGTGTATAGTACAG








CACCCTACTGCTCCCTTTGG








GCAGCAGAATTTGTCCTGAC








CTCTTACCCACCCGAGTCTG








CGCTTTTGTTCCACCTCGCT








GTCTCCCTGCTGTGCTGTTT








TTCTCTCAAGTGGGTTAAAT








CTCAACATGATTATCTCCCA








CGTTTCCGCTCAAGGGCAAT








GCCCAACATGACGGAGATCG








TTGGTGCATGGTAGTCACGA








GACCATCCGGACCCTCCAGT








GGTCGCTATAGTCATTTTGT








GTTGCATGGGGCATGCTGAG








TCACTTAACCGAAAGACTGT








AAATAACTCAAAAGAGGTAC








CCTCCGGGGTTCGGTAAA








(SEQ ID NO: 1335)






R2
R2-
.

Ixodes

GTTCCAAAGGAAGGCACTCC
TAGTGTGACGGAGTCCTCAA
MQCTSRLADAPRFARVGVEG



1_IS


scapularis

TTTGGTTCGTGATGAGATGT
GCCCCCACAAGTGCCTGCCA
EGVGASGNGTDAQLWYGCTG






TCATGGTGCTTGCCTAGCTG
GGTGGCAGGAAAGGGCAACT
CDEAFSSLRGLRIHAAQKKH






GAGAAATCCGACTCACACCT
ACTGGTGAGCGACCCAAGCA
GNQDGLLRLPAGRPRKRRVG






GCACGTGGTCCCTGCCGCCT
AGGCGGAGCCAAGACCAAGC
KSTTAGASDRVTTDPVPAPV






GCCAGTATGCCGAGGAAACG
TGGAGCCAAGAGCAACTCCA
PESPGLLPGLPGPSLPGCSD






GGTGCAACTTAATCCGTGGA
GGAGGCAGGGGTGGATATCA
LPPGVLPGGWSASPGPLSWP






TACTGGTAGCAACGTGAGCA
AGAGCAACCCCAAGGGACAC
PSLDAGPLPGPSRVSPGPSR






ACGGTACGGTCCTTCGCGGA
AGACCACGGGCAACTACTGG
PSPGKPTGPPSLDAGPLPGP






CCACCCTGGGCGTTCGGGTT
TGAGCGCCCAAGACAGGGGT
SRVSPGPSRPSPGKPPGTPE






GCCAGCCCGTTCGCCCGAAA
GGATATTAAGAACAGCCCCA
PLPGSPGGRRGVSPGQPGSR






TATCTTGGCCCTGAAACTAA
CAAAGTGTTACCTATATTAA
TDPSSSAGAGHFVCPQCSRA






AAGAAAA
CAATAAAGTTGAAGCCTCAA
FSSKIGMSQHQKHAHLEEYN






(SEQ ID NO: 1213)
CCACGCATTGCGGGTTAGAT
AGINITRTKARWDPEETYLL







GGCGTGGCTTGGCCCGCCGC
ARLEATLNPDHKNINQTLHA







CATGATGAGCTGGAACCCTC
ALPRGSCRTLESIKAHRKQA







CACCTGGTGGGCCGCACGAG
AYRDLVTSLRSARESSEAQH







ACCACCGGCTCTTTCTACTA
VPDRPLETPEPQTPANPQRD







AGGCCGGTCTCCGTGACTGC
SKQAVIEALQSLIGRAPPGS







GGTTGGGATAAACTCCAAGC
FQGARLWDIARQATRGTNIL







ACTGAGCGGTAAAAAAAAAA
PLLNSYLRDVFTLPTKPTRK







AAAAAAAAAAAAAAAAA
KPAVRPARSRRKQKKQEYAR







(SEQ ID NO: 1336)
TQDLFRKKQSDCARAVLDGP








TSSSVPGTGAFLQTWREIMT








GPSPALEAPPLPTRGEVDLF








FPATAQEIQSAEIAVNSAAG








PDGFSARLLKSVPALLLRVM








VNLLLLVRRVPAALRDARTT








FIPKVPDAVDPSQFRPITVA








SVLQRLLHRILAKRALEAIP








LNFRQRAFQPVDGCAENIWL








LSTALNEARTRRRPLHMASV








DLTKAFDRVTTDAILRGARR








AGLSGEFIGYLKELYTTSRT








LLQFQGESLLVEPTTGVRQG








DPLSPILFNLVLDEYLSSLD








PDISFVSGDLRLDAMAFADD








LIVFASTPAGLQDRLDALVE








FFDPRGLRVNVKKSFTLSLQ








PGRDKKVKVVCDQIFTIGGT








PLPASKVATPWRYLGMTFTP








QGSINKGTSEQLDLLLTRTS








KAPLKPQQRLVVLRNYLLPR








LYHRLVLGPWSAALLLKMDT








TIRGAIRRWMDLPHDTPLGF








FHAPVTEGGLGINSLRASIP








AMVLQRLDGLHFSTHPGAEV








AIQLPFLTGLHRRAEAAAQY








QGQRLLSKADVHRMWSARLH








GSCDGRPLRESKRVPAAHRW








AAEGTRLLSGRDFISITKLK








INALPTLERTSRGQHKDIQC








RAGCQAVESLGHVLQACHRG








HRGRIRRHDNIARYVCGRLT








QIGWAVKWEPHYSVAGRTLK








PDIVAHRGAETVVLDAQVVG








TSMRLGFHHAQKKEKYSLPD








LLHQVCEGRRDAARVSTITL








NFRGVWAPESAQDLKSLGLT








DNDLKLLTVRCLQGGAQCFR








LHRRMTTVVKATGDEANALP








AHSGLPPTQLGGRTLGPSAH








NQSARTT








(SEQ ID NO: 1458)





R2
R2-
.

Mnemiopsis

TGGGGGCCCCTTGGACTTGC
CCATGCTCTTACCAGGCCAC
MSNTSHSKLNLKMDNKLKTS



1_MLe


leidyi

TCCCTGGGGCAGGACACCAG
TTGACGGCGCTACACCGGTC
LETPSGVRADSIITRVRTSS






TGAAAGGAGATCCTCAAGAC
TGTAGGAGGGTATCTCAGCG
NRGEHSNGVTYPRCEQGVAP






AGGACAGAGAGACAGGCACA
ACTTGGACAAAATGGATCTT
LDTHGGICDAPPQVTVPATE






CCAACCCTTCGAACCTGAGG
GACTGCCTGAGGGCGCTTAT
TDKQKKCEYCEFTYLKPRQI






AGACACCCAGATCTGGTTAC
CGAGTGACCCAGAGTAAGCT
GTHMRKRHPQEWNDIKRTKF






CCCATTCCTGTAACCATGGT
GGTAGGAAGAATCTTGCAGT
LSEKRQKRWLDEDFELLCIG






TGCTCTCCGTGCGCTGTTAA
TGAGAGGGCTAGTAGGGCCA
QEEYLVLSSIGKQGKGINQY






GGAAACCCAGCCTAGTACCC
GCCTCTGCTCGTCAACTGTC
IQTKYFPTLSTDAIKSQRKS






TCGGGGAAAGGTTGCGTAGT
CTAGGCGTTAACGTGTGCCT
RRFSEYSEKRSRELQPCNTS






ACTTAGCAGTGTGCGGATCA
CCCTATGAGGTAACCGTGAA
SDPEELPNEAVTENSPLSFD






AACCTCTACCGGTCTCTCTA
GTATTACTTGTTTCCCTGTA
PLDRDVVKKISSKDHGDQIL






GCGATGAAAGTTTCTCCGAC
GAGACCATATAACTAGCTGA
LVQEHLINGRYQEANTLAKA






TGGAACTTGAGGGACTGGCT
GAAACGCCCTGCGGGTTAAT
IFEKLSGKFPNLKTGDHRPG






AGCCCAGCTAGCCTGTAGAG
ATATAGTGACGAGAAAAGTC
KQQTARKVGKKRVRGSGKKL






CAATGCGTATACGATGCCTT
GCTTTATCCTTACATAACAG
SPSKQNRRELYAIVQKQWRT






GGCGACAATGGCGACCGCTG
TGTGATAGTCATCCTTATCA
KKRSKVINQILTGNLNKEQS






CTTAGCAGACGGAGGTTAGT
CAACTGTCCTGGCGAACAAA
YTHTPDQLAQFWSTLFGRVS






GAAAGGGCGACTTGCTGTTC
ATGGCAGAGGGATAGTACTC
PRDDRPINHRRSVIPELDKP






ATAGTCACGTGAGTCGTCTA
GGTCCAACCAGAAGGAAGCC
LSVEEVEAALKGAKDAATGI






GAAACTTATACGCACCCAGA
CACACGATGCCAAACTTGCT
DGVPISHLKHLGSAALTILY






GACTGCACCGATGCTGCCCT
GTAACCCACGTGAGCGGAAA
NGLYVTGSIPDPWKRARTIL






CTGTTCCAGGAGAAGGACAG
ACATCCTCCGAGTATAGTAT
IPKSNPPASPGDYRPISISS






TGGAGGACTAAATCGTAGCG
GATGGAAGGATACAGGATGT
YFYRIYTSAISKRLASAVSL






CGAGCGGTGTT
GGACCGCTCTAGGCGGCGGA
DDRQKGFIKEDGIRDNLSLI






(SEQ ID NO: 1214)
CGGTGTAGACGGCGACCTTA
DTLINETKAGSKSLFMTFMD







CCATAGCTGCGACTGCTGTG
VKKAFDSVSHYAIARSLEWA







CGATCCGGAACGGGCCTTTC
GVPDGMRSVIADLYQDCTTD







TCACTTACCTCAAGCACCGT
ICGRSVKVTRGVKQGDPLSS







GTCGGATCGCGGGGTGGGCG
TLFNLVIEMVMSNVPERLGI







CGGGATTCCTGCTGGGAAGT
QFQGHRLFYLAFADDLVLLT







TGTCTGCACCCAAGTCGTCA
RGPTANQKLVSLVHEQLARV







GCTAACTGTCGGTCAGTTAC
GLELHPGKCKSIAIMADPKR







CGCCGTCCTGCATCCCCGAG
KTTFVDQGSSVLIGGEPVSS







TTGTGACCAGCGTACAGCCA
LGPQEWYKYLGIKLGSGGMP







TGTACCACGGCACTTCCAGC
QGIYRDQLADLLAKTDSAPL







ATTCTCTGGTCTAAGAATGT
KPQQRLYILRSHILPKFNHR







ATCAGCTGGCCCGCCGAAAG
LMFERVTCQTLEGLDKLIRT







ATAGAGCGACCCCGCCTCTA
HVRKWLKLPKDTPGPAFYAD







TCACTAGAGTAAGCAGGCAC
KGSGGLGLITLRYRVPLLKL







GCGAAATATATGCAGGACGC
RRHKKMADSPDPVIRLIPNA







CTTGGCTCAGTAGCTCTGGT
EPTISLLARWTKMCSLYGKQ







ACTGAGTATAGCATGGCTGA
YQHKSELSKIIRDKYWTMCD







ACGCCCCTTTAAGTTCGGTG
GKGLRTEVPPDTAKKTLSLL







GCCCAGAGACTCTCCGAGAC
FEDRTPLKPGQLIGAIGVRL







TTCCCAGTCCTGGAGAGGGT
NTLGTPARNNRAKGYSPEAN







ACACCTTTACCAGAACTTTC
ICDKCPGNRQATLGHISQTC







TGTGGTCGGTTG
PATHGRRVKRHDKIVNRIAK







(SEQ ID NO: 1337)
ALKERGSVKNILTEPHLRHD








KLPLRKPDLIVHTEKSVEII








DVQVVADQGISRHEDEDQQK








KIVKYDVDGYKRAAYKMLGI








DYGSIPCNVSAFTITWRGNL








APHSLKLASRLQFSPVLKYI








VADSLVDTWGAFLIWGKTS








(SEQ ID NO: 1459)





R2
R2-
.

Petromyzon

CTATTAATGGGATGAAGAAG
TAATTTAAGGTAAAATCGTG
MNERLTDELTTEFILSDMFL



1_PM


marinus

GGGGACACGAGTTTGTGTGT
GGATTGTTTTGATGGCAATC
WDYPCTDQNKCYPCNLVFLD






GCATCCAGTTTCCATGGTGC
TGCCTAGTCGCGGCCTTCCA
HRTWSSHMARVHPHANKTYK






ATGCAGGAGTGGTGGTTTAA
TTTTGGGTAGGCAGCAGACC
CRICNRTADSIHKIASHYGR






ATGGCGAGACTCTACAGGGC
CATCTATATAACAAACTACT
TCKSLIGKTNAITTTIDETL






TTCCATGGCTACACGGGATG
TTGCCTTTCATAGGGGTACC
FSCLHCSRGFTTKTGLGVHT






CAAGGCATCAGACATTTTGG
CGACCCTACCAACTTTCGGG
RRTHPTEHEAILQQNTPGRK






CACAGGCAATCCTTTTGGTC
GAAGTAAAAGAAA
VRWGEEEVEIMAHKEAQQKD






TCTACCGCAATCATGTCTTA
(SEQ ID NO: 1338)
EDINMNQLIQNSVMPHRTLE






GACCTCAGTAGCGACCACTA

AIKGKRRNIKYKELVRTLKE






CAACCACAGTGGTGACTGCT

TTYKVENQCLVNLVLPTTSE






GTTGAGTGAAGGACGACTGA

ITTTPSEGDQPAIRAEKEQS






GCGCTGGATAACAACTTTCT

PTAAEDLQVIINDLKSQNFS






TGCGTGGCCCAACATCGAAG

HNQALLLLNSHVEKFLNRSK






CAACCACTTCGGAGCTGGCA

PIKRKDHVNQQEIDENRHRR






CAAGGCAAGAGGGCAGCCCA

QSKQTKYRRYQYLYHTNKKA






AGGTGTGAATCATCTCAACT

LLDEITSDRSGPSIYPTEES






TCACTGCAGGAAGAAATGCT

IRGTFVTLFESNSPPDNIPS






GTGCAAGGATGAGTGTGAAC

KLKNDQSCIDIVKAITLDEL






GACACCAACGGGATTGTTGC

IKTLAIMKDKSPGQDNITLS






TGACCAGGAGGTGCCAACCA

DLRTLPIKYLLDILNIILYI






AATTTGAATGGATTGACTTT

QDIPQIWKQHRTRLIPKTKE






GGGCCTGGTTTCTCCTGCGT

ELEKPSNWRPITISSIVIRL






GTATTGCACGGAAAAACAAG

LHKILSYRLGQQLKLNYRQK






TGGCTACACGTGTGGCCGTC

AFLPVDGCFENSALLHFIIH






GTGTCCTGGGGTTTCGCAAC

NARQKHENTQIVSIDLSKAF






ACAACTCCACAAGATCGACA

DSVSHESIIRALNRFNLSKE






ACTATGAGGATGACAATGTA

SITYLTNIYKCNLTDIVFGS






CTTAAAGAACAAAGAGACTG

TIMRNINLKRGVKQGDPLSP






ACGCCAAAGGGGATTTAAAC

LLFNMIMDELLDNLPTYIGV






CGCCAAATCGTACATTGGGT

NVGNQKVNSMMFADDLILFA






CTCACTACAATTTTTTTACG

ETECGMNKLLDITTKFLDDR






TGTATTTATTTTCCTAAGTG

HLKININKCNSLRFIKYGKQ






TCTGTACTTGCCATTCTTCG

KTFSVATTSSYFINNEPINP






CTGCTTTTTCTGCATTAATT

VSYVKGFKYLGIEFDPRGKR






GCATATCGTATGCAAATAAG

SISCNLLAAMLNKLTRAPLK






CGAATTAACCACCACCGTGC

PEKKVYLINNNLIPRIIHQL






AACTATATGCAGATGTTACA

VLGKVTKGLLMSLDSEIRKT






GCTGAGCCCTCTATCATACC

VKLLLRLPHDTPDSFFYTSV






GGTGTACTAATCTGGTATGG

SNGGMGIRNLCDSVALSIIN






TGTTGGCATGCTATGCTTGC

RHNKLITSDDLVIRALSQQS






GTAACGACCTTTGCTGATTG

YTIATLKQAHIIAGSKFPSK






GTTCAGTCGGCTGATGGTGG

SLNQNKWSNKLYQTTDGRGL






GTTCAGGCGAAACATTTGTA

VYCQSQTENNSWITGNHRTI






TATTGGTTTAATCAAACCGA

KSYNYIDMVKLRINALPTKS






AACACTAAAATTTTGAACAC

RCNRGTLETKQCRFKCRSIN






AGTTTTCCATTACACCAGTT

NQISEETLAHILQKCDRSHY






GTATTGCTAGAAGTGCAAAT

SRIARHDSLVQFLATAAQKL






CGAAGGAGTCAATTTTGACC

NWEVIKEPTLPSDTNKAKPD






GACGATTAGCTGCCGATGTG

LILVRDSHVLIVDVAVPWES






CGGTGAAAAAGCTGATCACA

RSLAHAYDFKVKKYATDKKM






ATAGCATACACTTGGGCCGA

QAYLKTIYPEKEIRTEALII






CAACCCCGTGTGCTATAAAC

SARGGWCALNNMVTKKVGLS






GTAAGTCGCGAATTATAAAG

SAWVKLALIKVMEGSVKIWR






AAAACAAACCGGACGGACTA

SWSKG






CTCGGTGACGAACTAACATC

(SEQ ID NO: 1460)






GCTC








(SEQ ID NO: 1215)







R2
R2-
.

Schmidtea

CAGTGCTATTCGAATGTCAA
GGCCACGCGCGTCGTCCTTG
MKKVLNNETEKLPGSNLTFM



1_SM


mediterranea

TGTGAAGAAATTCAACTAAG
TTTGATCACTAGTGGATCAA
CGFCDREFDTARGRGVHESR






CTCTGGTTAACGGCGGGAGT
CCTTCGACTCCCCGGAACTG
GHLVERDAAVQSRVKAVVSK






AACTATGACTCTCTTAAGGA
TGGGAGTGGCGGAAGAAAGG
KYYYSNEEDVALAKMQLXHA






ATTAAGAATTTACCTGCCKT
CCAGAGGATGTCCTGAAACC
DLAKSEXLEAMYLALGKGRT






AKTAAAARTGAAATCMGTTG
ATATATTTATTTATAGAAGT
REAIEQHIRKSLRYKGVLEE






TTCATWGCAAGTGGTATTGT
TTTACTTCATCCTATTTACG
QRKLLETARGNVRQNNVGVP






ACACCTTCCCGCGGTGCTAG
TATTTCAGTATGAAAATGAG
ASNATKNLORFLESLPLGTN






TCGTTTAAAACTAAGTTACA
TAAAGTTCTCGACTCGATGA
RREERLDRIIRSNSIESQRL






AACCACGAGGGGCGTCCTGA
GTTGGGGGCAACCATTGGGG
ELIHYCNDMCQDFVQLDCQX






CGGACTGSAAAAGCATTGAG
GTCCTGAAGAGAGGCTCTCA
NPINAIRRRNPKRLSKKQLK






RGTCMTGAAGAGAGGCTCTT
CTGTAAAAAATCTCTTCGTG
RAKFSALQRLWIRDRKAAAQ






ATTGTACGAATCTCTTCAAC
TCTGTTTATTCCTAGGCACN
LVLKDKLDSLLSNKEDSKDL






GATCGAAGTCTGGACCGATA
TGCTGCATTATGAAGCGGWG
GSYWQQVFERESELDRRPIP






TGAGAACTAATACATTAGTT
AAAGTAAAGTTAGAGCTGAG
QVVENEELNSPVLEKEVEWA






GACAGGTGAAAAATACTGTT
AGATAGGTACTTGCTGCATT
VKNIKKSTAAGPDGLTALAL






GATTACTTAGTTCTCAGTCA
ATGAAGCGGAGAAAGGCCTC
KKIPYSELVKLFNIILLVGF






TGTGGTATATTGCCAGTCAA
GAATAAATAGGGTGTTAGAG
LPDVLKNSRTILIPEVDNPQ






TTACTACATWAATATTAGTG
TTATTGATGGAGAGTATACT
GGGDYRPISINSVLTRTLNK






TGGCTCTCAAAGGAACACGA
AGTAAGCTTAAGCTGCGCCT
ILAKRVSEGDFGINGQKGFK






TTGRTCGGCAGTCCAATGCG
CGCGCGGTGCCCAAAAATAT
SVDGCLENLATVESILADAR






CGACTGGCGGGCTTGTTGTT
ACTTAATGAGAGCAATAACT
MKKNKLAVVFLDMSKAFDSV






TGCATTTGTTACCGGCTACT
CAAGGNGAGTTTAATTCATA
NHESIVRAGEIKGYPKLLMT






TGAAAAGGTTATATATAGCA
TGCGCATGCGGCACCAAGGT
YVKECYNDATTNVAGVTAKF






GACGCTTAAAGCGCGACTGT
GCTGAATGGCATCGATTAAA
NRGVKQGDPLSPALFNNVID






AATTTACATYTCATTGCCCA
CCTCTCCTGTTGTAGAAGCA
LAIERVSGTGIGYNMGGKKY






GTATTTGTCTTTTGTCAGAT
GGTCATAAATGGAGGRGGGC
SVVAYADDLVLFGESREGLQ






TTAGCAAAATTTCATATTTT
AACCACTGAAACTTATGAGC
IALTALLEELKLNGLTPNPA






GTTAATTACCTTAACTGGTT
CAGAAGAAGCTTAACTACAW
KSASLTFERSGPHWFASTDT






AAACGATCCCATAATTGCTT
AAGTTTTAGGCAATTACTGA
VTALGDQIPAMGNIETYKYL






GCAATTATTATAAAGTAATT
ACGGAGTTAACTGTTAGTTA
GIKFNSCGVVKGSLPGIYTK






CAGGTAAAAATTACATATCT
ACACTACCATGTAGTTGTTT
KLELISKAPLKPQQRLAMLT






GGCTGATCCTGCCAGTAGTC
ATAAAGCAAATATCAGGTTT
DFLIPGVLHQAVFGQTNAGD






ATTTTACTTCCGCCGCGCTA
CAGTCTATATACTAAAAGTA
LRSLDKRTRRAVRSWCHLPS






TAAAACAGTTTAAAAACTGA
TTTTTTGATACCGTGGTATA
DTSTAFIHAKAKDGGMGIPS






ATAGGAATCAAAAAGAACAT
TAGGCAACTAGTTAGGAAAT
IRAEVQFGKLDRFGKLPNVK






GGCAAGCGACTATATGTAAC
AGTAACATATGGTGGTGCCT
DERSKVLADNAHIKKKMLEK






TGGGCATTCAACATTCCCTA
GGGGGGATGACGCATTGTCT
LGVGIPIKGVRCKNKLEFYN






TTAGTATCACCAGTCATGGT
CGTCTGTTTTATATAATGGG
KMREELIKSNDGIGLKEASL






GCCATATCTTTKGATAAAGA
TCTTTATGGTACTGAGGAAA
VPSANTWLKLSDLHMSGRTF






TACAGTTTAAAACTGCGATG
CTTATCGACTCGCGAGTACC
VGCLKTRGNLMATVTRTSRG






ATACTAATAGAGATCCTCTT
CGGGAAGTGGATCTGGATGA
GQNPGIELNCKKGCQYQGSL






AGACCTTCGTAAAGAAGTGG
AACCCGTAAACCGATCGATY
NHIVQKCPVVKGLRIKRHDE






GGATTGATGACATTAGCATT
TAGCCTATAAGTACCAGCGA
VVKYVEEITKKAGWSATMEP






GGAAGAATTAAATCTCCAAG
CAGTTAAACCATCTTACGCG
IIPFEGSHRKPDLVLVRGDL






GAAATGGAGTAACTTCAATG
AGGGGTAAAACCTGAGGACC
GKVVDIQIVSDHCGLDEKNS






AAGTCCCACAACCCCGTTGA
GATTATGGTATAACTTCTCA
CKIGKYDNDIIRNYVRGLGP






AGGGCTGGGTTCGAGTATCG
AGATTAGCACAAAATGCGAG
SRVEVAAITLNWRGVWSRDS






AGAGAAAACTCTAAATTCTC
TGCAACTTGAGGAGGAGGAT
FNLIKRLGMTEMDAKIISMR






TTCGGTTMTGTCCAACGGAG
TTGAGTGTTAATTCATAATG
VLASTAKMFKTCKKVLEPVC






GGGACATTACTGTAAAATAT
TACTAATCTAATTAAACTGT
RTKTADCDGYGPEETSARPC






CCTCTAAAAACAACT
GACGGGAATTGCAGCTTCGG
HELNLKESSGT*






(SEQ ID NO: 1216)
CTGTAATTACTTTGAGGCCT
(SEQ ID NO: 1461)







ATCACGGATTGTAAGGAACA








TATTGACACCGTAAGTCTAA








CGTGTTCCCGATTTCCAACC








AGGTCATATGAAGGGCTGCC








CTTGATAAGGCGGATTTGAC








CCAATTCTTCATATGAGAGG








CTTATTCCAGCCTTCCCGTA








GTACCGTGAGGTTTTCCCGC








CTCGAACGGAACAATGTTGC








AGGGTAATTAAGTACATCGG








GCTATATMGCGATATTTAAC








GTTTTA








(SEQ ID NO: 1339)






R2
R2-
.

Strongy

TCTCGCGACGCGTTCTTCTG
TAAACCTTGCCTCCCCGGGC
MENSFAWEGTSSAEGRTTVE



1_SP


locentrotus

CCTGATGAAGTCACGTCAGG
CCCCCTCAGTGACTAAGACA
DSPSSSDDFVSNVGFKVAKA






purpuratus

TAATAGACTTAGAAGGTTGA
ACTTTCACCGTAATAATCAT
DPTVWEEANMSEDNTIIEDP






TGAGCGTTCCTCTCCTGGAC
ATATTTGTATACCATGTATT
PSSSDDFANNVGFKVTRADP






CGGGGGTGAGGATGTGTTCT
AATCTAGGTAACAATTGAAA
TAWEEASTSTETEDLPSSSN






ACTGAATCATCGTTCCGGTT
GTAACATTGAACCGTATTCA
FIDNVETQIDMAGPTAWEDA






GTGAGGTCCGCTGCAAATAG
TACTTTAATGAGTACAATAG
DSNEDNIDEGTPNNINNNLA






GCCTTGGGGTGGTCTACCTC
TGAAGGGAACTGTATATTAC
IVRGRADAYACSCCERNFIS






CGGGTCGCTACTCCTTTTGA
ATACCTCGAGATAGAGGTTT
LKAIGTHLKETHNKKVVFEC






GCTAGTCGATCCAGGTGAGA
TTGTACCTTAAGGGTTCGTG
AKCQHTFVKAHGLACHVPKC






GTCGGGGAAGCCCACTTAGG
AGAATCCATACTGCATAAAG
KEDADTPMLNRLLHGCGECG






TGGGCCAGCTAAGCAGATCA
GGGTTAACTTGTAACTCAAC
LAFNTRRGLSQHERHRHPSA






CCCCCCCAGCACGACAGTGC
CCCAGGGAGACAAGCCGACA
CLSTRRRSRLDGIARKKSLR






GCTGTAATATAGCCTGTTGA
AATTCGGCATTACTATGTGA
NRRDIWTNDEIRLLKQLMIQ






GAGTGCACCCATTTATAA
GGGTCATAAGTGTTAAAGGA
YEHAKKINIKIAEHFNHKNA






GTTATAAAAGTATAAATGGT
CCCATTGTATATAACTTGTA
KQVMHKRRSLREKDMALGAP






TCTTAGCTAACCTATCCAAT
AATGTACAGAGAATTCGAGT
HDAPPPLAEEPIIEVVEGAR






GATTTTTATTGTTATGTATA
TGAGTTCGAAGAATAAACAA
EELEQAPVDVILPDLEALTV






ATACTGACCACTTTAGTCTA
ACAAAAAAGAAAACAAAGAG
NDGRGGGSPVLTEGGESTRD






GTGTCATGTAAGGATCCGAG
AATTCGTTCGTCCATAGAAG
MEENGGTDTRSPSPREERAG






ACAATAGCCTATGTCTCTAG
CGGAAAAAGCGCAAAAAGGC
STPWERGWQPRVDRGRGEYK






TCTAGCAATAACACAAGAGG
GTCCTAACCTCACGGTCTAA
GYGGERGDRHTVSLSQRGES






GATAATCCACCTCCCTACTC
CCCAGAAACATTTAAGGGGG
GVDPLTPGGVVEDYDDSYLE






CAGCCACCACCTTTACCTTT
GAAGGAGCTCATTACTCCAA
DYFPGWDEDEHMHIIGRLDL






CTTATCTTCCATATAGATGC
TCCAAAACCACGTTCCCTAG
SDESEQGEAAVSPRLGSFGD






CAACCAGACGGGTCGATGGG
CCCAAAAGCGCGCTTCAATG
LLEEVNAMEGKDNLSEALAE






CAGTGAGAAACAAGAATGGG
AGGGAGCCGATATTGGAGGA
TLGLVLHEGHRVEYIKEKMN






GATAATTTGGAATTTGACAT
CAGAGTTCATCAAAAGCCAC
INVKQMATEILAHGANKGNP






TCTTTCCTAAAGCAGACCTG
CTGTAAGTAGGCCCATTTCT
KRRKEAVAAKRNPGTRLDRA






AAGGGTTAAGAGTCACAAGC
GCCAAGGACATGCGCAAAAG
QRDNQAKAKAKEKKRIFSET






GGAGTCCGGACTGTCTTTAT
GAAGCAGATTATCAAACCAG
QTQYKKNPHRLVEKLLDGKG






AAACAGACAATATTTTCTTA
TCAAACAAGCACAAACATTG
DERCSVSLEVIQRTYMNRFS






TTTTACCACCTATATGGGGA
GGGGGATGGGATAACCCCGG
RESKEVDIGAYVDPETVEDN






TATTTCCCAACTCGTAATAT
AAAGAGAGGGATCTTTAGAT
QGIVDPISKAEIERAISTTK






AGGGCCCACTGTCAAGTGGC
AGTGGATGGAAGGGGTGGGA
KGSAPGPDGVTYDALKAYGN






TGGTATAAGTTACCCTGTGG
CGTTGAATCCAACCATGCCG
CQLYLLIMYNTWLAMGKVPS






GTAACAAATAAATTCAAACA
TTTTTATGTTCCCGATAAAG
EAKTYRSILIPKGQGDPMDI






AAGA
AAGGATAAGGTCACTCCAGC
NNFRPLTLANVISRLYSKIL






(SEQ ID NO: 1217)
CTGACACACAAAGTGGGGTA
TRRLDGAVSVCPRQRGFTHK







AAAGAACTCCGCTCGTACGG
ASIEDNTLILRELIMKSKRN







ACTCCAAATAGAA
KECLAVVLLDLAKAFDTVSH







(SEQ ID NO: 1340)
DLIIKALRRHRVHEHLISVI








MDLYEGGTTSFTTDEGTTCP








IAIRSEVKQGDSLSPVLFNL








ALDPLLATLEQRGKGVEIGG








HTFVSLAYADDTALVSSSHL








DMTANLDITVEYLNATGLSL








NVRKCQGFLLTPINKSFLVN








EAESWVVEREAIPWVEPGDT








AKYLGVQVGPWSRPWPSIQP








VIKRLTAYCESIDKAALKPR








QRIHILTTYIAPRIAFEIAE








GGYSTLVDCRGGIQYTRIRE








VDMTIRNYVRKWLFLPACLS








NSFLYTRRGEGGLGLVSFYD








YVPTERMRKLVRVCDSEDPV








IAGAAASLGLRERAAKISAQ








TGLPVPVKPKGAHNAWRKVQ








KKKWKAQPTQGKGVSCYQHR








LGNKWLGAPSFLTENDYIWA








IKLRTNLVPTREAMGRGIIG








RNQVECRHCHTTIETMGHIS








GYCQMVLDIRLIRHNRICKA








LIKAATATGLRVTEEPRIVG








TDGKNYLPDLIFSAGAGEPC








YVVDPTVVWDDDPKNLREAW








RGKVRKYTPIIPAVEAMLHP








SSVQIFGFVCGARGTWCPMN








DDIAKIVGLKNSGISRTLQI








VLCDTIRMVKAFMAR








(SEQ ID NO: 1462)





R2
R2-
AGKD

Salmo

AATCTTTAACCCCGGACTCT
TAGATCCGTTTGTTATGATT
MSGKRIVEMSGCDEKICQNK



1_SSa
01072

salar

TGGGGTTCTTACGACTCTGT
GGAGGGAGCCTGCCGAGTGG
HCLKRRWAWISGPKGETSPP




455

ATGAGGAACAGTCGAAGAGA
TATGAGCGCTCCAACTATTG
RKRGTCENVSFQDKSHASDP






GGGCGCTACCAATCCAAGTA
AACCCATATGATTCCCGAGG
DPLKAPEAREDAGSVAPQWV






TATGTCCCAAGAGGGCTGGG
CCTGGCCAGACGCCTAGATG
GEIKTPSLTSRDGVSEVVLP






ACAGGGTGGAAGAGTGCACC
CCTGCCACAATTGAACGCAG
PQPVHAEGVSPASDSKDKAT






TCGCGATCTGGGGCAGGGAA
CCCTAGCTTGCTAGGAGATC
KITLLISLPVCDLRCGRCER






GGAATGGAGAGAAGTCGAAG
CATAGGAACTGGCCTATGGG
PLETVGKAVRHFAVAHPTVS






AAGGCTTGTAGAGAAGGGGC
GCGTCATGACGGTTGAAGTT
VVFKCQKCEKSSKNSHSISC






TCTCCTAGATCCTAACCTGT
CCTCCATAGCGTGCTTGGGA
HIPKCKGMTETRTDVEGDHG






ATGACGCCCGTAAAACGGTG
GGGGACGACAATGACGAGTC
CDHCQEKFTTAMGLTQHKRH






ACCCCAGTAGCGAATAAAGG
ATGACGTACCGAGAGAACCC
RHIVQYCKEKEGEMTARRKG






AGGCAGGTGACAATAGAGGG
CAACCCAGGTTGGGGGAGAG
EVEAVKWSEWEESEVARLSD






CAGGGCCGACTTCCCAGGTT
AGCCAGCAAGAGCGGAGATG
GLAGLKMINRRIADSLGTGK






TACATTGTTGTACTTGTCAA
CTTGGTATACCAAGCTAGCA
TAEQVRQKRRRMRPEKVRCD






CATAAAGAGGTGTCTCAATA
GAGAGAGGGTTGAAGAGGAT
KPKEAKDKSNLIKMLSIPSA






GTTTGAATCAACAAGGGAGA
GACTACTGGGCTCAGAGTCA
TPTPQTGLKGFLLGELNGVA






GGAATACCGACCTGCTCCCT
TCTCACCCTAAAAGGCGGTG
TKGEVQIGGVTLSLRGVEQD






TGGGGCGGGGGTACTGGTCT
GGGCATCGGTTGAACACCTA
SALLNTSALELQRLLGGRAG






TAGCCCGGTTCCCCGCAAGT
CCCATACCGGGATGGGAGGT
SANPLSLQRERETTLPSERR






TTCCTTTGCCTGGGATGTGC
GGTAGGCCGAAAAAGAACAG
KTKQGEYRRVQKMFRSNEKK






CTGACTGGCTCCATCCCCTT
GAAGATGGTGGAGTAAGTTG
IAKYILDGNGDGEAASPPLE






TCCCCATTAGGCACGGCTAG
AGAGCGGTTGCTCGGGAAGT
IALAFKSRWEEVETFHGLGQ






ATGACGCACCGATGGGCGGG
TATGTTGTGATAACTCCATT
FYSRGEADGVVFRSLISMSE






TGTGTAGGTCGCTACCGAAG
AAGGCCGGTGGGCATGGTGC
VCENLGAIKNNTAAGPDGIT






GGGACTGGGGGTGTCCGGTG
GGATAATGGAAACTATAAAA
KPALLEWDPTGAKLAAIFSI






AACCAGGACTTCCCAAAATG
ACAATAAAAAGAAAGACCAA
WLTSGTLPGPFKKCRTTLIP






GTCTCACATTTTTAAGCGGC
AAAAATGTTCTGTTATGATG
KTDDPILLTQVAGWRPLTIG






TTGAGTATCGCCCAGTATCC
CCTTACACATGTCTGGGAGA
SVVLRLYSRILTHRLERACP






TCGCGCGGCACTGGGAACCC
CCCCATAAGGGTCTCCCCTT
INPRQRGFISSPGCSENLMI






AGTCAACCGCTCTGTGCCCC
ATACTTCACTGGGAAACCCC
LGGLIKRSWAKGERLAVVLV






GGCGCAGGCGGGGGTTTAAT
ATAAGGGTATCCCCCTATAT
DFARAFDSVSHSHILEILRQ






GTCTCCCCGGCTTCACCGGC
TTACTGGGAGACCCCATAAG
RGLDEHIIGIVGDSYTDVTT






GCTTCGGCGACGACGCAGAG
GGTCTCCCCCTATAGATGTA
TITVSGEQSPPIDMRVGVKQ






GAGCACCCGGAGGCCCCCAT
GAGCGTAAGGGGTCTCCAAA
GDPMSPLLFNLALDPMIDTL






GAACTTAAACCAACCTATCT
GTACCGGCCGATATGGCCTT
ERYGLGYRMGEQQITALAFA






TGAAATATGGCCTCTCGTTC
ATGGCAAACTCTGGTGGTAG
DDLVLVSDSWEGMACNIRIL






GGGTGAAGGGCAGGTGGGAA
GGACAAGGAGGTAAGGGCAG
EEFCRLTGLRIQPRKCHGFL






GAGAGGGCTGCCTCACGATA
TGCCAACCCCTACTTGATCG
IQKIQRARSVNLCKPWIVCG






AACACCTAGTCAATAGCCAG
GGACCATCCAGGGAATGCCA
EELHMVGPEESVSYLGMKVS






TCGGGAAAAATGTGGAATGT
TCCTCCCGCGAAGGTGATGT
PWHGIMEPDPVERLCNWISS






TAGGACAGGGAGGTAAGGGA
GGTGAGGTAAGGGGGGAGCC
IGRSPLKPSQKVRMLNVYAA






GGCGGCTTTTTCGTAAGGCT
CGTCTTCGAGTTTCCCCAAC
PRMTYQADHGGLGPIVLNVL






CCTTCAACCCCTACCTGTAG
CCCTACCCACAGGTGAGAGG
DGMIRKAVKVWLHLPLCTCD






TCCACCTATTGCAGGTGTTG
AGGAGAAGAGGAATCTGTCC
GLLYSRCQDGGLGIVKLACQ






ACAACATGCAAGATGACCTG
CCAACGGGAGGAGGGTGAGG
IPSIQARRVYRLWHSKEAIT






CCTCGTTACGGGTCGCGTAT
TGTAAGGGGGAGACCTTCTA
RVVTRRTVEAEEYRGMWLRA






CATTGCTACAGGTCGTGTGC
GTAGGGTCTTCTCAGTCGCC
GGSEAGLPPLEDREEGAVQC






CGCTTCTAAGAGGATAGTAA
TGACGTCCTGACTGTGGGGT
TDTAGSVKPKNPVIPDWRRA






GGAGAGGTTATAGGGAGGTC
GGATCAGTACCCTACAGGTG
EFLKWQNLTAQGVGVQVFGG






CTGTTAGGGCTTCCTCAACC
AGACCGGTGAGGTAAGGGTG
DKNSNHWMANPETLGSKERH






CCTCTCTATGCGATTCCTTA
TGGCCCTCTTGAGGGCTGCG
YIAGLQLRANVYPTREALSR






CAGGAGTGGATCGAGAAGTC
CCAACCCCTACTCGAGGTAA
GRPDLPKVCRQCLAGTESCA






CCGGACGTAATACACCCTGG
CCTGAGGGAGTGGTGGAATG
HILGQCPAVKDSRIRRHHKL






AGGTAAGGGAGTGGCCTTCT
GCGGCATGTTAGTGCTGGGA
CDLLASEAESAGWTVIREMC






AGTAGGGCTGCTTCAACCCC
CTTGATTGCGAGGGTTTAAT
CRTRAGALRRPDLVFVKTGF






TCTGATGGGAGTGTACCGGG
GAGAGTGGCCTGCTGAGAGC
ALVVDVTVRYEMAYDTLMGA






AACCTCGACTTGTAAGCACA
AACACTTGTGGTGCTTAAAG
AAEKVARYTPITPYVAMTLK






GGTTAGTATGGGAGCAGAAG
CGGGGCGGCCCATGACCACC
ARRVKVFGFPLGARGKWPGS






GGGGAGCCGTAATGGGCTTC
GTGAGATAGGACACTGCACA
NDRLLKAMGVGGGRRKQLAK






TCTTTCACCTGCTTACATAA
GTGCAGCCATGAGGTTCCTG
LFSRRALLYSLDVLRDFYRA






TACCTGTGGTGCATGTATCT
GAGGATGATGCGATGAGGTG
EGETGDLDDESVDDHL






AGGTCTTGGCGGGAGAGTAC
GGGGCCTCATCAGCCCCTCC
(SEQ ID NO: 1463)






TGAGAGACAAGGTTGAGACC
TGGCAGGGCGTCGGCCAGGG







CCAAGATTGGGTCTCCCTAG
AAACTAAATGTCTCTAGCAT







CCTCTATAGCTGCGACTCTT
GTCAGTGCAGTGAGGTAAGG







AGCGGGGATATGGAGTAACA
GGAGAGCACTCTAGTAGGGC







TGTACCAAGGGAGTGAATAA
TCTTCCAACCCCTACCTGTA







TAAAGGAATTGACGGGGTAC
GGTCACCTGGTCCAGGTGTC







AAGTGACTGTTGGCCCGAAT
GATGATGTGAAAACAAGAGC







CCTAGCCACTTGATGACCAG
TACTTTGGTACCGGTCTGTT







GGATATATTACAGACTGAGA
GCAAAAAGGGTTCTGCAGAG







GCGAATCTAGAGACGTGAGA
GACGACGGCTATCCCTATCG







ATAAAGTGAGAATTGGTTGA
GGAGGGAATAGTCGGTCCCA







GCGAATACAGAGGAAG
GGTAGTGGAAAATGGGGCTT







(SEQ ID NO: 1218)
TCCACTGAGCATGAAAATGT








GGTAGAGGTTGCGTCCAACC








CAATGATTTGCAGCAGAGCT








CTTGGACACGAAGTCTGTAT








AGTCCCATGCAGGCAGCCAA








CCAGAGAATGGTGGCAAGAC








CCCAGCTCCGTATGGGAGGG








GAGGGCCAAGATATACGGAA








CGGCTGCTAAAGCGTTCTGC








CGGTGTCAGTCTAATCACAG








ACAGCTGTGACGAAACAAAG








TATGGGTTCCGACATGCTTG








GTCAGCTCTTAGCCGCAAGG








CTTAAATCGAACGCAGCCCG








CCGAGAGTGAACATTAAACG








GGGATGGAATGTGTCTAGCG








GTTACGTACTACCAGGGCTC








AGGTTCGCCTGAGCCGAGGC








TCTACACGTCATGGTGGGAG








TTCTCCCCACGCTCGTGAGG








GCATGTAGTGGGATGGCATG








TGGCGGACCATCAGCTGGCA








CTACCAGGCCTCGGGCTTGC








CCGAGTGCGGGACCTCACAC








ATTGTAGGTGTGCTTGTCCC








CCCTACGTTCGAAGACTTGA








GGCGGAGAATACTCATAGGC








CCCACGGCAAAGGGACACAA








CACGGAGGCTTGTGTCCGAC








GAGCCGTGGACTCCTATAGA








CAGCCCGGGATATCACTGGG








CACGCTCATACTGAAGAAAT








TCGATGAACCGGGCCTACCG








GAGCAAATGCACTCTAATCG








CCTTTGTGGGCGACTGTGGC








CCCCTCATGCGAGTGAGGAA








TATCATAAACTGCAATGGTT








CAAAAAGTGATTCCTATGGC








TCGTCGGGGAGGGCTGACTG








GGGCAAGCAAATGATTGAAA








GGGGAAGAACCTTTTTCAAC








TGTTTCTTGCCAAGCCCGGT








TGATGGTGGCGCTAGTAATT








GCGACGGGAAAATGCGGTTT








AAGTCTCCGAAGTAGTGCGT








AGCACCGGATGTCGACCGGG








TGTAAAAGCCCTTCGTAAAG








TCCCTGGGGAGGTCAGTCCT








GGGGCTACTGATGCGCAGTA








TGTAATTCGCAGAATAGGGC








CATCGATACCGCCTGCGTGA








CTCGACTGGGTTTCCACTTG








AGGATATCCGACCGTAGCGT








GCACCCTCTTGTAGTTGCGC








CGGAAACGGCTGTGTTCCCT








CACGTATGTGAGGAAACTCA








ACAATGTGAGTGGGTAAACG








GCGGGACGAACTATGGCTCT








CGT








(SEQ ID NO: 1341)









R2
R2-
.

Tribolium

AGTCATAGAGCCAGAACCTC
TGAAAAGAGTGGCAGTTGTG
MSRRPGKSNEPPVRSRAMGL



1_TCa


castaneum

CTCGTGGTCCCGCTGGGCAC
GAGACTTCCTTCGGACGTCG
TTLSGTKTSNSGAQGPSTSA



s


AGGGATTAATTTTTCTGTGG
GGTCGGATTTTCGGACGCCA
PMQNMAGGFVCDCGRSYALK






CAAATTTGACTGGCTTCAGA
GGGTACCTCCACCGCTGGGT
TSLARHKKECGKNNAECRWC






GAGCGTTTTTCGAAGTGGWC
TCACAAACTAGGCGAACATC
GTRFNTLAGTRQHERKAHFV






TGTGTGACTGCGTTCCCCCC
TGCCGATACCCTCTTTAGGT
QYQSDLAKALPQPESELMEK






TTAGTTGCTATWTCCGCTKM
CATAGGACCACATGTCTCTG
IAIVEARSXNGIFYKEMMAS






GATTAACATCTCACCTCGAC
CACGAGATTAACCCA
TGLTHQQVRSRREKPEYKGF






GTWTAAGATCATT
(SEQ ID NO: 1342)
LERARRSLAQTNIRAGSISP






(SEQ ID NO: 1219)

ASTXAGSLESASPKAGCSSS








ASPGPTTRSRAPTKGVPXRS








SNSARIVVEAQVHTRAPPNT








GETEVALRESRRTVPRLGXN








PSRPCGISPLMAIAIDEDSV








LGGLRVQAGPSPTAVHSVEA








FPGTSSMTPMETDRVHNKSG








IDPILEHNGTRQVRREESST








REDPVEQWSPNYPKTPVTMP








NITTTADAXXTSYNRTPQTL








PGNRRRRSRSLPPVQRKSAS








DXLESVDSLGPWAVFLQDQV








DAGSLSGNDSLADLVRVALT








KSDRGVLNDAVNRYLAQRAE








SLRIRKRGSKGKRKSKTGRH








YGQTTSGSGQRAALFKKHQD








LFLKNRRGLAETILSGKEDF








GPRPEPPVTSVEEFYGGIFE








SPSPPDNEPFEVRATGVEDP








PPLTSPWTKRTLRSPCYWRR








RPPPTYITMDEIKAARAGWQ








ISAPGSDQIPVAAVKTMSEL








ELAILFNIILFRNVQPSAWG








VLRTTLVPKDGDLRNPANWR








PITISSALQRLLHRVLAARL








SKLVSLSSSQRGFTEIDGTL








ANALILHEYLQYRRQTGRTY








XVVSLDVRKAFDTVSHCSVS








RALGRFGIPSVIREYILATF








GAQTTIKCGSVTTRPIRMLR








GVRQGDPLSPVLFNLVMDEL








LEKVNEKYEGGSLOSGERCA








IMAFADDLILIADRDQDVPA








MFDDVSTFLERRGMSVNPAK








CRALIAGAVSGRSVVRTGSS








YKIHNTPIPNVDALDAFKYL








GLEFGHKGVERPTIHNLSVW








LNNLRRAPLKPDQKCLFIRQ








YVIPRLLYGMQNPQVTSRVL








READRLIRRHLKTYYHLNVH








TPDSLIHASVSDGGLGIMEL








RKAIPRIFLGRLVKLLNKNK








DSVLSSVLQSNRVRTLMGKL








STMAGEVPESTFWRNQIASG








PLSKGLEQAAEDSASRLWIS








EKPSGWSGRDHVRAVQLRTG








NLPTKAIPSVPVGQRRCRHG








CACDESISHVLQMCPLTHAD








RIRRHDEVVKKVARHCTSRG








WTVEVEPHIRSRCGRLFKPD








LAVHQPGGAIVIADVQVSWD








SESLTVPYERKRAKYDVPQF








HQAAQHAWPGKALTFAPVIV








GARGIWPRINNDRSAALQIP








PVVRRACVNSVVKWGSSIHA








TFMRSVWANRLNPRPLRA








(SEQ ID NO: 1464)





R2
R2-
.

Tinamus

CTGGGGACCGTGGTTACAAC
TAGGGGGCTTGGCATTTCTC
MGSWIVNFVSVATQTGEFPV



1_TGu


guttatus

CCGGGCTTAGCTGCAGAGAC
ATTGCCTGCTCCTGAAAGGA
DTARRAPVPVTSYPESECHX



t


AGTACCTCCCCGTGGTTCCC
TATGGGTCCTGCGTCGCGTG
PLPLTFCNSDVTIWGGVRPE






GCCGGACCCCGTAACATCGG
GTAGGCAGACCCATTCGTCC
PVDCLGDLPEXYDALPGVAG






GTGACTGAATCTGTCTCTGC
GAGTAGGGGGCTTGGCAGTN
PREXVGGSPPGEGVRSPGIA






CCCGGGAGTAGTTCCTCCTT
TCCATTGCCTGTGCCCGAAA
SPSGTAVQHDFGSPILVPGA






GCCCTATTGACCAGCGGTCG
GGACGTGGGTCATCTGGTCT
EAAEVSTPVVKVPQDHPACP






CCGGCTGCTCAATAGTATTC
GTCTGCCTACACCTCTCTAG
CCGTRVVKVTALSEHLRRAH






TAGGCGTGAAATATAGCGAT
ACTTGTAACATCTAGTCTGT
GRKRVLFQCSRCGRMNEKHH






AGTCCTAGTGGTTGTCTTAC
CAACAAGATCAAAATTCTTC
SIACHFPKCRGPPVEEGPLG






TGGGCCATAGCCCCTTGCTT
ACACAGACGACCGAGCTTGC
APEWCCEECGQKFNTKSGLS






CAGGGGTCATTCGCGAAGTC
TCAGTCTTCCTGTACCCGCA
QHKRSVHPLTRNVERIEAAR






TCTCAGGAGAACTGGGGGTG
GAATTTTGCTCTTGCTCTCC
PKGKGKRGAHKGCWTEAEVA






GTGTTCTTCTGGGTATAGCT
TTTGGCTGTGTCCTGGACGT
QLIELEGRFKNQRFINKLIA






AAACCCCCTAGACTGTGTCC
GGGACTATTCCATCTCGTCC
EHLPSKSAKQISDKRRQLAA






GATCC
CAAATGCCGCGTCCAATTAT
ATKTSSPEKRVTSSTSGESS






(SEQ ID NO: 1220)
ACCGGATTTGACAAAGCGGA
PEVEKVEGIKREYRRRVGEW







CGGCCCGCTTTATAAGCCGG
LCAGSLXDQTSFQKILEDVE







AAAAGGTGCCTTGTAAAATT
SGSEIVTGPLEELASFARGK







GCAAGGTTCATTAAATAG
LAAARVRHHRKHPAEAVPAR







(SEQ ID NO: 1343)
EEQRWMKRRVGRRGLYLRFQ








RLFALDRRKLAGIILDDVES








IKCPLPMEEVADVFRRRWEE








VAPFTGSGSFRSLGKADNGA








FKPMISAKEVMKNVXEMSRR








SAXGPDGLSLRDLMKIDPQG








SRMAELFNLWLLAGRVPDQV








KAGRTVLIPKSADPGKIGNI








DNWRPITIGSVXLRMFSRIL








SARLRRACPINRRQRGFIAA








PGCSENLKLLQALIKSAKRD








HRTLGVVFVDLAKAFDSVNH








QHIFQVLVQKGVDGHIIDIL








RDLYTNAGTYLESGSQRSGF








IKILRGVKQGDPLSPILFNL








ALDPLLCRLEDRGLGYKYGD








QQIXSLAFADDLALLSDSWE








GMQQSIRVVEEFCQRTGLRV








QAPKCHGFLIRPTKESYTIN








DCDPWTIADMQLDMIDPGSS








EKYLGLGIDPWIGLSRPELS








EVLTRWVKNIGGAPLKPLQK








VDILRSYALPRLLFIADHAG








LSATCLHSLDLSIRSAVKGW








LHLPPSTCDAIIYVSYKDGG








LGLPRLASLIPNVQARRLVR








IAQSEDDVIRSVVLQEGIQE








EIRKVWISAGGRPEKVPSVT








GEFPVMEAQAADEALSEWER








RAPRTIYPIPCKWRKREMEN








WTNLKSQGHGIRNFENDRIS








NDWLLHYGRIPHRKLITAIQ








LRANVYPTREFLARGLGEGA








PRGCRHCPAEWESCSHIIGY








CPAVQEARIKRHNDICGVLA








EEARKLGWVIFIEPHLRDNT








NELFKPDLVLVKGSCAKVVD








VTIRYESGLTTLSDAAAEKA








RKYQHLAGEVRALTSATTVD








FLGFPIGARGKWYVGNNGLL








SDLGFSTSRVVRIARALSKK








ALLSSVDIIHIFASRARQAQ








TSE








(SEQ ID NO: 1465)





R2
R2-
.

Trichinella

CTCCTGACTAACCTGATTTC
TGAGGTTTTTGTTTTCTTTT
MSNRLANTAAAGGVPEKTSG



1_TSP


spiralis

GTCCGTGCGGCGGCGTTTTC
TTCCTTTTACCATTCTTGTT
TLDIPGQPSSSGEKRAISYP






TTTTCGCTCTCCGCTCGTCG
CCATTGTTGTTATTTGCTTT
GPFGCNSCSFTSTTWLSLEL






AAATTTGCTGTAGTTGATTC
AATCCTGTATTTTACCGCCG
HFKSVHNIRDFVFLCSKCKK






GCTTTTCTTTGCGTTTTCTT
GCAATTCCATTGTTATTATT
SWPSINSVASHYPRCKGSVK






CTACTTTCGCAGTTTTTTCT
ACTGTTACTGTTATTATTGT
AAVVPTSLANTCTTCGSSFG






GCATTGCCACG
TACTATTGTTTTTACTTTTA
TFSGLQLHRKRAHPDVFAAS






(SEQ ID NO: 1221)
CTTACTACTGTTATTATACT
CSKKTKARWSNDEFTLLARL







TTAATTCGTTAACTTACGTT
EAGLDPACKNINQVLAERLM







ATTGTTACCACTACTTACTT
EYNITRGVEMIKGQRRKDQY







TGCTCTCTCGCAAACGTTCG
KALVRQLRSNSETQQCVGLA







TTGTTGTTTCTTTTGGACCA
GSMDSNVPANDTSSSVASEV







GGTTTAGAGAAATCGCACGC
SITYPEYGAVMSCDLIKEAT







ACAGCGGAACTGGACCGCTT
GMAIVDINELQSNLRKAFLS







AAGCCAGAAATAGTAAAGTA
GRKLPMKFHGARETAQKKMA







ACAA
NPRVAKFKRFQRLFRSNRRK







(SEQ ID NO: 1344)
LASHIFDKASLEQFGGSIDE








ASDHLEKFLSRPRLESDSYS








VISGDKSIGVAHPILAEEVE








LELKASRPTAVGPDGIALED








IKKLNTYDIASLFNLWLKAG








DLPASVKASRTIFLPKSDGT








TDISNCRPITIASAMYRLFS








RIITRRLAARLELNVRQKAF








RPEMNGVFENSAILYALIKD








AKVRSREICVTTLDLAKAFD








TVPHSRILRALRKNNVDPES








VDLISKMLTGTTYAEIKGLQ








GKLIPIRNGVRQGDPLSPLL








FSLFIDEIIGRLQACGPAYD








FHGEKICILAFADDLTLVAD








SAAGMKILLKAACDFLEESG








MSLNAEKCRTLCITRSPRSR








KTFVNPAAKFIISDWKTGIS








SEIPSLCATDTFRFLGHTFD








GEGKIHIDTEEIRSMLKSVK








SAPLKPEQKVALIRSHLLPR








LQFLFSTAEADSRKAWLIDS








IIRGCVKEILHSVKAGMCTD








IFYIPSRDGGMGFTSLGEFS








LFSRQKALAKMAGSSDPLSK








RVAEFFIERWNIARDPKVIE








AARRVYQKKRYQRFFQTYQS








GGWNEFSGNTIGNAWLTNGR








ARGRNFIMAVKFRSNTAATR








AENLRGRPGTKECRFCKSAT








ETLAHICQRCPANHGLVIQR








HDAVVTFLGEVARKEGYQVM








IEPKVSTPVGALKPDLLLIK








ADTAFIVDVGIAWEGGRPLK








LVNKMKCDKYKTAIPAILET








FHVGHAETYGVILGSRGCWL








KSNDKALASIGLNITRKMKE








HLSWLTFEIIFITQISRIYN








SFMKK








(SEQ ID NO: 1466)





R2
R2-
scaffold_

Tetranychus

CTCTCTTATTTTAACATATT
TGATGTATCCCTTCAATATA
MCILGGLTSHSREGGLSRGS



1_TUr
6

urticae

CGATGTACTCGTACATTGAA
TTGTAATCCTCATTCGTCCC
SQLKTVKPQNEEDNGTTQLK






TATGCTTTTATTTTTTTTCA
TATCCTTTCATTTGATAAAA
AGSADSFPRPSGDLNPEEPL






AAGTTTTTTGGGTGCATACC
GAAACTTTGTTGCTCCTTTT
SIDICPVCFROMKSYLGVRV






CCTGGAAAATTCTGAGATGT
AATAGTTGGTCCCTCCTGTC
HMQKMHLEEYNASIPDPVVS






ATAAATCTCCCATCAGCTTT
CCTTTTCTGGAACCTGGTTG
HTRWSDEEAAQLAFTEAKIE






GGCTGAAACGTTGGCTAAGT
TATCGATTATTGAAAGTTGC
VDKLLPRGKGINKFLLELLP






TTTGTAGGTTGTTTGCCCCC
AATAAACGGATTTAA
GRTLESIKSHRKRQSHKDLV






TACTACTTAGTCGCAAATGG
(SEQ ID NO: 1345)
RKYVKEFVDTLAADNDDDTI






TATTTGCTAACAGTTGTTAA

ICQDNGDIFNDPIVGATDSQ






ATTGTTACATTTACAAGTCC

SETETVADPAEFKTFIELAD






TATCCAGTGCCTCCTCGTGG

DPTKPKVVAKLRNLIKDKPK






CGCTACCCGGTAACACTTAG

SEILGSDILVRILRRTLHGL






AGTAATCTGAGTGGCTAAAC

PVEDELDQYLEVYFTGKIKQ






TGGAAGGGCGGAAAATGCAA

RRSKTQTALSKKQIKQRDYG






ACAGGCGGTTGGTAGATGCT

RLQELYSRSRKRCANEILNP






TCGGCATTTTGCCAAAAATC

TSMSGGFGHQELSEFWTKTF






CACGGCTTTTTAGCCCAACA

GPDEQPTLGEVEIIPKENCW






ACATCAGGGTGATGGACCCG

WDIFSPISSDEIKASYPSIG






CCAGCTTGTGGTCAGGATCC

KAAGPDNFSAYQLRKVPVWH






CATCCATGAATAAAGCATGG

LECLYNIFAFYKDIPSRLKD






CTCTGCTTCTGGTGCATCCT

AKTILIPKKDNAESPGDFRP






CAACGGGATCGGCTTCGGCT

ITLSSIITRHFHKILATRVN






GGATGTAAGTCTTGCGGAGG

NFVRFHPMQRGFIQSDGCLE






C

NTALIQTVIREAKVRRKQVH






(SEQ ID NO: 1222)

ITFCDVRKAFDSVRYDSIIA








AIAKKGAPGSFIMYLSNLYR








GNKTTLLTAGGETRITPTRG








VRQGDPLSPILFNCVMDQIL








TALPSRTGFTLSAGDESVNV








NCLAFADDIILISKTKNGHQ








ELLDVTQRILKENGLDLNPD








KCCSLSLIPHSKTKKIKVVR








ADFVVNGVKVRSMSIGDSTC








YLGVSINVTGQVAPVKMYQA








LCEKLDSAAIKPHQRLYILK








HFVITKMFHPLILSTIAAHK








IKNLDLISRRYVRKWLHLPH








DCGSGMIHAKVSDGGLGVPL








LFRTIADLKVRRKEKLQVHE








NPIFRILAKLSTVSKELENC








KKIASKTTDIQEKTFKEMLA








TYDGLSLKEARAVPEVHKWV








DSYDKRYKFAGRDFVQVIQA








RFNALPTRSRVWRGRGADEK








SLRCRAGCNARETLNHVSQS








CFRTHRVRTARHDKILDFIC








ERLDVVGVKYVREKPISFPG








KKLIPDLIVENTDQALVLDL








QIVGDNSELPLDERGKNKVI








KYNCSEMQELYKRKKKTLAV








KALTLHYKGLMAPETSNILR








SFGFKSKDLEKMAYMALFGT








VAAWGIFNRSTETMRSVANW








PRPEEL








(SEQ ID NO: 1467)





R2
R2-
.

Drosophila

GAAGCTGGGAAGCTGGGTCG
TAGATGTACTAACCTCTAGT
FERRSNSWGYQNLEPSNVGQ



2_DWi


willistoni

GATGAGCGCAGAAGGGGTGT
TTCTCTATACTTTTGCCTGC
DMNTVPRINNTTTTPATSRP






TCTTCGGAGCACTGTAATTC
TACCTTGGCATTACATCTAA
GDQPREAIAVVNLAGEIPCA






ATAAGTCGTAAGTCTGATCA
AAAGGTACAAACATCGCATT
VCGRLFNTRRGFGVHMSHQH






AGTCGACTCGGAACCTCTTC
GGCAAAAAGAGGTGGTTTTA
KDELDTQRQREDVKLRWSEE






GTGGTGTTTCCTGGGTGCTG
GTACATAGGCGCTGTGGGAC
EAWMMARKEVELEASGNLRF






TTGAGTTCCTAGTCTCTAGG
TTCATTGTCCCGATGATGCA
PNKKLAEVFTHRSSEAIKCF






TTCTCTCCAGTAGCTAA
GCGAATCGTGCATACGAGAT
RKRGEYKAKLEQIRGQSTPT






(SEQ ID NO: 1223
TGTCCAGTAGTTGGTTGCTC
PEALDSITSQPRPSLLERNH







GTATCTTTAGAAGATTTCCT
QVSSSEAQPINPSEEQSNWE







TCCTCGGCGATCAAAANAAA
IMRILQGYRPVECSPRWRAQ







AAAAAAAAAAAAAAA
VLQTIVDRAQAVGKETTLQC







(SEQ ID NO: 1346)
LSNYLLEVFPLPNEPHTIGR








SNLRRPRTRRQLRQQEYAQV








QRRWDKNTGRCIKSLLDGTD








ESVMPNQEIMEPYWKQVMTN








PSTCSCENTRFRMEHSLETV








WSAITPRDLRENKLKLSSAP








GPDGITPRTARSVPLGIMLR








IMNLILWCGKIPFSTRLART








IFIPKTVTANRPQDFRPITV








PSVLVRQLNAVLASRLASKV








NWDPRQRGFLPTDGCADNAT








LVDLILREHHKRWKSCYLAT








VDVSKAFDLVSHQAIIKTLQ








AYGAPTNFVSFIEEQYKGGG








TSLNGAGWSSEVFIPARGVK








QGDPLSPLLFNLIIDRLLRS








YPREIGAKVGNTMTSAAAFA








DDLVLFAETPMGQTLLDTTL








GFLASVGLSLNADKCFTVSI








KGQAKQKCTVVERRSFCVGE








RECPSLKRTEEWKYLGIRFT








ADGRAQYSPADDLGPKLLRL








TRAPLKPQQKLFAHRTVLIP








QLYHQLTLGSVMIGVLGKCD








RLVRQFVRRWLDLPLDVPVA








YFHAPHTCGGLGIPSIRWIA








PMLRLKRLSNIKWPHLEQSE








VASSFIDDELQRARDRLKAE








NVQRCSRPEIDSYFANRLYM








SVDGCGLREAGHYGPQHGWV








SQPTRLLTGKEYLHGVKLRI








NALPSKSRTTRGRHELERRC








RAGCDAPETTNHILQKCYRT








HGRRVARHNSVVNAVKRGLE








RKGCVAHVEPSLQCDSGLNK








PDLVGIRQNHIYVIDVQVVT








DGHSLDQAHQRKVERYDRAD








IRSQMRRFFGVTGEIEFHSV








TLNWRGIWSGQSVKRLIAKD








LLIAEDTKLISVRAVNGGVT








SFKYFMYCAGYTRS








(SEQ ID NO: 1468)





R2
R2-
.

Petromyzon

CGGTGCGTTCCCTTGGGTAA
TGAGAATAATGAGGTGCTAA
RPQTKLMTDKLKFSSQLARG



2_PM


marinus

GGAACACGAGTCTTAGTGGC
CCTCCCTGGGCCTGACCAAA
LAKQRAMDGARVGDPPITVR






CTTGACCTCCACGTGGTCCC
CCCAGAACACATCACTGGCC
PTETDLCNTEGSWGRRPMKL






GCTGGTAACATCATCTCTTG
AAGATGATTTCCCGCAGCAC
LFVSVSTQTQNEDALWASDV






ATGATGGCTAACAAGGCTAA
GTTGCTTTTCTCTCTCGATA
AKPMASRSALKMTSIPSMTF






TGCACCCATTCCATCTCCTA
CCCGAGAATGTTCTGCGGAA
HNSSLEKEEEMNYDFYEQIK






TCTCGCATGGAGGCCGCTAT
CTACGCAGTCTATGAACAGT
SLVESDDSSDDFTEDDEDVE






GCGTGATTACTAG
CACAGACAACCTCTGATCCA
ESFLDISAEEPVLGKFPIDT






(SEQ ID NO: 1224)
AG
KGTITVVLPSLEYICVICKQ







(SEQ ID NO: 1347)
HMGKASELVAHFNIKHRDIP








LVFKCAKCDKTNSNHRSIAC








HAPKCGGIKLTEESLPMVCE








CCQARFATLSGLSQHKRHAH








PVTRNEERIKDGIKGTSQRG








VHRSCWSLKEVEQLALLELQ








FQGKKNINKIIAEALGTKTN








KQVSDKRRDLSKKTGAPMSD








SLHFSSRPLETLSPPPNVTT








GTSSILAQAAERLTNENSGT








LEKPAMEAIKAWLNGEGQHD








ALVETATALMLCPMRLVKNK








GKRSKPENDIIKPRILPTRS








WMKKRAEKRGSFMKHQKLFF








KNRSLLASLVLDGTERHECR








IPNADVYRFYCEKWEKVLPF








NGLGQFKSSGVANNEYFEPL








ISVEEVQTAIRAIKPTSAAG








PDGLTRAAICAADPEGRTLT








ALFNAWMITGIIPKELKKNR








TILIPKVMDDEKLKELGNWR








PITIGSMILRLFSRIMTARL








ARACPLNPRQRGFIAASGCS








ENLKVLQDLMRHAKKLHRPL








AVMFIDIAKAFDSVSHAHIL








WVLRHKKVDEHVVGIIQNAY








DRCTTSFKSNGESTREISIR








VGVKQGDPMSPLLFNLAMDP








LICTLESHGVGYSIDTDHVT








ALAFADDLVLVSESWVGMAA








NLAILESFCGLSGLEVQARK








CQGFMISPTKDSYTVNNCDP








WTIKNKDVHMIQPDESTKYL








GLKICPWTGIIRSDLHVQLK








TRISKIDEAPLKPTQKVELL








NAYALPRLLYPADHSDCKQS








TLRVLDQEIIKAVKGWLHLP








ASTCDGLLYARARDGGLAIL








KLENAIPSVQVRRLQRIANS








SDAIARNIASSQGVEEEYRS








LWVRAGGDSEAIPTFFLRGS








ESKEPVYPRPCDWRKRESRR








RCEKPVQGRGIVNFAQDRIS








NAWLGPRCGFKQCFFIAALQ








LRANIYPTRESINRGRDGAS








RSCRKCSARLESLSHILGQC








PAVQKFKDCATQTEAEMVPH








GPAGNALPGWSLSRTFLVNV








PQYKNSRIARRNKISDILAD








EAARLGWWVYKEPRFTSEAG








ELRKPALVFAKGEEALVIDV








TVRFELSRKTSSEAASHQVA








YYTPPCDQVKVLTKASNVTF








FGFQVGARGKVAP








(SEQ ID NO: 1469)





R2
R2-
.

Schmidtea

AGTCATAGGGTGAACTGCAA
TAGAAGGGAAACAAAGGAAA
RKELVTIKNLFEESGATAPA



2_SMe


mediterranea

TTCTGACACGATGACCGAGC
AACGAAATGACTGGAAACTA
PVPLEVAVEVHQSSSVPEIT



d


TGTGTCAGTTTGCAGCTAGT
TGAAGGATATAGCTGAAAGC
DESTTTQEGSYSEPPIHRCE






CGCTAAAGACTCGATCAGTC
CGCAAGGAAGGCTAAGTCCT
NCGREFRTRAGVQQHRRKAH






CGCCAAGTGAGGTGGCCGGG
GAAACCGATCTACATCTTCG
TNEFMEEKEKAAPTKKLRWT






TATCTGCAGCACTAGAGCCA
ATCCCAAGAGGAACTGTGGG
DEEKEILIESEIKIIKEGSL






CTGGTATCAAGAGCAGAGAT
TTAAGCTTGAGCCGACGGAA
KEQHEINKILASRMPGRSQD






ACGCGAGTGGAAGTTGAGTA
AAAGCGAATGCATGTTAGAC
GIAKIRQKQEHKAEIQRRLH






CGACTACCTTCACGGGGTCC
GACGAGGTACAGTCACCTCC
GTVTTNETRGNRTSEITEPI






TCCTGATAACCACAGTGGAC
TCGTGGTATTTGGCGGGCAA
RSLPINTKTWSEDEMKRMLA






TGTGGGAACTAAATGTGTGC
TGCTCACTAAATTAACTGTG
EEVKLRTKNEKDINKKLAEI






TCAGCGTTCCCTACTTTCTC
AGTAGCTGAGAACTGTATGT
FPNRTMGSIKSKRTKDKDYQ






GTAGGGTAAAGGGTATGATA
GTATCATGAAAAAAAAA
DLVKLTMQTISENPDNETDF






ACCCAGAGAATATCCCATGG
(SEQ ID NO: 1348)
NTSNTENNSTDAEKEVKNYL






GAGATATCCATGGAAAAAGC

NMLLLTINEEEWLTSTLKEA






ACCACGTTAGACAATCCGAT

ATLALQGKKTEASEKLNEYA






GGTCTAACTCGGCTCCGAGG

SKTLFPGLKITNQTRKREKK






GGCTAACTATCCCAAAGGGC

ISKRETRRQEYAEIQKLYKK






TTAA

NISSAAEKAINGKWSIKPEE






(SEQ ID NO: 1225)

EYHNNKDLIKAWKPILEAPP








FSDCRPIENIKEMDYALMEI








STAEIFLAIRAMGKTAPGPD








GIKYSKLKKNIQSMAILFNT








CLLTSFLPLPLKIARTILIP








KQENPGILDYRPLTIASVVT








RVFHSILAKKLDNNAQLSQR








QKGFRKCDGVAENIVILETI








LTNSRSEKRPLCMAFVDLRK








AFDSVGHESIIRGAKRVGVP








PMLLEYISSSYQNASTNLFG








EILNSRRGVRQGDPLSPILF








NFVIDEALENLNRNIGYLLK








EEKVSCLAFADDIVLIAETK








GGLENHIEKLLEKLNGAGLE








LNASKCATLMVMKNGKEKST








YISTKAIKIKENDIPTMKAT








ETYKYLGLOMGFKAREQNAN








EVITEGLENITRAPLKPQQR








IHILRDFLIPRLIHKLVLGR








VAKKSLKRIDQNIRKKVRNW








LHLPKDTTAAFIHADAGDGG








LGVPALEHTIPLLKRERITN








LRKSNDPVTKECLRMEYTKQ








VLGKWSRPTKIGETLATNKS








QLKEAFRKQMLITLDGKGLK








DHHETPTIHKWIRRGENMTG








KQFITAVKIRGNLVATKSRN








SRGRPEQEKLCEAQCGRPDS








LGHILQGCWRTHGMRVERHN








NICRRIKAIMKGKESEVVEE








PRLQTNEGLRKPDLLICHKG








KIIICDAQVVADSSNCSLES








ENQRKIDYYKKDSVVSEARK








LIGRVDEDIIIMAVTFNWRG








AISKTSIRDLDMLLDIKSKE








VIKMSRKIIRDNSIMVEMHR








NRTEKRR








(SEQ ID NO: 1470)





R2
R2-
.

Tribolium

TGGAAGACCCCGCCCATGAG
TGATGCTCCTTTGGTTTTAC
MKSRSFRRIGDCAAGSSRRG



2_TCas


castaneum

GCTTGGAGAGTGTGATCCTG
CATCTGTGGGGGCATCGGTC
VRLTGKAGREGRFAASPHLS






ATCACACTTGAAAAGTTATG
CTCACGGTTTCCTCGGGTTT
PRYLAGSVSGNVPSVPPGPG






CTGAGTACGTCGTGAGAGTC
CCTATTGTTTTTCCTAAACC
LGAGAPAFAAGRNADGGPAQ






GGTAACTGTCCCAGGATGGT
CGACAAGGAGCCCTTTGGCC
NPCPYCARSFTTANGRGLHI






CTGGGATAGGCTAAACCTCA
CTCCTCCTTAAACACCTCTC
RRAHPDEANNAIDIERIHAR






GCAGGGGAAAGTTGTAGGGG
CTTCATCCTSTTAGTCCATT
WSHEETAMMARLEAGAIQRG






CCTGCCACCCCTACACTTTT
CCGGCTAAAATGATGAAGAC
GVRFMNQFLVPRMPGRTLEA






ATAGATATGGCATTCGATAC
CGAGGAGTGTCACTCTCTTG
VKSKRRDATYKALVQRFLQA






CTCAAATAGAGCCTCGGACT
GCGGGGTTAACCCGTCCAAG
PQINLPELRDGDAPRQPDPQ






TGGAGGAGCATGGTTCCCCT
TGTAAATGTGACCTCGCCAT
QENPPEPPSFDGAIRGAVAD






CCTCCTCGTACTAGACCTGG
TCGGGCTCTGATA
LVGGVDWQRLGFQGDRLCNI






AACCAACGGTCTTGACAACC
(SEQ ID NO: 1349)
ARRACDGGDVSGQLLGWLRD






CCATTGGACCTACGGGAGCG

VFPVKRVSTRGDQSDLDVDG






GACCATGCTCATGGACATGG

ALVSRRTARTREYARVQELY






ATTCCGAAGACGAAGCGGGG

RKEPKACLARILGDRREGAN






GAACACGGACCCCCCGCCGA

RAPNRDPAFIDFWRGVFSEA






TAATGCTCACTTAACGTCAG

SAEVEGWAEEVSDHGELARR






GCGAACCCATCGAAATCATC

VWDPISVEEVGRSRVRNGAA






TTGATGTTACCCTTTCAAAG

PGPDGIAVSVWNKLPPEAAA






CAGGTCATGCGGCATATGTC

LLFNVLLLGRCLPAELTRTR






TCAATGCCGGAAAGGGTGGC

TVFIPKTDAPRTPADYRPIS






TCCCGGGCGTAACGGACATC

IASVVARHFHRVLSARVQRI






TATCGCTGATGGAASCTAGC

PDLFTKYQRGFLSGVDGIAD






TCGCTCTTGAAGAACGGCGG

NLSVLDTMLTMSRRCCKHLH






ACGGGGACTTGGAATCGTGG

LAALDVSKAFDTVSHFAIVR






TGTGGTTCTGATGTAAGTCC

ACRSIFGSAETVLEEGGRRH






TGAAATTATGGCGTGATGGC

FVQVRXGVRQXDPLSPLLFN






CCGCCCGCCCGACCGGAGGG

LVLDRALKRLSTDVGFRLTD






ACTTAGAACCCCCTTCCGCG

ATKVTALAFADDVVLCATTA






AGGGTCCTGTCTGTAGGTCC

RGLQTNLDVLEAELRLAGLL






WCCATCTCCGTAAAACGAGT

LNPNKCQALSLVASGRDHKV






TGGAGGAAACCGCAGACGGG

KLVTKPTFKVGQNTIHQVDA






G

SSIWKYLGIQFRGSGMCGCG






(SEQ ID NO: 1226)

SEGVAAGLKRITCAPLKPQQ








RMHLLRVFFLPKFYHAWTFG








RLNAGVLRRLDVVVRTSVRT








WLRLPHDIPVGYFHAPTKSG








GLGIPQLSRFIPFLRLKRFD








RLGRSAVDYVRECAFTDIAD








RKIRWCRERLSGIVDQVAGG








RDALDAYWTAQLHQSVDGRA








LRESASVASSTQWLRCSTRA








IPASDWLHYTAVHIGALPSR








VRTSRGRRGGQDVSCRGGCL








LDETPAHCIQVCHRTHGGRV








LRHDAIAKRISADLMELGWI








VTREVSFRTTAGVFRPDMVA








VKEGVTVILDVQIVSPAPTL








DEAHRRKVAKYRDRADLARY








LAEAAVARGRAPPANIRFAS








ATISWRGVWSAESVGSLREL








GLSARHFDRYTTMALCGSWR








NWVRFNASTASRMGRGRGDA








SPRRHENQQ








(SEQ ID NO: 1471)





R2
R2-
.

Megachile

TCTAGTTAGCAAGCGGCCCC
TAAATGTCGAACCGAATTTT
SGPATSTFGETKSRLCEPTS



7_MR


rotundata

CTCTAA
GGGTAACGTGCACCCCACCA
ALGCRPGAVVIQWAQIHKEK






(SEQ ID NO: 1227)
TCCTTAATCGGCAGCACGCA
RKRIVGWPLGHLGSPTSLKL







ATAAAGCCGTGGGCAGTGGT
RHPRLQAKRIVPVLAELMQC







TTTAGTGGGTAGTCATTAGG
LCARHVSGRSPQKSAWAFTL







AGTCCCACAGTACCCAGCGA
EERTSACGVDAMFVCSTCQR







ACATCTTAGTGGGTCTGCGT
SFATKIGLGVHVRRAHVEVA







AAACGCATTTCCACTGCCTA
NAAISVERVKDRWSEEERRI







TCCTCCGGGAAAAAAAAAAA
MAAVEVRGVLSGARFINEYI







AAAAAAAAAAAAAAAAAAAA
MSHLQTSRTLESVKGTRKNP







AAAAAAAAAAAAAA
KYKELVATLLEEARTSVREE







(SEQ ID NO: 1350)
SPRSAVNDSATQPSGPSDTR








SLRTEHLFTESTEPFEHRIR








ELIGDLEGVTDFRAELLVSI








AEQQLQGDEVAESLTRWLGE








VFKPENQQQQVQRKRRRQRK








APVSGQLPKWRERRRDYAAM








QTLFHRNPSLAAGRVLDGKN








ESRPPDLPEMTAFWEPILTE








QSAEHRAVGPASEKSELCSV








WGPVEKEELLSSVPPLDTAV








GPDGVTARQWRAVLPAVRAL








LYNIILKRGSFPASMLESRT








VFLPKKQHSVNPADFRPISI








ASVVVRQLHKILAMRLRRTN








LVDERQRCMDDGCAENITVL








ASLLDDARHGLKELHLVSLD








CAKAFDSVSHHAIDATLKEC








GLPAGFVQYISRTYSDSSTR








LEVGRNRSEPIKTNRGVRQG








DPLSTLIFCLCFDRVARTLS








PHIGYDLNNTRISTLLYADD








AFLVSTTAPGMNILLRSVEE








SAGEVGLSFNTSKCSALSLI








PSGKEKKMKVGTTPTFKTSQ








GFITQITPSQEWRYLGVDFQ








YSGPKKASRSLKIELERISK








APLKPQQRLLILRVYLLPRY








YHHLVLSRTTLGHLRGLDLQ








VRAAVRRWLSLPRDIPIAYF








HTTAKEGGLGLPAFETSIPC








LMLARLRSMETSTCKAARAA








VQGFWVQKRIHWATAALTKN








GEALTCKADVDRWWASRLHK








SVDGRELRECSGVGSSSTWV








NSALNITGRDYVQYHHVRIN








SLPTRIRTSRGVRREGMEVT








CRAGCQVTETAAHVIQSCHR








THGGRILRHNAVCKVLASGL








RDKGWEVREEPKLRTRQGLR








KPDIVAIKDGVARVIDAQVV








SGSGPLDEAHETKRKYYSDN








GDVTAAIARECNIAPSNVAY








SSCTISWRGVWSPRSAADLL








QVGLSKKLLGFITLRVLRGS








HLNWTRWNKMTTMRVHHQRT








GIG








(SEQ ID NO: 1472)





R2
R2Amel
.

Apis

TGGTAATCAAATGCCTCGCT
TGATCGTTAAAAGTAAAAAT
MSSNEEGASDTGAPGPGVPV






mellifera

ATTTTAGTAGCGGTAGCGCT
CTATTTATTTATTTTTATTC
ADVSAADGRATYDDHGMSTD






CCGCCCGCGCAGGAACCATT
CTATATTATAACACATTATT
YEKQTIELPLNGQIQCLWCH






GACGCCGCCGTAGTGTGGGT
TATTTATTTACTTATTGTTT
IEGRNQRFLQESQYLKHKDT






GATTTTATATCCAACCAATC
TAAAGATGACGAAGCCGCAA
QHPKGEIIWRCAACQKEFEK






ACGTCAACTACGATCATTTG
GGCCAATCCAAATTTAACAA
LHGCRCHLPKCKGRKEAKGV






TAATCACCGACGGTACTTGG
AAGAACGAGACTACTGGTCG
AKFKCDSCEESFLTQRGLSM






TAGGGGTACCACATGGGCAT
ACATTAAAAAGACGAAGCAG
HELHRHPAIRNLKRTQGTSR






TCTTGCTCATTCCACAACGC
CTGCCAGCTGATAAACAACA
GNTRPINRASVWSKEETDLL






CGCCTCCATCATGGCAACAA
GAGCCCGTCTCGGCCTTTAC
IKLNERYKHLKQPNVALKEY






TTTAAAATATATATAAATTC
ACCGAGCGGTGCAAGTCCTG
FPDKTLKQISDKRRLLPVQE






TTAAGGTTTGACCGTATTCA
ACGTACTATTGTACGTCTAG
PEDVATTDETGPPPSDSSEE






TATATATATATATATATTTA
GGCGCGGGGCAGATTCTACC
SIYESATEDEGGGDMQQTAP






ATATTACAACCATAAATCTT
GTGTAGAATCTGGGGCGACG
NDSWKEPFIQSIRTNHLEEE






ATATCGAGCCTTCTATTTGG
CCTCCGCGAGGCACTCCCTG
DSLRKVEEAIERMAMNEGVT






TCTCAAAAGCAATACGTTGT
GACAACGTACGCTAAAGCGT
EQEVGTLLEQFVDSLTQSPT






CAGATCTTGTAGAACATCAG
ACGGCTAAGTGCGCCTCCCG
TERKGSRRKSQKTTKRKTTH






GAGTGAGCGGTGCGCTGTGG
AAAGGGTCCCCGTTCCTAAT
NNRKKFLYAKHQELYKKSPR






TATCCGTGCTTTGTGCCGCG
TTTTCCGAGCCCGCGGGCAG
RLLELALSGESSSGREVVNL






GCGACAAACCAATACGCTGC
ATCTCGTGGCAGTGACGCTA
PEADSVGPLYKSLWGQIGPE






TGCCTGTCGCAAAGCAATAC
GAAAGTTAAGTCCGCGGACA
KTHRNQPMCNNIDMSEIWTP






GCTGCTGATTCTCGGATGCG
TATAAAATTACAGCCTTAAA
IALESLVEKFKKIKSDTAAG






GGTGTCGACGGTCACGCAAA
TAATGAACCCCACGAAGGAG
ADQIKKFHLRKKGALHVFAK






GCGATACGCTGGTGGGGTTT
GTATCCTCGAAATTCCGCCA
LCNLLMLHRIYPAQWKTNRT






CAAAACAATACGGCGCTGGT
CGATCCTTCTGATCGTAGGC
TLIPKPGKSAEEVENWRPIT






GCTAAAAAGCATTATGCCGC
GCAAAACA
IGSLLGRIYSAMIDRKLRSK






TAACGGCTGGATTGTCGATC
(SEQ ID NO: 1351)
IKQHIRQKGFTQEDGCKNNI






GCCGCTGCGGGGGCTAGTGG

AILSSALTKMKEDSGGIITI






CGCACCCAGAGAGGTGCGAC

IDISKAFDTVPHGEISQSLM






GCGCAAGCATTGGTTCTGTG

NKGVPSPICEYIQKMYIGCK






CGAAGCGGAGTTCTTGAGAG

TIIYCRDKKTLPVDILRGVK






TAATGGTTGCTGGGGGCACA

QGDPLSPLLFNLIIDPIIGT






AAGCGCAACATATAGCCTCT

LDETTEGIKLENENISVLAF






TATGCCTCAAGTCGTAGTTC

ADDLVLLAKDKETADKQNRL






GTACCTCCACGTGGTCCCGC

INEYLDDLKMKVSAEKCTTF






TGGAATGCCTATCGACTCCT

EIKRQNKTWFLGDPQLTLGQ






CCCCGGAGGATCATAGAGTT

QRIPYADPEAAIKYLGTNFN






CGAAACCGGCTACGGCGAGG

PWRGLCKTSIKEIIDAARTV






CAAGGGCGGTGAGGTGCACA

KQLKLKPHQKINLIRTYLLP






CCGATGGGGAGCAGCGACCC

RYIHKLVANPPPLGTLDLID






CACCTACCCTTAGCTAAGAG

KELKTIIKEILHLHPSTTDG






AGCAGGCGATCCGCCAACTG

LIYTDKSHGGLGIQRVANIV






TCAGCACGAAATAAACTAAT

KLAKLKHSILMTRSEDNAVK






CATATGTATACGAGGGAGAA

IALNGQEGMVKRYATSIGLQ






TTTACAACGGGTACCTTGTG

WPCGIEEIEETRKKLKRADT






CCCGAACCGCCTGTAGGTAT

NKWKTLISQGQGIKEFFGDK






CACCTACAGGTGTTAAAATG

TGNAWLYNPEMLRPSRYLDA






AATCTGATAGCTGGCGGATC

LKLRTNTYGTKAALHRAKRD






GTCGACCCTCTTTGATGGCT

IDINCRRCGVQVETLGHILG






CTGCGCCAACGACTGGAAAG

LCTHTKNKRIKRHDEICDLI






AATAGGAACGGAAGTCTAAT

AKNVSKEYVIFREPEVEVNG






GGAAGGAAAGTGTCGGGAGC

DRRKPDMVIKDHDKVYVVDV






ACTATAAATTCCCAAAGAAG

TVRYENNDSLNKAYKEKENK






AAAAGAAAAGAAAAAAAATA

YKETAEIMRRDLKAKESRVL






AAAAACCCAAATTAA

PVVIGSRGAVPRATIENLKV






(SEQ ID NO: 1228)

LGLQTKHALTASLIALRSSI








EMANEFLDYDHTT








(SEQ ID NO: 1473)





R2
R2B
.

Nasonia

GACTAGACTATGGGTTCAGT
TGACCTGAACAAAACGTGTT
TFAPTHPMVRSGPCRKTKRP



_N


vitripennis

CAGTCCCAAATAGCCGATCC
GTCTTGTCTTGTCTAAAACT
GSDYRESLIMDSGNNVASEP



Vi


TGGCGCGTCCGGCAGTAATG
ATTTATTCGAAATAAGGGGA
RGAVDVTSAAPIGAELNAEP






CCACGTATGAGTCGGTTACC
GGCTAACTGCCTGCAAGTTG
CEGRNORREAALSAQTRRRN






CATCTCTAAACGCGTAGAGG
AACGCGAAAGTTAGACCTTC
XARRARNAQQADEPGDDEEI






TGGGGAGCTAAAGGCCAGGC
CCACCTAAAGCCCAAAAGTG
ETHGPLTIRTXEPMEIVAIA






GGTTTACCCGACGTCGAATT
ATCGGGGAATGAATCCGCGG
KNPQACPKCLQGGTQLLCMG






TCTCCAGGTCTGTGTCAGTC
GTGACCCCAGAGTTGGGTAA
SWELSRHINKEHPSVDVTWV






GACGGAATAAAGGTACTACA
ACCCTTGAAACGTTGGAGAA
CGACQRRCTTLRSWSCHVLH






ACATCTACTATCTATCGGGA
GCGGAAGAGAGTCCCGCCAC
CKGRQEPKDLPFKCEHCSLS






TCGGAAGACGCCTTACAGCG
CGAGCATCGAGTGCTGCGGC
FDSQIGLSQHERHVHPEVRN






TTTTCCGATTTTTGCTCTTT
GCCCGAATGAAACCGATCGC
DKRAAEANKPKGKSGRRPSI






GAGCATTTTTCTTCAAATTG
GGATGGTGCAAGTCGTAGGA
WSDEDLLLIRELESEYHGAR






CGATAACCGACCCGATCACG
CGGGGCACGACCTAAGCCTC
NINEKIAEHFPDRTGRQVSD






CGGGGCTTTGACAAAGCAAT
TGTCACGGCGGCGAAGCCAG
ARRRKDYAALRGRGGPQGPA






GCGTGGTCGGTAAGATGGTT
GAATCACCATGCAAAGGTGT
EGVEAIEEVDEGEIPEGEEL






GCAATCTTTTCCACCTCGTT
GAACTGGGGCGGATACCTCC
VATDGAALESGPPENGGSAP






TCTTTTACGGAACGAAAGCA
ACGGGGTTTCCCTGGGCATC
AEQVNAPALESSSQQDRECS






ATGCGTGTGGGGAACGTTAA
GCGCGAGCGATGGCCAAAGT
PAVGSDEQIEDSSDDDEFSD






AAACTCCCTTCATGCATCCC
CCGCTTTCTCAGCTACAAAA
ALGEISLPEPLSVERTTISP






AGGATTTATCCTGCTTACTG
CAAAAATGGTATGAGACTTC
PPRDDWKGPMRWEICNASEE






CAAAGCAATGCGTGTGGAGC
GTTAACACTAATTTTTCCGA
AGSYANWVTGLQELVRNNAL






GACTTTACCACGAGTCGCTC
GCCTAGCAGGCTCCCTTGAC
SEIGLDSLYDQLIQIMRHPS






CACCGCAAAGCAATGCGTAT
AACGCTTATGAATCTGGAAA
DDNEQDRLQLNARGPPRRGH






CGCGCAAAAGCAATGCGTGT
AGGACACAAAGTGGAAAAAG
RKNRRRRRLTAADRKRFAFA






GGGGGACTTGTCAAAGATCC
CGCTGATGGTGGACAAAAGT
RCQDLWNNNPKKLAELVIAN






CCCGCCGCAAAGCAATGCGT
CAGTTGAGACTTGATATCAG
DLSILQRRQAPGRTETQTLY






GTCGGCACCACGTAGAGCAA
TTGTTTTGACTAAGAATTTT
NELWGRVGPNIEAPRRTEDP






AGCGTGTAGGCAGACTTTGT
ATTATCGTTGACTTTTAAAT
IPVSRIFTPITPQEIMGRIR






CAAAAGTAGTTCTGCCGCAA
ATTTTATTATTGACTGTTAA
RIKNDSAAGPDGVTKDDLRG






AGCAATGCGTGTGGAGATCT
TATACTGACTTGGGACCAAG
RGVSIALSKLFNSILLAGYY






TCGCCGGTGAAAGCAATACG
TCATCTCTGTTACCCGGTAC
PKAWRENRTTLLPKPEKDPA






TGTGGGCGAACTTAG
CGGTTCCTGTCATCAAACCG
DVKNWRPITISSMVSRVYSG






(SEQ ID NO: 1229)
GAAAGTCCGTCCCACGTAAT
LLDQRVRAVIKQCDRQKGFT







GTGGTAGACGCAGGAG
EENGCFSNIQLLDDAVSNAK







(SEQ ID NO: 1352)
KAGGVITILDVSKAFDTVPH








AVIQGCLEKKGIPETVAAYI








SSMYRDCSTAIRTRSGDVKI








GMKRGVKQGDPLSPLIFNLV








LEPLLERLQETSGVEIEGMN








LSCAAFADDIVCFANTAPEA








GRQLRMVADYLGRLDMSLSV








SKCIAVEYVPHRKTWYTKNP








GLEVNGNAVPSISPSETFKY








LGAKVSPWKGLLEGFESDAF








REVISRVQRLPLKPMQKVDL








LQMYIFPRYTYGLITSPPAK








AVLKTIDRIIRTRIKEILHL








PESVSSSFLYTPRKQGGLGL








LEVEKMVLIAALRNGLRARQ








SHDPVTRAAMNSNAADDRLK








SYADALRLHWPLTTKELDTY








KYQLRLSYAQKWAEQKWQGQ








GVEEFAQDPVGNSWLQRYDL








LPASRYIDAIKLRTNTYPTR








ALMKIIDGRVDSSCRKCQGS








SETLGHILGRCRYTKDKRIS








RHNEIKDLLKARLAKNHQVM








DEPQITVRGQRFKPDLVVKT








NEGRVHVIDVTVRYEHRTYL








DEGRTEKIGKYRQILSTLRR








DLHSNAEEVIPIVIGSRGAI








PRETRKALSKLGIGKSDWLT








ISLIALRSSLEIVNAFMDD








(SEQ ID NO: 1474)





R2
R2Ci-B
AB097

Ciona

CGACGGTGAACCACCTTGTC
TGACAGTAATATGAAAACAT
MGEWPWVSWSLTVLVEKWRP




122

intestinalis

GCGGTGTAAGAGCTTTAGTG
CACATCTGACCGGCACAGAA
FTILQPYPMPGQLRVDVYLP






TCTCGAACAAGAAATAGCTT
TCACCATGCCGTAATGCACC
RKTSYLMDKNIYENTTSPGG






GTGTGCTGTCCTTCTGGGCG
CAACTAAGGATTCCAATGGG
GPLCGEKTHRSDVIIPPPGF






GTGCACATACTTCTTAACCT
TAAAAAAAAAAAAAAAAAAA
APSTDTASNTLGENVDASAT






CCCGAGGCCATGCCGGCGGG
AAAAAAAAAAAAAAAAAAAA
TSSANPLSQEPGWCESCSKL






GGCTTTAGCCCCCGGCAGGT
AA
FKSQRGLRVHQRSKHPELYH






TTTACCATGCCGGACGGGTT
(SEQ ID NO: 1353)
SQNQPLPRSKARWSDEEMVI






CGAGAGGTAGAGGCCAAACT

FAREEIANRKIRFINQHLHK






AAGAGTTCACCAGCAGACTT

VFPHRTLESIKGLRGKNVRY






CGCACGCGGCTGGCCACTGG

ARIMADLEAEMTSQPEAATS






CCGAAGTTTAAACAACAGGG

LCTETSENLASSNVLPQTRG






CCGCATCTTCCCAAACTCAA

WAENLVENIDTAHLANLGPL






TATATGGTGTTAAGTGAACC

SQFEPGKPSSSTKEAINTEY






GTGCCG

NDWISKWLPSGAAHRERRAN






(SEQ ID NO: 1230)

PPSTKLNARATRRLQYSRIQ








NLYKLNRSACAQEVLSGAWK








VQSGELNLKEVQPFWEKMFR








KESAKDRRKPKPTGEVLWGL








MEPLTIAEVGSTLKSTTPSA








PGPDKLTLDGVKRIPIAELV








SHYNLWLYAGYQPEGLREGI








TTLIPKIKGTRDPAKLRPIT








VSSFICRIFHRCLAQRMETS








LPLGERQKAFRKVDGICHNI








WSLRSLIHNSKDNLKELNIT








FLDVRKAFDSISHKSLGIAA








ARLGLPPPLITYISNLYPNC








STKLKVNGKISKPIEVRRGV








RQGDPLSPLLFNAVMDWALS








ELDPRVGVQIGEQRINHLAF








ADDIILVSSTKIGMVSSINT








LSRHLAKSGLEISAGKEGKS








ASMAIVVDGKKKMWTVDPLP








RFKVNSQKIPALSITQQYKY








LGINIDAQGARNDAARILTE








GLAELSRAPLKPQQRLYLLR








VHLLPKLQHGLVLSSCAKRA








LTYLDKSVRSAIRRWLTLPK








DTPTAFYHAKACDGGLGITR








LEHTIPILKRNRMMKLTLSE








DPVIMELVKLTYFTNLLHKY








SNVKLLNSWPVTDKDSLARA








EASMLHTSVDGRGLSNCSDV








PRQSDWVTNGASLLSGRDFI








GAIKVRGNLLPTKVSAARGR








QREITCDCCRRPESLGHILQ








TCPRTWGPRISRHDSLLKRV








RNQACLKNWTPIIEPSIPTN








IGLRRPDLVLAKGNIAFLVD








ATVVADNANMQLQHEAKVEK








YNNSDIKEWIKVHCPGVDEV








RVTSLTANWRGCLYGGSASF








LTEDLGLPKAELSLLSAKIN








EKGYYLWCAHYRGTARLWNR








PLRS








(SEQ ID NO: 1475)





R2
R2C_N
.

Nasonia

CGGGTTCCCCCGACTTCGGC
TAGCGGACTGGACTGTCTGG
WVTSPRRPRYVGPQKKKASD



Gi


giraulti

TTGCCGTGGTCTGGGGCTCA
AGGAGTGTTTAACTCGGGTT
GNDGRAAARAEPTNPGGPDR






CTGCTTTTTGTGGAGTCATG
CTCATGGGAACCCGACAACG
ADDDEGDVKFWCEFPGCDRF






GTTACATGGTGACCCTGGTT
TTGTTATCTTGTATGACAAT
FMTRSGRGLHHKKGHPDWND






CCTCGCACCCCCGCTGGAAA
TCATAAAAAAAAAAAAAAAA
QRNLAGKQHRKEIWSEEERL






CTATCTGGGGAGGCCATGAT
AAAAAAAAAAAA
LLAKKEAELAISGARFINVE






TGGGTAACGATAAAGGTCCT
(SEQ ID NO: 1354)
LRDFTARSLDAIKGQRKRPD






GGTCGTGTCCTCCTGAGATA

YKILVEKFVRELRVRGIRQG






GGCTGAATGGGTCACTAAGT

VASRSQQARAMAVAGAPAAT






GGCACCTAA

SSGAPPVATQPPPSGRVLRS






(SEQ ID NO: 1231)

QVVEAPAMEIPVAESEGDSS








GDELFEDVEPVRLSDLPPDR








FTIYFAGLEIPGTEDIYAHR








LHTICLMTTWRTKEEVRLEL








GLFLKDLFPSKGSQERPERT








NLPDPRNRIERRRGEYKKCQ








DLWRRNKSTCVQRILKEDLS








QGECLPRELMEPFWNATFTQ








NPGTAPVLPPPTEVYSSVWE








PIRPENIKGNYPPQNTAAGI








DGLTVGDLKGVSREMLARIF








NLFMWCGKLPEHLCASRTIL








LPKKPGAKVPGEFRPITVTS








VLIRTFHKVLAERLKVVPLD








PRQRGFRESDGCAENVMLLD








MTIRYHHERRRKMFLALLDM








AKAFDSVSFESMREVLTTKG








IPTPFIEYFMTHLEDSFTVL








QHGNWQSGKIHPTCGVKQGD








PLSPPIFNFIMDEMLKRLPK








EIGVNLDGLFVNAMAFADDL








SLVANTEQGLQILIDEATSF








LGLCGLRANPNKCVTLAIKT








IPKEKKTAIDPSSHFRIGNA








VIPSLKRTDEWVYLGIKFNS








NGRLISDAKPKLIKDLELLT








KAPLKPQQRLWALKVIVIPG








ILYRGTLGSSTAGYLRSLDC








VIRAYVRRWLRLPGDCPNGY








FHAAVADGGLGVHPIRYKAM








VDRLARLRKLEKSAYITGPE








AARYLQRQVSIAENRLRDGA








NRIMSDASMLREFLRELLYK








SFDGRPLENSSKVPGQHRWV








EEPTRFLSGADYMNCIRARI








AALPTAARCARGRLKDKHCR








AGCGNVETLNHVLQFCHRTH








GTRIGRHDAVVKYVVGGLKK








RGYAVKEEPKIVLQDVVYKP








DMVATKEGKTLILDAQVLGD








QRDMRLAHEDKLRKYGAPEF








KRKIRSETGSATIKSLSVTL








SWRGLWGPDSVKGLLEEGVI








LKKDLKILSTRVLIGALAGW








RRFNERTSMATSGRREEVTT








RMVRRWKRRERVGVG








(SEQ ID NO: 1476)





R2
R2La
JN937

Lepidurus

GGGGTAGCAATTGATCGATT
TGATAATCGCTCCATCCTGC
MSGKSSKPRTVSSGSSSQET




617

arcticus

CCCGCTTCCTCGTGGCGCTA
AACTAATTATGAATGCAAAT
PPSGSNACDICGKCFMKPVG






CCCTGGGTAATACTATGAGG
CTGTTAAGTGACATTAGTGA
LSRVHPSQYHARLEKNQPKA






AATTGATCACACCGTAGCGA
TACTTACCTGATACTTACCC
KKFRWTDEDLYFLAKKEAEL






ACGTCATCAGTCACAGCTGC
TGGTATTTATTTGACCTATA
LHLGSIKFVNKELAEFFPEK






ACGAATCCAGATAGAAATAT
CTTACCCTGGTATCTACCTG
SVDQIRGQRRSETYKQQVLS






AACAGACGAGTAATTCTTTT
ACATATATTTATCTAACCAC
IHSELLKLQTVADSPPPSRI






AAAAGCTCGTCAGTAATCTT
CTACCTATGATGACTCCCGC
PAKEVSAWLDFFLALPKTKN






CCCAG
GGAAACTCTCACTTACCTTA
KFSEDKLDQLIRTAQDGTLI






(SEQ ID NO: 1232)
TTACCCACTTGGTCTTTTAT
LDDLDLYLREVLVQPTSQGE







TTTCTCGTTCCTTATTACTT
KQAKLLPPPKSSREKRDREY







TGTTCCTTTGGTGTAGGGTT
ARAQNLYRKNKTACVNAILD







CTCTGGTTTTTGGAACGGCT
GNKKCENKIPDIDDFWKTIF







TCCTTAGCCGGAATTTTGTC
ESHSPPDAEPVCYVVDEEPT







TGATGTATCTTGCTTGTGTC
NIWSWISFFEMNHNYPDSST







CTTGAAATATACGACCCAGG
SPGPDGVTARMLRSIPARVL







CTTGCGTCATTTAGGCTCTG
NKLLNLLLFIEDLPAVFKCH







GGAAA
RTVLIPKIDNPTSPGEFRPI







(SEQ ID NO: 1355)
TISSIVVRQLNKIIAARVSE








GVPINPRQKAFRQIDGCAEN








VFLLDFILRDAKTKIKSLSL








ATVDIKKAFDSVSHHSIFRA








IRGARCPENLVNYIQNSYSG








CTTQISVGGSISASKIPMNR








GVKQGDPLSPVLFNLVINEI








IRKLPASIGYPINSELSINC








IAYADDLILVTNTREGLKLL








LGLLNEELPKRGLELNASKC








FGLSLTALGKLKKTHLCTSD








QLDLHGTLIKNLTAEESWVY








LGVPFSHIGRSKSFSPDLEA








LLNKLQKSPLKLQQKLFALR








VYLIPRLLHGLVLSRVAIGE








LKIMDKLILKHLRVWLRLPK








DTPLGFFYSPVKLGGLGIKN








LRTNVLKCRKQRIERMLVSP








DDVVRLVAESEIFLKETDKL








KDLLTINGMCLDXRNVPRTG








KNNKFWSERLYTSFDGKTLA








YSEYFTQGGGWIREDKILQP








AHVFAECIKLRINALPTKSR








VAHGRPTKDRSCRAGCLDVQ








KVPTIETINHIAQVCPRTHG








ARIKRHDRLVQFLSLNLRKN








PKRNVLVEYNFRTVAGIRKP








DIIVIEDTRAVILDVQVVGD








SSNLEMEYLEKSRKYSNDAN








FINALQKLYPTVTNLTFHAV








TFNNRGLIAKSTVAALRMLG








VPPRCIMILCVISLEKTLEV








WRMFNQSTASARK








(SEQ ID NO: 1477)





R2
R2LcA
.

Lepidurus

TTTGGGGTAGCAATTGATCG
TAGTGCTTGAGTGATGCCTA
MSEESRPKQTASKRGAAVEK






couesii

ATTCCCGCCTCCTCGTGGCG
TCCTTTCTTTGATTAACTCT
TMMSGTYVCTLCGRSFEKSV






CTACCCCGGGATAGCCTCAA
TACCATATACTTACCAGTTC
GLSLHTNRMHPEAYNKLKEA






AGAAATTTGACGGTAAAGCA
TTACCCGTACTTACCCTGTA
KKPVLKKARWSEEEVFLLAQ






AAGAGGAATTGATCACCCAA
TACTTACCTGTGTGCGTACC
KEAELSFIGGIKFMNIELHK






GGCAGTACATCGGCCTTCCT
TGTGTACTTGTCCTTTAGCC
IFPERELEGIKGQRKNPTYK






GCAGGAGCTCTGATAAAGAT
GCCTTGTGTTTTTACCATTG
AQVVSLLAEIRESKANDSSS






ATTAGTGAGTTATTCTGTTG
GTACTTACCTTGTGTGGTTG
SSSSSSSCDSASLGISNWLE






AAGCTCGCTATTTCATTCCC
CCCGATACTTACCTTGTATT
FLLALPKTSNQFQEGRLDRL






CTG
TGCCTTGTAATTCTGCATGA
ISDALRGVDVLENLDAYLLE






(SEQ ID NO: 1233)
TATTTATTGTGTAGGTTCCT
VFAKPMAQNPCPKPPPPAKN







GATGCTTACCTGATTTGTCC
SRERRDREYSRVQNFYKKNR







CCCTCATCATCTTTAGTTTC
SACINSILDGNTRSQNVIPG







GTTCTATTTCACTCCATTAT
LTKFWTETFEKNSPPDDEAP







GGAGTTCCGTTTGTTTTTTG
DQFVADEPRDMYKWITFYEM







GTGGAGGTACAGCACCCTTT
SQDYLDSSTAPGVDGFSAKQ







AAGCTGGAATTGAGTGAGTT
LRSMSPRVLNKILNLLLLSE







TATGTACTTTGGATGGTTGT
NLPNSFKMHKTVLIPKIDDP







AATAAACTACCCGGAGGCAT
KSPGDFRPITISPVLARLLN







(SEQ ID NO: 1356)
KILAARLSKLVPISQRQKAF








LPVDGCGENIFLLDYILRSS








KKSSKSVAMAVLDVKKAFDS








VSHHSILRALNEAKCPINFI








NFVRNSYDGCTTKLTCGGTS








FPDSVRMNRGVKQGDPLSPV








LFNLIIDSAIRKLPDSIGYV








IRDGLKINCLAYADDLILVA








SSRAGLKTLLNIVAEHLSLR








GLDLNAAKCHGLSIIASGKA








KTTYVSAADSLDLDGQPIKN








LGVLDTWTYLGIPFSHLGRA








EKVSPDLTNLLNKLQKAPLK








LQQKLYAVRNFVIPRALHGL








ILSKTNLKELNTLDRAIRVF








LRTLLYLPKDTPLGFFHSPI








KSGGLGITCFRTSVLKCRLQ








RIARMRSSCDGVIQAVAESD








IFADEYAKLRDLIRINGNVL








DTTESIKRYWAQRLHSSVDG








KTLAYMDYFPQGNLWMSEDK








VSQRSYVFADCVKLRINAIP








TRVRVSRGRPNKEMCCRAKC








FDSQRMPAFESLNHITQVCP








RTHGSRIQRHDKIAKFLFKN








LNNCPSRSVLYEPHFVTVDG








LRKPDIIIYDDSHMVVLDVQ








VVSDSANLEKEFECKAKKYA








NDVALRSAMLIKYPFIKSFS








FVAATYNNRGLIAKSSVQVL








RQLGLSPRSIMVSILICLEG








TLETWRIFNQSTMNAH








(SEQ ID NO: 1478)





R2
R2LcB
JN937

Lepidurus

TTTTGGGGTAGCAATTGATC
TGATAATCGCTCCATCCTGC
MSGKSSKPRTVSSGSSSQET




619

couesii

GATTCCCGCCTCCTCGTGGC
AACTAATTATGAATGCAAAT
PPSGSNACDICGKCFMKPVG






GCTACCCTGGGATAACCTCA
CTGTTAAGTGACATTAGTGA
LSLHMSKVHPTQYHARLEKN






AAGAAATTTGACGGTAAAGC
TACTTACCTGATACTTACCC
QPKAKKFRWTDEDLYFLAKK






TAAGAGGAATTGATCACACC
TGGTATTTATTTGACCTATA
EAELLLLGGIKFMNKELAEF






GTGACGAATATCATCAGTCA
CTTACCCTGGTATCTACCTG
FPEKSVDQIKGQRRSETYKQ






CAGCTGCACGAATCCAGATA
ACATATATTTATCTAACCAC
QVVSIHSELLKLQAVADSPP






GATATATAACAGGCGAGTAA
CTACCTATGATGACTCCCGC
PSRIPAKEVSAWLDFLLALP






TTCTTTTCGAAGCTCGTCAG
GGAAACTCTCACTTACCTTA
KTKNKFSEDKLDQLIRTAQE






TAATCTTCCCAG
TTACCCACTTGGTCTTTTAT
GTPVLNDLDLYLREVLVQPT






(SEQ ID NO: 1234)
TTTCTCGTTCCTTATTACTT
RQGERQAKPLPPPKSSREKR







TGTTCCTTTGGTGTAGGGTT
DREYARVQNFYRKNKTACVN







CTCTGGTTTTTGGAACGGCT
AILDGNKKCENKIPDIDEFW







TCCTTAGCCGGAATTTTGTC
KAIFESQSPPDAEPVSYVVD







TGATGTATCTTGCTTGTGTC
EEPKNIWSWISFFEMNRNYP







CTTGAAATATACGACCCAGG
DTSTSPGPDGVTARMLRSIP







CTTGCGTCATTTAGGCTCTG
ARVLNKLLNLLLFIEDLPAV







GGAAA
FKCHRTVLIPKVDNPALPGE







(SEQ ID NO: 1357)
FRPITISSIIVRQLNKIIAA








RVSEGVPINPRQKAFRQIDG








CAENVFLLDFILRDAKTKIK








SLSLATVDIKKAFDSVSHHS








IFRAIRGARCPENLVNYIQN








SYSGCTTQISVGGSISTTKI








LMNRGVKQGDPLSPVLFNLV








INEIIRKLPASIGYPINSEL








SINCIAYADDLILVANTREG








LKLLLNLLNEELPKRGLELN








ASKCFGLSLTALGKLKKTHL








CTSDQLDLHGTLIKNLTAEE








SWVYLGVPFSHIGRSKSFSP








DLEALLNKLQKSPLKLQQKL








FALRVYLIPRLLHGLVLSRV








AIGELKIMDKLILKHLRVWL








RLPKDTPLGFFYSPVKLGGL








GIKNLRTNVLKCRKQRIERM








LVSPDDVVRLVAESEIFLKE








TDKLKDLLTINGMCLDXRNV








PRTGKNNKFWSERLYTSFDG








KTLAYSEYFTQGGGWIREDK








ILQPAHVFAECIKLRINALP








TKSRVAHGRPTKDRSCRAGC








LDVQKVPAIETINHIAQVCP








RTHGARIKRHDRLVQFLSLN








LRKNPKRNVLVEYNFRTVAG








IRKPDIIVIEDTRAAILDVQ








VVGDSSNLEMEYLEKSRKYS








NDATLSMRINALQKLYPTVT








SLTFHAVTFNNRGLIAKSTV








AALRMLGVPPRCIMILCVIS








LEKTLEVWRMFNQSTASARK








(SEQ ID NO: 1479)





R2
R2Nve
.

Nematostella

GGTTGGGGCCTTCTCGTGGC
TGATGGTGGGTTACTCGCCT
MLRGTGNMNDKRDGSATADP



c-A


vectensis

GGAGTCGTGAGTAAGGGGTA
CTGTGTAACAGGCAAATGAA
TSALLGAVGDGSLVCNLCGL






TAGGGGTAAGGGACACCACG
AGCTGCGCAAGCAGTCGATG
ACKSRGGLSIHRRSKHATVY






GACCGAGAACGGTTACCGCT
AGCCAAAGCCGCACAGCCCC
HAERQPAPRAKARWTNDEMI






CAAGGCGAGTGGTGGAAGGC
CGACTGGGGTACAGGGCAGC
LVARKQIASEKSRCSAVVEG






ATAAAATCGTAACGCCGCCC
CCTGGGCTATGCCCGAAGTC
MREAVPHRTFDAVKSLKTKN






TCCGACCTGCTCCTGAAACT
TTTTGACAGGTCCAAACTTC
RNYTRILEQIRAECSEEEVI






AATGCCAACCAACTGACTGT
ACCCTTGCCGCCAGTAGGGC
ESGVLKDRTENVCVQTTSNV






GGGGCTAACCTCCCCAGAGT
ACCAGGGCCAGGTGCAGGTG
PGSAGRAASVELEGNIQVGH






CAGG
GGCGCTTTGTTCTATTTGGT
QLAQKTMAGNNSRKQPANHT






(SEQ ID NO: 1235)
TTCGCTTAATTTTTGTTAAA
NWAEFNIEEGNITLRKSKRK







TTTTTCCCGTGCGCCCACCC
ANGMPDATHRPGPPTVDSLK







TTTTTAACCCTTTTAACACC
HPVCLLQGAADKRDEPHTVE







AATTTTTTTTACAACCCTCT
QLYYNIEEGMPLAEEQQWSE







ACTCAATAATCCAAATGAAT
KLFDAIDSSLLSVEVELGRI







AAAAGCGGCATAACAGGTGA
VPGCPDEETRQLIDREFLDF







ACAC
IHSYSREKPPQRGLAKSKPP







(SEQ ID NO: 1358)
PKGPKSLRRQQYRQLQRLWD








KNRSAAAEQALTGKWQEVRT








AAGVPLSLMEVPWREIFETP








STTDVREPAPAGPVLWQLLR








PVTIAEVEDAISSKKSASGP








DGVPCAALQTMGAASLAAHF








NLWLLAGTQPKRLTECRTIF








VPKEVNTHLPLHHRPITIGS








VVVRLFHQILGKPMEAVLPL








GSGQRGFRKGDGICQNIWLL








HTLIRRSTDLLRPLKLVFLD








VKKAFDSVSHESLLIAAKRL








GVPGPLLTYINELYSRSETV








FEVGGESSGSVKVSQGVKQG








DPLSSTLFNCVIDWAVSDLD








PHIGVLLGESRVSFLAYADD








LVLLSETEAALTSQLNSIEK








SLAHCGLKLSTGDSGKSASL








NIVIDGKAKRWVVNPTPFLR








ASGGEIRSLVANETYKYLGI








NIGAQGVKAAEYNAFKEALD








NLSRAPLKPQQRLFLLKTYL








LPQLHHSLVLSRTTGKLLNS








LDALVRKAVRGWLKLPHDTH








RAFFYAHQADGGLNVPSLYH








LIPLLRRSRYERLTRVEDPE








IREVSRTDYFKRVLGAAAAA








TTVAGHRIDSKVTLRLAWRE








ALYASADGRGLSQCPLVPEV








HSWVTDVSGLQTGSQYISAV








RLRGALLPTAVRKSRGRGGV








NSNCDCCGRGQPEFLGHVLQ








TCPRTWGSRISRHNHVLSLI








AKACRSRRWQVLEEPIIQTP








AQLLKPDLVIWNHQAAYVVD








VSVPGDNTPLSTCHNRKVAY








YSGESVREWVRSKTGHNPTV








SSVVINWRGAMAKESYRLLT








KDLRLAKTLPRLLVLRVLEG








GHGIWLNFHRSTFAVGVT








(SEQ ID NO: 1480)





R2
R2Sm-
.

Schistosoma

ATGTTTTAATTTATTTTTGA
TTGCTTATCTTCATGTTTTT
MPVSTGAETDITSSLPIPAS



A


mansoni

ACTACTACTGTCTGAGTGCT
GTGTTAATTGACTGCTCTCT
SIVSPNYTLPDSSSTCLICF






TCTTACAACCTGAAGGCTCA
TCTGGGTTGATGTCTGATTG
AIFPTHNILLSHATAIHHIS






GAAACTACCCACTTTTTGCT
TCTCTCTCTCTTTCCATATT
CPPTPVQDGSQQMSCVLCAA






GTTTATCCACAACAACAGTT
GCTTGCTCTCCCCGCTTACT
AFSSNRGLTQHIRHRHISEY






GTGAATCTATTCTCCAAATA
TCCAATAGTTGTCATATTAT
NELIRQRIAVQPTSRIWSPF






TTCCTTGTGCTTTTGTCAAC
GTCTTTGTTTACTTGCCATG
DDASLLSIANHEAHRFPTKN






ATTATTCTATACCAACTGTA
TCTAACGACAATTACTTTAT
DLYQHISTVLTRRTAEAVKR






CCACCTACTTCTTCATCTCA
CTACCTTAGTTGGTCCTCTT
RLLHLQWSRSPTAITTSSNN






CGTTTTAATTCTGGTCTAAT
GGTTTGGTTGCCTTCATGTG
HTTTDIPNTEARYIFPVDLD






TTTCTCATCATTAGTCACGG
TTCATGGCGGAATCTGATGT
EHPPLSDATTPDASTHPLPE






AGAGGGCCTATGAACGGTCC
TTATAATGACTATTCCTACT
LLVILTPLPSPTRLQNISES






GTGACGCGAAATTCAATCCA
ACCACCATTACAACTATTAT
QTSHESNRNSMHTPPTYACD






CGAATTCGTCCTCTTCTGCT
TATTATCACTATTATTAACA
SDESLGVTPSSTIPSCFHSY






AGTGGTCCCCGAAATACGGT
TTATTATTACTTCTACAATT
RDPLAEQRSKLLRASASLLQ






TCCTCTGGCCTGTCAGTTGT
AGTATTATGGCTACTCCTTT
SSCTRIRSSSLLAFLQNAST






GTTAAAACTATATAATAACG
CAGCACACCAATAAAATCTC
LMDEEHVSTFLNSHGEFVFP






(SEQ ID NO: 1236)
AATCAAACATCTCACTTATT
RTWTPSRPKHPSHAPANVSR







AAACTCTCTATTTCCCCTTC
KKRRKIEYAHIQTLFHHRPK







GTTATAAACTTACAATTCAG
DAANTVLDGRWRNPYVANHS







TTTAACCGAATATCTCTCTT
MIPDFDCFWTTVFTKTNSPD







TTACAAATCTTAAGTATGTA
SREITPIIPMTPSLIDPILP







ATTTTGTGCCAAGCCCATTT
SDVTWALKEMHGTAGGIDRL







GGGTCTGTACAATTTGATAC
TSYDLMRFGKNGLAGYLNML







TTAAAAATAAATGTTAT
LALAYLPTNLSTARVTFVPK







(SEQ ID NO: 1359)
SSSPVSPEDFRPISVAPVAT








RCLHKILAKRWMPLFPQERL








QFAFLNRDGCFEAVNLLHSV








IRHVHTRHAGASFALLDISR








AFDTVSHDSIIRAAKRYGAP








ELLCRYLNNYYRRSTSCVNR








TELHPTCGVKQGDPLSPLLF








IMVLDELLEGLDPMTHLTVD








GESLNYIAYADDLVVFAPNA








ELLQRKLDRISLLLHEAGWS








INPEKSRTLDLISGGHSKIT








ALSQTEFTIAGMRIPPLSAA








DTFDYLGIKSNFKGRCPVAH








IDLLNNYLTEISCAPLKPQQ








RMKILKDNLLPRLLYPLTLG








IVHLKTLKSMDRNIHTAIRK








WLRLPSDTPLAYFHSPVAAG








GLGILHLSSSVPFHRRKRLE








TLLSSPNRLLHKLPTSPTLA








SYSHLSQLPVRIGHETVTSR








EEASNSWVRRLHSSCDGKGL








LLAPLSTESHAWLRYPQSIF








PSVYINAVKLRGGLLSTKVR








RSRGGRVTNGLNCRGGCAHH








ETIHHILQHCALTHDIRCKR








HNELCNLVAKKLRRQKIHFL








QEPCIPLEKTYCKPDFIIIR








DSIAYVLDVTVSDDGNTHAS








RLLKISKYGNERTVASIKRF








LTSSGYIITSVRQTPVLTFR








GILERASSQSLRRLCFSSRD








LGDLCLSAIQGSIKIYNTYM








RGTQRLNE








(SEQ ID NO: 1481)





R2
R2Tc
EU854

Triops

TTTTTGGTCTGGCATTTGAT
TAGATGACTGCCCTACCCCT
MSQKRRPEKAVPDEGATAHD




578

cancriformis

CGTTTCCGCCTCCTCGTGGC
TTGCTGCCGAAGAACTACTG
VAQPDKSKCSVCGETFKGPA






GCCAGACTGGGTAAGCTGAT
AAGACTATTGAACTAACCAA
SVTMHMVKKHPVEFNELKMA






TTATCAGTGAGCTAAGAGAA
CTGTGTTAAGTAAGAACTAA
KKPVPKKVRWSEEEIFQLAR






ACGATCACCGCAGGAGTCCA
TGCCTCTTTTTCCCTGCATG
TEAELTLQGVRFINVELQKI






TCTACCTGCGCTGCACGTGA
TATCCCCTGATCAGTGACTT
FPAREIEGIKGQRKLAKYKE






TTTCATTGTGCACTTGGCGA
ATTTTCTTTTCCTGTTGCGC
LVKDQLDEIGRAPNPPEQEI






GTTATCCCTTGTGGAGCTCG
CCTTTGTTTAGTTATTTCCT
GEDVPSPFKAWLELLLALPK






CTCGTTAGCTTTTGAG
TTAATTACTAGAATTATCTT
TPNDFLEHKLDNIIVQALKE






(SEQ ID NO: 1237)
TTCGTTCTCCGTCTAATTGC
DVNSDQVFNDLNSYLKLILE







TTT
PSGRAKSVPGEIIHGDPSGS







TTCGGTGTAGGAACGGCTAC
AKTSVTKAPKPATVSSSRKK







CTAAAGCTGGAAGTGGGAAG
RRDAEFARIQRLYRKNRTSC







TGTTTTCAATGTACTTTGTG
INTILDGNTREHEAPKNMEG







ATTATAGAAATATATGACCC
FWREIFERESPDDPDDPDIF







GAGGTGCATTGTTTGGCATT
LEEEASDIWKYISFYEMCNL







TCCTCGAGAAA
YPPPSTAPGPDGFSSKDLRR







(SEQ ID NO: 1360)
MTPRVLNKILNLLLHLRDLP








QILKSHRTVLIPKTDLPTKP








GDFRPITISNILVRHLNKIL








ANRVSHLIPINERQKAFLPI








DGCAENIFTLDFILHHARTK








IKSLSMAILDISKAFDSVSH








HSIFRALREARCPIGFIKFI








ENCYGGCFTKLFCGGVKYPS








EVSMNRGVKQGDPLSPVLFN








LVIDGLIRQIPSALGFNVSD








QVKVSCIAYADDLILIATTR








AGLKTLLDLTNSYLAKRGLS








LNPDKCSALSIVASGKQKLV








YIASSEHFDLAGQKMRNLNV








GDSWRYLGIQFSHLGRAEKV








TPDLTCLINRLQKAPLKLQQ








KLYALRIYLIPRLIHGLTLS








KTNLGELKTLDKLIRKYIRA








WLHLPDDTPMGYFYTPLKAG








GLGLPSLRLVILNNRLERIL








RMKASQDIIVRTIAESETLG








VEIRKLHDLLSIDGTILDTS








VKIHSFWAERLYSSYDGKCL








CNSANFPPGNKWIGEDSLNQ








RSHIFADCLKLRINALPTRS








RTARGRPLKDKPCRAGCRNS








DGVKVIETLNHITQVCERTH








GARVKRHDRLVDFAVKGLQR








PHRVVLKEPHYKTVNGVRKP








DIVIKIPDHTYICDFQVVSD








TSCLELEFRKKALKYAEDKG








LCDQLTRDHPGELSFTAITF








NTRGLIAKSSVTALRKLGMP








PRSIMTLQKICMEGSLEIWR








IFNQTTAMARN








(SEQ ID NO: 1482)





R2
R2_DA
.

Drosophila

AGAATATGGATTTGATTGTG
TAGCCAATGCACGGGTTCCA
FERRKDPWGYRPPGTLKQIG



n


ananassae

CAGAGGGGGTGCTATACCGT
GATTAAGCTTGCTGCCGAAG
ATENNEPRNLNRFVRGESTA






AACTCGTAAGCCATGCAATC
CATACCATCAAAATCGGCAT
SSLESTQFGTSAEVNLAGRV






AGATCAAGTCGACTCAAAAC
AAAATTCGCTTAATAAAGGA
PCTICEMTFSSKRGLGVHMS






CTCCTCGTGGTATTCTCTGG
GGTGGTTTTAGTACGTAGGC
HRHKDDLDAQRLRVDKKARW






GTGCCAGTATTTACTGGTAG
GTCCCGGGACTTGTCTCGGG
SEEETLMMARKEVELAASGV






CTGA
ATGAATCGTGCATGCGTATA
RFLNKKLAEIFTHRSADAIS






(SEQ ID NO: 1238)
ATTGGGATCGATAACAAATA
SYRKRSEYKAKLEQIRGQSV







CCAACTAAGTTATTACTAAT
PTPEAEEINTTQRRPSNSEQ







ATATCGAAATACATAAATAT
NRRVPRSEGGPIAPTEQTNN







CCCGTCCTTACGTATCTTTG
EILRVLQGLAPVVCLPRWRA







AAGATTTCCATCCTCAGCGA
EVLQNIVDNAQVSGQETTLQ







ACAAAAAAAAAAAAAA
SLSSYLMEIFPPRNEPHILT







(SEQ ID NO: 1361)
RPRTEPRNMRQRRRQQYARV








QRNWDKHPGRCIKSLLEEDD








ESVMPNQEVMEPYWRRVMTQ








PSSSSIKRDMFNMEHSLERV








WSAVNQRDLRATKVKLSSSP








GPDGITPKTARSVPEGIMLR








IMNLILWCGNLPYSIRLART








IFIPKKATANQPQDYRPISV








PSVIVRQLNAILASRLSAAI








NWDTRQRGFLPTDGCADNTT








IVDLVLREHHKRFKSCYIGT








LDVSKAFDAVAHEAVYNTLA








SYGAPKGFINYLRKAYEGGG








TMLAGNGWVSEAFIPARGVK








QGDPLSPILFNLVIDRLLRS








LPSEIGAKVGNAMTNAAAFA








DDIVLFAETPMGLQKLLDTT








VCFLSSVGLTLNTDKCFTVS








IKGQAKQKCTVVERRSFLIG








GRECPSLKRTDEWKYLGIKF








TAEGRARYDPAEDLGPKLLR








LTRAPLKPQQKLFALRTVLI








PQLYHKLTLGSVTIGVLKKF








DKLVRYTARKWLGLPVDVPV








SFFHAPHKSGGLGLPSLRWT








APMLRLKRLSNIKWPHLERS








EVASSFVEEEMRRARDRLQA








GSEELLTRSQVDSYLANRLH








MSVDGCGLREAERFAPQHGW








VSQPTRLLTGKEYTDGIKLR








INALPSRSRTTRGRHELERR








CRAGCDAPETTNHILQQCYR








THGRRIARHNGVVNFLKRGL








ERRGCVVHVEPSLQGETGLN








KPDLVAIRQNRIYVIDTQIV








TDGHSLDQAHQRKVGKYDTP








DIRTNLRRSFGAFDIEFHSA








TVNWRGIWSGQSVKRLIASD








LLSSGDSNIISVRVISGGLW








SWRQFMYLSGYTRDWT








(SEQ ID NO: 1483)





R2
R2_DM
X5196

Drosophila

TTGGGGATCATGGGGTATTT
TAGCTAAATCGTTTGGTTCA
MTTRPSVDIFPEDQYEPNAA




7

melano

GAGAGCAGAGGGGGAGTATT
AAACATTTGCTTGCTGTCTT
ATLSRVPCTVCGRSFNSKRG






gaster

CTTCTGTAATTCGTAAGTCA
GGCATAACATCAATAAAGGC
LGVHMRSRHPDELDEERRRV






TATCATATGATGTGCGGAAG
ATAAACATCGCAAAATAATG
DIKARWSDEEKWMMARKEVE






GGGAATTTTACTCTGTAACT
GTTATAATTAAATGGCTATG
LTANGCKHINKQLAVYFANR






CACAAGTCTCTCCTTTACTC
AGGATGGTTTTAGTACGTAG
SVEAIKKLRQRGDYKEKIEQ






AAGTCGACTCAAAACCTCCT
GCGTTGCGGAACTTCGGTTC
IRGQSALAPEVANLTIRRRP






CGTGGTGGTCCCGGTAATGC
ATATAGAGCAATGAATCGTG
SRSEQDHQVTTSETTPITPF






TAAACTCGTTTAGCAGCTAA
CATGCTAGGAAAACTGACCA
EQSNREILRTLRGYSPVECH






TTTGAGCGGAAAAACTTTTC
CACACAGTGTTGGCAGACCT
SKWRAQELQTIIDRAHLEGK






CGATGGGCTGGTTCCCCAGA
AGTATCTTTCGAAGATTTCC
ETTLQCLSLYLLGIFPAQGV






GGAAATTTATTCATATTGGA
ATACCTCCGCGATCAAAAAA
RHTLTRPPRRPRNRRESRRQ






ACTACAAGCACAAATAACGA
AAAAAAAAAAAAAAAA
QYAVVQRNWDKHKGRCIKSL






GCCTCGGATACCTTTACACA
(SEQ ID NO: 1362)
LNGTDESVMPSQEIMVPYWR






ATCTG

EVMTQPSPSSCSGEVIQMDH






(SEQ ID NO: 1239)

SLERVWSAITEQDLRASRVS








LSSSPGPDGITPKSAREVPS








GIMLRIMNLILWCGNLPHSI








RLARTVFIPKTVTAKRPQDF








RPISVPSVLVRQLNAILATR








LNSSINWDPRQRGFLPTDGC








ADNATIVDLVLRHSHKHFRS








CYIANLDVSKAFDSLSHASI








YDTLRAYGAPKGFVDYVQNT








YEGGGTSLNGDGWSSEEFVP








ARGVKQGDPLSPILFNLVMD








RLLRTLPSEIGAKVGNAITN








AAAFADDLVLFAETRMGLQV








LLDKTLDFLSIVGLKLNADK








CFTVGIKGQPKQKCTVLEAQ








SFYVGSSEIPSLKRTDEWKY








LGINFTATGRVRCNPAEDIG








PKLQRLTKAPLKPQQRLFAL








RTVLIPQLYHKLALGSVAIG








VLRKTDKLIRYYVRRWLNLP








LDVPIAFVHAPPKSGGLGIP








SLRWVAPMLRLRRLSNIKWP








HLTQNEVASSFLEAEKQRAR








DRLLAEQNELLSRPAIEKYW








ANKLYLSVDGSGLREGGHYG








PQHGWVSQPTRLLTGKEYMD








GIRLRINALPTKSRTTRGRH








ELERQCRAGCDAPETTNHIM








QKCYRSHGRRVARHNCVVNR








IKRGLEERGCVVIVEPSLQC








ESGLNKPDLVALRQNHIDVI








DTQIVTDGHSMDDAHQRKIN








RYDRPDIRTELRRRFEAAGD








IEFHSATLNWRGIWSGQSVK








RLIAKGLLSKYDSHIISVQV








MRGSLGCFKQFMYLSGFSRD








WT








(SEQ ID NO: 1484)





R2
R2_ DP
.

Drosophila

AAGATATGGATCTGAATAAT
TAGCCTATACACTATGTTGG
SSFGLIVTNLNSETVLWGCQ



e


persimilis

AGCGTAGAAGGGGAGTCATT
AGAGAAGACGCTTGCTACCT
PLGQFSLIGTNMQNTTPRII






CCGTAATTCGTAAATCGTAA
AGGCAAAATGTGAAATTAGG
NTNSLTNQIPTVSSLGAQSE






AAATCAGATCAAGTTGATTC
TATAAACATCGTGGTTGTAA
HSAQVNPNSGYQCTICESSF






AAGACCTCCTCGTGGTATCT
AACTTGAGGTGGGTTTTTAG
RSKSGLGVHMSRRHKDEFDQ






TCTGGATGCTATTAGACTGA
TACGTATGCGTGATTACTTC
LRLRTDRKAQWSEEELSMMA






(SEQ ID NO: 1240)
GTAATCATGAATCGTGCATG
RKEIELAANGERYLNKKLAE







CTAGTGGGGTTTGGCCTCCA
VFTNRSVDAIKKCRORERYK







CTAGTATCTTTGAAGATTTT
TKIEQLKGQAVPLPEALESE







CCTTCCTCAGCGATCAAAAA
TIQRRPSIRERDLLVTPPNT







AAA
LGTTPTELSNSEILAVLQGY







(SEQ ID NO: 1363)
PPVVCNDQWRVEVLQSIVDG








AQASGKEITLQRLSTYLMEV








FPSQNDRPIQTRPPRRPRNR








RQGRRQQYALTQRNWDKHKG








RCIKAILDGTEGTATMPSQG








IMGSYWRQVMTQTSPTYSGT








NTTFRTEHPLEGVWSPITLG








DLRVHRVSLTKSPGPDGITP








RTVRSIPSGVMLRIMNLILW








CGKLPVSIRQARTIFIPKVG








NASRPQDFRPITVQSVMVRI








LNAILASRLTSSVDWDPRQR








GFLPTDGCADNTTIVDLILR








DHHKRCKSLYIATLDISKAF








DSVSHAAVSATLTAYGAPKE








FVDYVQNSYEVCGTTLNGDG








WRSEEFIPARGVRQGDPLSP








IIFNLIIDQLLRSYPNEIGA








TIGDHTTNAAAFADDIVLFA








ETRLGLQTMLDTTVDFLSSV








GLTLNSDKCFTVGIKGQPKQ








KCTVVIPETFRIGSRSCPAL








KRTDEWKYLGITFTAQGRTR








YSPADDLGPKLLRLTRSPLK








PQQKLFALRTVLIPQLYHKL








TLGSVMIGVLRKCDILVRST








VRKWLGLPLDVSTAFFHAPH








TYGGLGIPSVRWVAPMLRMK








RLSNIKWAHLAQSEAASSFL








TDELNKARGRTLAGLNELTS








RTEIETYWANRLYMSVDGRG








LREAGLFRPQHGWVCQPTRL








LTGQDYRNSIKLRINALPSR








SRTTRGRNELERQCRAGCDA








PETTNH








ILQNCYRTHGRRVARHNCVV








NNLKRILEEKGHTVHVEPSL








QLETSVSKPDLVCIRDNHAC








VIDAQIITDGLFLDDVHHRK








VEKYKRPEVISALRREFGVS








GNVEVLSATLNWRGIWSNQS








VRRLIAKGLISSGDSNVISA








RVVTGGLYCFRQFMYLAGYT








RDWT








(SEQ ID NO: 1485)





R2
R2_DP
.

Drosophila

CAATTGGAAAGATATGGGTC
TAGCCTATACACTATGTTGG
SSFGLIVTNLNSETVLWGCQ



s


pseudoobscura

TGAATAATAGCGTAGAAGGG
AGAGAAGACGCTTGCTACCT
PLGQFSLIGTNMQNTTPRII






GAGTCATTCCGTAATTCGTA
AGGCATAATGTGAAATTAGG
NTNSLTNQIPTVSSLGAQSE






AATCGTAAAAATCAGATCAA
TATAAACATCGTGGTTGTAA
HSAQVNPNSGYQCTICESSF






GTTGATTCAAGACCTCCTCG
AACTTGAGGTGGGTTTTTAG
RSKSGLGVHMSRRHKDEFDQ






TGGTATCTTCTGGATGCTAT
TACGTATGCGTGATTACTTC
LRLRTDRKAQWSEEELSMMA






TAGACTGA
GTAATCATGAATCGTGCATG
RKEIELAANGERYLNKKLAE






(SEQ ID NO: 1241)
CTAGTGGGGTTTGGCCTCCA
VFTNRSVDAIKKCRORERYK







CTAGTATCTTTGAAGATTTT
TKIEQLKGQAVPLPEALESE







CCTTCCTCAGCGATCAAAAA
TIQRRPSIRERDLLVTPPNT







AAAAAAAAAAAAAAAAAAA
LGTTPTELSNREILAVLQGY







(SEQ ID NO: 1364)
PPVVCNDQWRVEVLQSIVDG








AQASGKEITLQRLSTYLMEV








FPSQNDRPIQTRPPRRPRNR








RQGRRQQYALTQRNWDKHKG








RCIKAILDGTEGTATMPSQG








IMGSYWRQVMTQTSPTYSGT








NTTFRTEHPLEGVWSPITLG








DLRVHRVSLTKSPGPDGITP








RTVRSIPSGVMLRIMNLILW








CGKLPVSIRQARTIFIPKVG








NASRPQDFRPITVQSVMVRI








LNAILASRLTSSVDWDPRQR








GFLPTDGCADNTTIVDLILR








DHHKRCKSLYIATLDISKAF








DSVSHAAVSATLTAYGAPKE








FVDYVQNSYEVCGTTLNGDG








WRSEEFIPARGVRQGDPLSP








IIFNLIIDQLLRSYPNEIGA








TIGDHTTNAAAFADDIVLFA








ETRLGLQTMLDTTVDFLSSV








GLTLNSDKCFTVGIKGQPKQ








KCTVVIPETFRIGSRSCPAL








KRTDEWKYLGITFTAQGRTR








YSPADDLGPKLLRLTRSPLK








PQQKLFALRTVLIPQLYHKL








TLGSVMIGVLRKCDILVRST








VRKWLGLPLDVSTAFFHAPH








IYGGLGIPSVRWVAPMLRMK








RLSNIKWAHLAQSEAASSFL








TDELNKARGRTLAGLNELTS








RSEIETYWANRLYMSVDGRG








LREAGLFRPQHGWVCQPTRL








LTGQDYRNGIKLRINALPSR








SRTTRGRNELERQCRAGCDA








PETTNHILQNCYRTHGRRVA








RHNCVVNNLKRILEEKGHTV








HVEPSLQLETSVSKPDLVCI








RDNHACVIDAQIITDGLFLD








DVHHRKVEKYKRPEVISALR








REFGVSGNVEVLSATLNWRG








IWSNQSVRRLIAKGLISSGD








SNVISARVVTGGLYCFRQFM








YLAGYTRDWT








(SEQ ID NO: 1486)





R2
R2_DS
.

Drosophila

GGGATCAGGGGTAATTGCGA
TAGCTAAAACGTTTGGTTCA
FERQNFSDGLVPQRKFIHIG



e


sechellia

GCAGAGGGGGAGTATTTTTC
AAACATTTGCTTGCTGTCTT
TTNRNNEPRSNLRNLMTTRP






TGTAATTCGTAAGTCATATC
GGCATAACATCAATAAAGGC
SVDIFPEDQYEPNAAATLSR






ATATGGTGTGCGGAAGGGGA
ATAAACATCGCAAATAATGG
VPCTVCGRSFNSKRGLGVHM






ATTTTACTCTGTAACTCACA
TAATATATAAATTGGCTATG
RSRHPDELDEERRRVDIKAR






AGTCTCTCCTTTACTCAAGT
AGGATGGTTTTAGTACGTAG
WSEEEKWMMARKEVELTANG






CGACTCAAAACCTCCTCGTG
GCGTTGCGGAACTTCGGTTC
HKHINKQLAVYFANRSVEAI






GTGGTCCCCGGTAATGCTAA
AGATAGAGCAATGAATCGTG
KKLRQRGDYKEKIEQIRRQS






ACTTGTTTAGCAGCTAA
CATGCTAGGAAACTGAAGTG
ALVPEVANLTIRRRPSRSEQ






(SEQ ID NO: 1242)
TTGACAGACCTAGT
NHQVTTSETTPITPFEQSNR







ATCTTTCGATAGATTTCCAT
EILRTLRGYSPVECHSKWRA







ACCTCCGCGATCAAAAAAAA
QELQTIIDRAELEGKETTLQ







AAAAAAAAAAAAA
CLSLYLLGIFPAQGVRHTLT







(SEQ ID NO: 1365)
RPPRRPRNRRESRRQQYAVV








QRNWDKHKGRCIKSLLNGTD








ESVMPSQEVMVPYWREVMTQ








PSPSSCSREVIQMDHSLERV








WSAITEHDLRASRISLSSSP








GPDGITPKTAREVPSGIMLR








IMNLILWCGNLPHSIRLART








VFIPKTVTAKRPQDFRPISV








PSVLVRQLNAILATRLNSSI








NWDPRQRGFLPTDGCADNAT








IVDLVLRHSHKHFRSCYIAN








LDVSKAFDSLSHASIYDTLR








AYGAPKGFVDYVQNTYEGGG








TSL








NGDGWSSEEFVPARGVKQGD








PLSPILFNLVMDRLLRNLPS








EIGARVGNAITNAAAFADDL








VLFAETRMGLQVLLDRTLDF








LSLVGLKLNADKCFTVGIKG








QPKQKCTVLEAQSFYVGSRE








IPSLKRTDEWKYLGINFTAT








GRVRCNPAEDIGPKLQRLTK








APLKPQQRMFALRTVLIPQL








YHKLALGSVAIGILRKTDKL








IRYYVRRWLNLPLDIPIAFI








HAPPKSGGLGIPSLRWVAPM








LRLRRLSNIKWPHLTQNEVA








SSFLEAEKQRARDRLLAEQN








ELLSRPAIEKYWANKLYLSV








DGSGLREAGHWGPQHGWVNQ








PTRLLTGKEYIDGIRLRINA








LPTKSRTTRGRHELERQCRA








GCDAPETTNHIMQKCYRSHG








RRIARHNCVVNRIKRGLEER








GCVVIVEPSLQCESGLNKPD








LVALRQNHIDVIDIQIVTDG








HSMDDAHQRKINRYDRPDIR








TELRRRFEAAGDIEFHSATL








NWRGIWSGQSVKRLIAKGLL








SKYDSHIISVQVMRGSLGCF








KQFMYLSGFSRDWT








(SEQ ID NO: 1487)





R2
R2_
.

Drosophila

GGGATCTGGGGTAATTGCGA
TAGCTAAAACGTTTGGTTCA
TCLAANLSGKNFSDGLVTQR



DSi


simulans

GCAGAGGGGGAGTATTTTTC
AAACATTTGCTTGCTGTCTT
KFTHIGTTNTNNEPRISLHN






TGTAATTCGTAAGTCATATC
GGCATAACATCAATAAAGGC
LMTTRPSVDIFPEDQYEPNA






ATATGGTGTGCGGAAGGGGA
ATAAACATCGCAAAATAATG
AATLSRVPCTVCGRSFNSKR






ATTTTACTCTGTAACTCACA
GTTATATATAAATGGCTATG
GLGVHMRSRHPDELDEERRR






AGTCTCTCCTTTACTCAAGT
AGGATGGTTTTAGTACGTAG
VDIKARWSEEEKWMMARKEV






CGACTCAAAACCTCCTCGTG
GCGTTGCGGAACTTCGGTTC
ELTANGHKHMNKQLAVYFAN






GTGGTCCCCGGTAATGCTAA
AGATAGAGCAATGAATCGTG
RSVEAIKKLRQRGDYKEKIE






(SEQ ID NO: 1243)
CATGCTAGGAAAACTGACCA
QIRGQSALVPEVANLTIRRR







CACGCAGTGTTGGCAGCCCT
PSRSEQNHQVTTSETTPITP







AGTATCTTTCGATAGATTTC
FEQSNREILRTLRGYSPVEC







CATACCTCCGCGATCAAAAA
HSKWRAQELQTIIDRAELEG







AAAAAAAAAAAAAAAAAA
KETTLQCLSLYLLGIFPAQG







(SEQ ID NO: 1366)
VRHTLTRPPRRPRNRRESRR








QQYAVVQRNWDKHKGRCIKS








LLNGTDESVMPSQEVMVPYW








REVMTQPSPSSCSGEVIQMD








HSLERVWSAITEHDLRASRI








SLSSSPGPDGITPKSAREVP








SGIMLRIMNLILWCGNLPHS








IRLARTVFIPKTVTAKRPQD








FRPISVPSVLVRQLNAILAT








RLNSSINWDPRQRGFLPTDG








CADNATIVDLVLRHSHKHFR








SCYIANLDVSKAFDSLSHAS








IYDTLRAYGAPKGFVDYVQN








TYEGGGTSLNGDGWSSEEFV








PARGVKQGDPLSPILFNLVM








DRLLRNLPSEIGAKVGNAIT








NAAAFADDLVLFAETRMGLQ








VLLDKTLDFLSLVGLKLNAD








KCFTVGIKGQPKQKCTVLEA








QSFYVGSREIPSLKRTDEWK








YLGINFTATGRVRCNPAEDI








GPKLQRLTKAPLKPQQRMFA








LRTVLIPQLYHKLALGSVAI








GVLRKTDKLIRYYVRRWLNL








PLDVPIAFIHAPPKSGGLGI








PSLRWVAPMLRLRRLSNIKW








PHLTQNEVASSFLEAEKQRA








RDRLLAEQNELLSRPAIEKY








WANKLYLSVDGSGLREAGHW








GPQHGWVNQPTRLLTGKEYI








DGIRLRINALPTKSRTTRGR








HELERQCRAGCDAPETTNHI








MQKCYRSHGRRVARHNCVVN








RIKRGLEERGCVVIVEPSLQ








CESGLNKPDLVALRQDHIDV








IDIQIVTDGHSMDDAHQRKI








NRYDRPDIRTELRRRFEAAG








DIEFHSATLNWRGIWSGQSV








KRLIAKGLLSKYDSHIISVQ








VMRGSLGCFKQFMYLSGFSR








DWT








(SEQ ID NO: 1488)





R2
R2_
.

Drosophila

CATAAGTCTTGCCTTTACTC
TAGCTTAAAACGTTTGGTTC
FERRIFPKGLVPLTKDNHIG



DYa


yakuba

AAGTCGACTCAAAACCTCCT
ACATACATCTGCCTGCTGCC
TTNLQNEPRIFTNDLLTTRP






CGTGGTGTTTCCCGGTAATG
TTGGCACAATATCAAAAAGG
SVDHVPEDQYEPNAAATLSR






TTAAACTTGTTTAGCAGCTA
CATAAACATCGCACATATTG
VPCTVCDRSFNSKRGLGVHM






A
GTTATTTACGGCTATGAGGA
RSRHPDELDEERRRVDIKAR






(SEQ ID NO: 1244)
TGGTTTTAGTACGTAGGCGT
WSEEEKWMMARKEVELMANG







TGCGGAACTTCGGTTCGGAT
FKHINKQLAVYFANRSVEAI







AGAGCAATGAATCGTGCATG
KKLRQRGDYKEKIEQIRGQS







CTAGGAACTGACCAAATAAC
ALAPEVANLTIRRRPSRSEQ







AGCAGCCCTAGTATCTTTCG
DHQVPTSEASPITPLEQSNR







AAGATTTCCATACCTTTGCG
EILRTLRGYSPVVCPSKWRA







ATCAAAAAAAAAAAAAAAAA
QELQTIIDRAEFEGKETTLQ







AA
CLSLYLQGIFPVQGVRHTLT







(SEQ ID NO: 1367)
RPPRRPRNRRESRRQQYAVI








QRNWDKHKGRCIKSLLNGTD








ESVMPSREFMEPYWREVMTQ








PSPSSCNGEVIRTDHSLETV








WSAITEQDLRASRVSLSSSP








GPDGVTPKTAREVPSGIMLR








IMNLILWCGNLPHSIRLART








IFIPKTVTAKRPQDFRPISV








PSVLVRQLNAILATRLTSSI








DWDPRQRGFSPTDGCADNAT








IVDLVLRHSHKYFKSCYIAN








LDVSKAFDSLSHAAIYGTLR








AYGAPKGFVDYVQKTYEGGG








ISLNGEGWCSEEFVPARGVK








QGDPLSPILFNLVIDRLLRA








LPSEIGTKVGNAMINAAAFA








DDLVLFAETRMGLQTLLDKT








VDFLSTVGLKLNADKCFTVG








IKGQPKQKCTVLEAQSFCVG








SREIPTLKRTDEWKYLGIHF








TASGRVRCNPAEDIGPKLQR








LSEAPLKPQQRLFALRTVLI








PQLYHKLSLGSVTIGVLRKT








DKLIRFYVRRWLNLPSDVPI








AFVHAPPKCGGLGIPSLRWV








APMLRLRRLSNIKWPHLVQS








EEASSFIEAEKQRARGRLIA








EQNELLSRPAIEKYWANRLY








LSVDGGGLREAGHYGPQHGW








VSQPTRLLTGKEYLDGIRLR








INALPTKSRTTRGRHELERQ








CRAGCDAPETTNHIMQKCYR








SHGRRVARHNCVVNRIKRGL








EERGCVVIAEPSLQCESGLN








KPDLVVLRQNHIDVIDVQVV








TDGHSMDEAHQRKINRYDRP








DIRTELRRRFEAAGDIEFHS








ATLNWRGIWSGQSVKRLIAK








GLLSKYDSHIISVQVMRGSL








GCFRQFMYLSGFSRDWT








(SEQ ID NO: 1489)





R2
R2_KF
GU949

Kalotermes

GAGAGGTCACTGTTGCTGAT
TAATGTCCCTTTTGGCTTGC
MEVPLTSSLGSGATQAPGTP




558

flavicollis

CAGCTGGACACCTAGCTGAC
CCCCACCTGCTTAAAGGAAC
ELLGEHTVERPGLDQGHSYG






TGCGTGCATGCGTGCACGCC
TGGCAGGAAAGAGAGTGATC
LLMDDVELPVRLPFFGPLIC






GCGCGCCGCCTCCTCGTGGT
CGTGCCATAGAAATATGGTT
PGCRTLLTSEETISSHHRRV






ACCGCTGGTAACGCAGGGTC
ATCCGGGGCAAGTCACTAGC
HPDARTRWVCYGCDSPFMTY






ACTGAGACTACCTGCGGCTA
AATATGGGACTTCTCCGGGT
RAIKCHLPKCSGRKVVTGDH






AAGGCGCGCGCGAGGGATTG
CCGTGCGGTCCTTCCAACAT
ICNGCTKRFESQRGLSLHKR






TAAGTCCACCACCTCCACGT
GAGCTGGACGTAGTCCACTC
RAHPGLRNEEMLEPPVRAER






GGTTCCCCGCGGGCAACGGC
TATGACTTGAACGATACGGG
RPNAHKSSIWSIDEIRILEQ






ATAGTTCATCTGCCGGCCAA
GGCGTATCTCCCCCGGAAGA
YEAAYVGDLHINMKIAAHLP






GCGTGCCGTTCCCTCGATCC
GGTCGCCTAGGCGACTTAGA
FKTNKQVSNYRNDRRKKSRT






TCCTCTGATTAGGGATAGGA
A
ATDASQQGLGPNDGNRGIVP






GGGGGGCGGTGGCTCCCGCC
(SEQ ID NO: 1368)
SGQSSPLFLEGSDAEGDEDV






GACGACCTGGCAAACACCTT

FNVLVPPTLGGLEPAGQVHS






GGACACGCTAAGATAATAGG

LSEGETSPLVGEADPCFMGG






CCGTCCTCCGGGCCGGCCGA

TPSAGEASGSTLLGPDPTPA






ATCATAAGCACACA

DGYSLVRKDLQLSVQTSPLL






(SEQ ID NO: 1245)

AVGSVGTESVQFERGVLSCG








TPPEFLHPEQFAHCANNDPV








LNASEEQVHAPLGEEANDLP








DNNHPSELGVDPEDPTCSPA








TEQVQPSSEEEADDPFAQFK








AWRRRVASYALKIETGVLPA








QVDDLLRRLRDGDTQSKVTC








EEVEEVVLSLTRTILGGTAP








KKRVEGRTKWTYKSRTNHEA








RKRIMYARCQDLYRRRPQRP








VERAVGYQAEESLLDNQDER








PSHGAFETFYTGLWGKSGQC








NITMPPGVPRHTGHVLREVT








PKDIYSRLRKLKKDYAPGPD








GVTKLKVQSMGAYPSLLAKV








YNLVMLTGYFSSCWKEHKTS








LIPKDRGSPMDVSNWRPITI








GSLLSRIYTGLIERRLRTVS








DIHQRQVGFMPVNGCAANLF








IFDECIRQAKKEGTIVGSLI








DVAKAFDTVPHEAILRALSS








QGVDEHTMAHIRDMYSGIRT








RINGKGSDIPLVRGVKQGDP








LSPMLFNMVMDPLIRDLQRK








GFRIGGHEIGALAFADDIVL








LADSIDGAQDHVDQVGRYMN








KLGMTLNPRKSSSFLITAMR








KTWICRDPGLSIGETKVPGA








RPSSALKYLGVNYTLSEGLE








SGALIDKLMQAVNRARGLAL








KPLQKVNLILERIIPKFLYG








IILGGPSLTRLHAADKCVRM








AVKEILHLHPSTTDHVLYAR








KKDGGMGIPRLAHLVRLASL








RSGLALLASGDVAVQAAGMA








GDLEGRCKKVANDLRLNWPV








TLRDVVRASNKFKSQESKDW








ERLASQGHGVKDFRNDRLGN








CWLYDPTVLSSSRYTDALRL








RTNTFGVNVALRRADKDLEV








NCRRCHGKPETLGHVLGECV








AGKGMRIQRHDKMAAFVATK








CEEKGYQTTREQLFSIEQGK








LKPDLVVIDGERALIVDVTV








RFESGNALSRGASEKIEKYQ








PLADYFVSQGAVREANVLPI








VVGSRGAITQATLKSLATLG








LDVERVGKYLAICAVASSVE








IACMHLDYT








(SEQ ID NO: 1490)





R2
R2_RL
GU949

Reticulitermes

CTCACTGCTGTCATGCCATT
TGAGTTAGATATATATGACG
MMADYNNSVDHALEDNTRLI




555

lucifugus

GTTAATGCGTTGGGTGATGG
ACTGATTCTTCCCTAACTAA
FARDAVLARVCGPFDNLECG






CGGGTGATGGGAGACGAGTT
AATATAATTTGGAATGTGCA
LCGVLLTSLQGVREHCHRAH






ACAGCAGAGCTGGCTTCACG
TTACTTATACTTTATTGTAT
HNLDLTFQCTKCDKGFSSYR






GGGGGGGCTGTAGTTGCCCG
TTATTGACATCCGTAGTAGT
GICCHFSKCIGARISVSEGP






AACCAGTCTGCTTCTGAGTG
CTGTTTAGTGGATTTAGATC
LSCSECEREFDSKRALSTHE






CCTCCACGTGGCCTCGCTGG
GTTAACCATTCTGTGACGTC
RHMHPGIRNAKRLKDFNPRG






AAACGTCGGAGCTGCTTACG
GCTGATGTCTATATGTACCA
GGGKTIHGNTKWTEEEVQLL






GCTAACCCGTAGACAACTCC
GTGGCAGAATGCTGACTAAG
VSLSKRFEGYKSINKEISLI






GGCGGCCAAACTCAGAAAAC
AACAAAATAATTTTAAACAA
LTSKTCKQISDKRRYLNLHN






GGCCTTACTACCAGGGTCAT
TAGACAGCACTAAGAAATTC
GNGGLAAAEAVLEFCEDSHP






CCCTCGCGGCGGCGCTAAGA
TGCAAACTGGAACGTGGCCC
EVTESDGAVLSEIMDEECHQ






CCTTACTGTATGTACTACGG
ACGGTAGGCCAATATACCGG
SSVTMRSSIVHGDIGREVQG






ACTACAGTTGAGTAGCGCGG
AAAGGGAAAATGACACATCC
KELVRIPPDNSVMGNCVVLL






GAGAGCACTTGATGTGAGGG
CCCCTTAAA
RKLATDKRSDLDLSKDKELR






TAGCAACTTGTTGCTACTTC
(SEQ ID NO: 1369)
IDIEKATKANRESADGRVIQ






GACTGTCTCCCTGAAGATTT

SVAGNEIDPDTFQWKELLLG






CCGAAGGGGTGCCGCAAGTC

QVRGFPRVDENSELFDLDDK






CATGGGGGCGGTCCTGCTGT

LTKELSSDSPVWNDNCELIV






GGGAGTTATCTTGCACCCGG

SDLCQVLCKKKYELGRQHHV






GAAGCCCTCAGTGGATAAAT

RKGKRHRGIHHKREKFRECQ






ACTCCAAAATGGCTTAGCCA

KIFRKSPRKLAEYLYRDKDL






CCTCACGTGGAGAGAGGGGA

SHISKDASTPQGIEQYYSQL






ATTAGGGGTTTACTCCTAAG

WGEPELLESNTIEEKLPSSS






GGCCGATGCCTCCAATCGGA

LFDCLPPITPEEVEGRIHKI






TTTCCCCGGGCAACGGCTAT

RPSSAPGLDGVRKIHLVGKG






TATCAGCCGGCTAAGTTCGG

ITLVLVKLYNLLFLTGGYPE






ACCCTCATCTACTGATGGGA

CWKRNRTVFIPKIGKDLSEV






TACTCTCTCTCCCCCCGCCT

GGWRPLTIGSLLARMYSAFL






GCGTGATTAGTTGGGAATGC

ERRIRRVTSLSLSQRGFTNI






ACGCGGGCGGGTGATTCTGA

QGCHVNLTILKEGIRQAKVK






CAAGCCCAGCCCAAAAGTTC

NGGVIVSVDIEKAFDTIPHS






TTACCACGACGTACCCGGGG

VIFSRLASQGVPPLLRKIIS






GTGTCACCCGAACTAATATA

NMYKDVYTVIEGQCIPIKRG






TTTTCCGCCGATCGGTTCGC

VKQGDPLSPLLFNIAIDPVL






TGTGGGGGGATGTCGGGGCC

RSLEEFQGGLPLGNSAIKIL






TGGTGGCCGGGAACCGTCAG

AFADDIILGASSAGQAQQMV






CCGCTCTTTGAGTGTCCTGC

DMLGIGLTSCGLGVSHRKCF






GTAACCCGGAGGCGGTGACG

GFQIVNKNKTWTIVDPMITL






TCGAAGGGCTAGGCTAGCAC

NGSSLPFSGPEDRLPYLGVD






GGTCCAGACCCCTGAACGCC

INPWDRKSRYDAGQRLISAA






TGGGGAGACGGGCCCACCAC

KRGSQLSLKPQQKINLITAF






CTGAGTAGGAGTGGGTCCTT

LLPKFLYILIEDPPSPAYLK






TACCTCATTTGAGGTGTCTC

SIDHDLRQIYKNILHLPNCV






CTCGTTCTTGTAGGGGGCGA

STAFMYSPKRDGGLGLPRLS






AGGACGAGGAATGCATCCGT

CLVPLAHLKAGIKLGSLQDS






CCCTCCAGGATGCTGGTGGT

LVREITTSDRFVRTMGSVAH






TTCCGTCTGGTGGGCTCATG

SLWASWSLTLQDIYKLKSAL






CTGAAACGCGCAGGTGCCGA

KRREAKAWESCVSQGQGAAQ






TTTGTCGAAGAGGACATATG

FRGDSIGNNWLHNPGTYRPG






GGGTAACCCATAGAACCTAG

QYIEALKLRANLTGVRVNLK






GCAGGAGTAATCCCTTAGCT

RSGYNVPITCRFCKDIPETQ






GGGGGGGTCAGCTGGTGGGT

AHVLGLCPKTKGMRIQRHDS






CCTGTCAATTTATCCTCCCC

IVNRVRDKLKTKSPVALMHE






TCCATGCCAGAGCCGGTCCG

QNFTVEEGQVFKPDIVTILG






AGGTTAGAGACGGACTCATT

EVGYVIDVTVRYDDRDYIKD






TTCTCCTTTTTATATGTC

ASVEKIRKYEALKGYLKDLY






(SEQ ID NO: 1246)

PQLNKVEVLPLVFGSRGAVP








GSTVHNMGLLGFTKREMVHI








SRKVIADSLIISNFLEVY








(SEQ ID NO: 1491)





R2
R2_RU
GU949

Reticulitermes

CACACTGCTGTCATGCCATT
TGAGTTAGATATATATAACG
MMADYNNSVDHALEDDTRFI




554

urbis

GTTAATTCGTTGGGTGATGG
ACTGATTCTTCCCTAACTAA
FARDSVLARVCGHFDNLKCE






CGGGTGATGGGAGACGAGTT
AATATAATTTGGAATGTGCA
LCGVLLTSLQGVREHCHRSH






ACAGCAGAGCTGGCTTCACG
TTACTTATACTTTATTGTAT
HNLDLTFQCTKCDKGFSSYR






GGGGGGCTGTAGTGGCCCGA
TTATTGACATCCGTAGTAGT
GICCHFSKCRGARISVSEGP






ACCAGTCTGCTTCTGAGTGC
CTGTTTAGTGGATTTAGATC
LSCSECERKFDSKRALSTHE






CCCCAAGTGGCCTCGCTGGA
GTTAACCATTCTGTGACGTC
RHMHPGIRNAKRLKDFNPRG






AACGTCGGAGCTGCTTACGG
GCTGATGTCTACATGTACCA
GGKTIHGNTKWTEEEVQLLV






TTAACCCGTAGACAACTCCG
GTGGCAGAATGCTGACTAAG
SLSKRFEGYKSINKEISLIL






GCGGCCAAACTCAGAAAACG
AACAAAATAATTTTAAACAA
TSKTCKQISDKRRYLNLHNG






GCCTTACTACCAGGGCCATC
TAGACAGCACTAAGAAATTC
NGGLAAAEAVLVFCDDSHLE






CCTTGCGGTTGCGCTAAGAC
TGCAAACTGGAACGTGGCCC
VTDSDGAVLSEIMDEEYYQS






CTTACTGTATGTACTACGGA
ACGGTAGGCCAATATACCGG
SLTMRSSIVHGDIGREVQGK






CTACAGTTGAGTAGCGCGGG
AAAGGGAAAATGACAAATCC
DLVRIPPDNSVMGNCVVLLR






AGAGCACTCGATGTGAGGGT
CCCCTTAAA
KLATEKRSDLDLSKDKELRI






AGCAACTTGTTGCTTCTTCG
(SEQ ID NO: 1370)
DIEKATKANRESADGRVIQS






ACTGTCTCCCTGAAGAGTTC

VADNEIDPDTFQWKELLLGQ






CGAAGAGGTGCCGCAAGTCC

VRGFPRVDENSELFDLDDKL






ATGGGGGCGACCCGGCTGTG

TSELSSDSPVWNDNCELIVS






GGAGTTATCCTGCACCCGGG

DLCHVLCKNKYELGRQHHVR






AAGCCCTCAGTGGATAAATA

KGKRHRGIHHKREKFRECQK






CTCCAAAATGGCTTAGCCAC

IFRKSPRKLAEYLYRDKDLS






CTCACGTGGAGAGAGGGGAA

HISKDVSTPQGIEQYYSQLW






TTAGGGGTTTACTCCTAAGG

GKPELLESNTTEEMLPSSSL






GCCGATGCCTCCAATCGGAT

FDCLPPITPEEVEGRIHKIR






TTCCCAGGGCAACGGCTATT

PSSAPGLDGVGKIHLVGKGI






ATCAGCCGGCTAAGTTCGGA

TLVLAKLYNLLFLTGGYPEC






CCCTCATCTACCGATGGGAT

WKRNRTVFIPKIGKDLSEVG






ACTCTCTCTCCCCCCGCCTG

GWRPLTIGSLLARMYSAFLE






CGTGATTAATTGGGAATGCA

RRIRRVTSLSSSQRGFTNIQ






CGAGGGCGGGTGATTCTGAC

GCHVNLTILKEGIRQAKVKN






AAGCCCAGCCCAAAAGTTCT

GGVIVSVDIEKAFDTIPHSV






TACCACGACGTACCCGGGGG

IFSRLASQGVPPLLRKIISN






TGTCACCCGAACTAATATAT

MYKDVYTVIEGQCIPIKRGV






TTTCCACCGATCGGTTCGCT

KQGDPLFPLLFNIAIDPVLR






GTGGGGGGATGTCGGGGCCT

SLEEFQGGLPLGNSAIKILA






GGTGGCCAGGAACCGTCAGC

FDDDIILGASSAGQAQQMVD






CGCTCTTTGAGTGTCCTGCG

MLGIGLTSCGLGVSHRKCFG






TAACCCGGAGGCGGTGACGT

FQIVNKNKTWAIVDPMITLN






CGAAGGGCTAGGCTAGCACG

GSSLPFSGPEDRLPYLGVDT






GTCCAGACCCCTGAACGCCT

NPWDRKSRYDAGQRLISAAK






GGGGAGACGGGCCCACCACC

RGSQLSLKPQQKINLITTFL






TGTGTAGGAGTGGGTCCTTT

LPKFLYILIEDPPSPAYLKS






ACCTCATTTGAGGTGTCTCC

IDHDLRQIYKNILHLPNCVS






TCGGTCTAGTAGGGGGCGAA

TAFMYSPKRDGGLGLPRLSC






GGACGAGGGATGCATCCGTC

LVPLAHIKAGIKLGSLQDSL






CCTCCAGGATGCTGGTGGTT

VREITTSDRFVRTMGSVSHS






TCCGTCTGGTGGGCTCATGC

IGASWPLTLQDIYKLKSALK






TGAAACCCGCAGGTGCCGAT

RREAKAWESCVSQGQGAAQF






TTGTCGAAGAGGACATATGG

RGDSIGNNWLHNPGTFRPGQ






GGTAACCCATAGAACCTAGG

YIEALKLRANSTGVRVNLKR






CAGGAGTAATCCCTTAGCTG

SGYNVPITCRFCKDIPETQA






GGGGGGTCAGCTGGTGGGTC

HVLGLCPKTKGMRILRHDSI






CTGTCAATTTATCCTCCCCT

VNRVRDKLKTKSPVALMHEQ






CCATGCCAGAGCCGGTCCGA

NFTVEEGQVFKPDIVTILGE






GGTTAGAGACGGACTCATTT

VGYVIDVTVRYEDRDYIKDA






TCTCCTTTTTATATGTC

SVEKIRKYEALKGYLKDLYP






(SEQ ID NO: 1247)

QLNKVEVLPLVFGSRGAVPG








STVHNMGLLGFTKREMVHIS








RKVITDSLIIISNFLEVY








(SEQ ID NO: 1492)





R2
RaR2
FJ461304

Rhynchosciara

CAGAACGTGGAGAAACGGAA
TAGAAATGTGTGCGATAAGG
MSNYNETNTSGGDNPRMATQ






americana

TAACTACCCAGATCCGTTGG
TGTGAATAGAAGGGTTCACC
TTGSLSSGPINQHTCELCCR






TTAACCGGTGGCAAAGTTAA
AAGAGGGAGACCTAGTTTGG
TFGTRAGLGQHVRKTHPIES






TCAAGGTTGCCATAGGCTTA
ACCTCAGAAATGGGGTCATA
NQSINVERKKRRWSPEEIRR






ATAACCCTATGGAAATGTTT
GGAGTGATAGGTTGTAAAGC
MANMEAQATINNIKHLTQYL






CCACACACCTCCACGTGGTG
CGTTGGGGAATCCGGCTACA
ATYLPQRTLNAIKGRRRDAE






CCTGCCGGAAATTGTTCTAG
CATGGTATCTCAGGAGCCAT
YKELVTGIIANLRSNSSTQQ






GGTGAACAAGCTAAGTIGTG
TCATGCGCTGATCTCATTAA
TNQVCNESEMSQRSKILQSI






AGAAACGGGCTCCACCACAA
GGCGTAATAAACTGTGAAAC
RESVRDLRSRRNKYAKALQE






TATGGAGCCTGCCAGGGCGC
AGATCCTGATAATGCCGTGC
LGEAALCGKMLNEEQLIHCI






GAGACTCAGGACTCTCCATG
TACCAAATGATGTAACGAGG
KSMFNTAKCPKGPRFRKTAT






TACAAAGTGGTTAGTTGCAA
CGGAAATAAAATTAATCTGG
HSGTNKQQRQQRYARVQKLY






AAAGAGTGCGCCTAGCATGA
GGCGTTCTGCGGAATGACTA
KMNRKVAAKMVLEETDKIQI






CTGATAATTTTTCACTGAAA
CTAAATATAGCGATGCTATA
KLPDHDPMFKFWESEFKEGE






TAACGTTGAACTTTATCTGT
TATACAAACGACTGATGGTA
GMPERMPKDLKESPDLKAIW






GTCATGTGCACAACACTATG
ACACCGGCCTTA
DPVTEEEVRKAKVANNTAAG






GTGTCTGATCAAGCACCATC
(SEQ ID NO: 1371)
PDGIQPKSWNRISLKYKTLI






AGTGGTGGACCTGCTAATGT

YNLLLYYEKVPHKLKVSRTV






ATTAGTAGAACGTGTCCAGG

FIPKKKDGSSDPGEFRPLTI






CGATAATGCACACACGGCCT

CSVVLRGFNKILVQRLVSLY






CCGGGCCCATCGCTTTTTTT

KYDERQTAYLPIDGVGTNIH






GAGATTCCCTAGAAACTTCA

VLAAILNDSNTKLSELHVAL






GTGTGTGCGACAACTGTATA

LDITKAFNRLHHTSIIKSLV






ACCCATAAGGGATGGACAAA

GKGFPYGFITFIRRMYTGLQ






GGTTATACTAGGGGGTAAAA

TMMQFEGHCKMTQVNRGVYQ






ACCCTAATCGGCTAATGGCA

GDPLSGPIFLLAIEKGLQAL






AATGGGATGTAGAAATGCCA

DKEVGYDIGDVRVNAGAYAD






AAGATTACTCGCACCGAATA

DTDLVAGTRLGLQDNINRFS






ATGGTGGCCGAAAAGCGGGT

STIKQVGLEVNPRKSMTLSL






AATCGAATGAAATGGTAATG

VPSGKEKKMKVETGKPFRAN






CTGTAGCGGAAACATGATCA

DVPLKELSINDFWRYLGISY






CATTCTGTGACAGTAAACCA

TNEGPERLSLTIEQDLERLT






TTAGACCTAGGGGGAACTAT

KAPLKPQQRIHMLNAYVIPK






GATTAACAAGATACCAGCTT

YQDKLVLSKTTAKGLKRTDR






ACATGGAAGCAATGAAATGT

QIRQYVRRWLKLPHDVPIAY






AAGTCACAGTAGTGATAAGT

LHAPVKSGGLNIPCLQYWIP






GGTGAAGAGTCTTGTAATCA

LLRVNRVNKITESQRSVLAA






CCGTAACTAGGCCAGGTTCT

VGKTALLTSTVYKCNQSLAT






GGGGATGCCATGAACTTAGG

LGGNPTMLAYRTYWEKELYA






GGGAGTATGGTTAGCAGATC

KVDGKDLQNARDDKASTRWN






TACCAGCTAACACTATTACT

GMLHSDISGEDYLNYHKLRT






GATAATATGTAAGCCGCAGT

NSVPTKVRTARGRPQKETSC






AGCGCTAAGTGGTGTACAGA

RGGCKSTETLQHVVQQCHRT






TTTGCAACCACCGTAACTAA

HGGRTLRHDRIVGLLQHELR






GTTCTGTTTCGATGGACTAG

RDYNVLAKQELKTGIGLRKP






GGGGAATCATGATTAACAAG

DLVLIKDDTAHIVDVQVARC






ATACCAACTTACATGGAAGT

SKLNESHVRKRSKYDKKEIE






TATGAAATGTAAGTCACAGT

VEVKSRYRVSKVMYEACTIS






AGTGATAAGTGGTGAAGAGT

YKGIWDKQSVMSMRRLGVSE






CTTGTAATCACCGTAACTAG

YCLFKIVTSTLRGTWLCWKR






GCCAGATGTAGTCAAAGCAC

FNMITSVRS






ATGTTTAGGGGGAACAAGGT

(SEQ ID NO: 1493)






TAACATTGGTAAAAGACCAA








TGCAACCTCCGTAACTAAAC








GTGAAGGCACAAAACTAAAA








GTCCAGTGGATGACAGGTGA








GGTCATCACTGGACCCAAAT








GTTTTAAGCTCATCATAACA








ACACGGTGAAAAATCCAGCA








TTTATTTGCCTGATTGAGTA








GCTTCCACACTATTCCAAAG








CCGAACCTATCTGGGTTTTT








CTTGAAAGGCCGTATAGGGC








ATATGTCGAGAAATAAGTCC








AAGGTGAGGTAGTGTGGCCC








TGTACCCAGGGGTAAGGTAC








TATACGGTCGAGTGGCTCAG








TAGGCCTAACTAGCCACTGA








GTCACTATAATGACTAGTT








(SEQ ID NO: 1248)







R2
YURE-
.

Ciona

CCAAAATTACTTCCAGCACC
TAAGATCCGCGGCTGTGGCG
MAGHKITMSEGKLLEVAVRY



2_Cis


savignyi

TCCACAGCAGACGAACGAAG
CCGAACGAGCACCTGCCCAT
GGVRNVSYECPVPDCTKTFS






AAAGAAGACTTACGAAATAA
TCTTCTTGTAGGGACTTTTT
QANNLIRHLNNFGNTKHRAH






GAATAAGCAGTTTAAAGACG
CACCCTCACTCCCCCCAATA
NFTYFFTCEKCKIQIHSNTK






AAGCAGACGAACACCACTCC
GTTTTTTTTCGTTTTTTCGT
HNISNHYKQCCATGGGPSCE






ACCAACGACGCTCTGCAGCT
TTTTTCACCCCCACCCCACT
TGQYFCPACEQAGLGNLESA






ACACCACCACCATCTCGCCG
CGCCTCTGGGCTGCACATCC
LRHFQSSHPEFNLPPRSQFS






CAACAAGAAGAATTTTCCGC
CACACGTAGGGACCTGTTTA
KSHPNSYTLSLKPKDHLMKI






TGCTCTCCACTCTGCTCCAA
TATTATTTGCCTTTTATATG
LYSGPLTPGQLVCPIKICLR






CACCACCTCTCCTGCTCTGT
TACCACTTTTTAAATATATT
SSAARLFHDVSKLRKHMLVD






GGACTGCTGCCTTGCTGCTG
TTTGTACCCCACAAGATGCT
HNRTLVYETTCGKCLRPVDT






GACCAACCTCTACCCGAAGG
TTTCGCCAAAAAAAAAAAAT
SKNMRKTTSHFEKCSGESFI






AACCCTTCGAACCCAGCAGC
TTTTGTATCACATTTTTATA
SSPSPIPQKTYKLDLPSTST






AAGGTACGTGTCACCACCTC
TTTTGTAAAACACAGATTTT
PPPRKSPKLQPYKPIRTFKN






TCCACAGAGCCAAGGCCAGA
TATAAACTTTGCACTATTTT
PLTKSSQSKSDNPPKPTPFF






GTGGATAGAGCAGCGGCCTC
TATATAAACTTCGCACTTAT
SPRTLERSASWPALSEVVDP






TACAACCAAGTTCTCCACTC
TTAAAATGAATCGCATCTTT
LPKLKEKHPSLPCALDKCPP






CGACGACAAAACACCTGCCT
TTTATATACACCAACACAAA
SPRIKPSTLVPPCHTANNSP






CGTGGCCAGAGTCCTGCCGA
CAGGATGTGCAGCTCAGGGG
KPTSPESPSTLKPLPRPIRP






AGAAGAATCTTCGACCCCAA
AACCAATCCTGCGTCCCTCC
SKPLEDWLTVRSVGPDREIV






CACCGTCTCGGCAGGGCTCC
TAGCGGCGGGAGGGGCGCCC
LNIGPRPRPGPAAGSRTTSP






AAGCAACGACATCAGCCACG
ACCTACCCCCACGCTCCTCT
PSTAPAKRVAANPIAAPLSG






AGTGCCCACCGGAGTGGACG
TGAGCACCAACAGGGACTCC
EPGATLDCGQTGRKVQPPKK






CGGCGAACCCGAGGACCGCT
CTTCCGGAGCCCCTGCACCC
RPTESAGSLPPPAEPATDLL






GTCGCCAAGAACAATAACAT
TCAACTTTTCTTATTTTTAA
TGREGLARLVEEYHLSGDFG






CACCGCCTGCAGCAGCTACC
AAAAAAAAAATCATATATAT
AFCRDLERWTALSSTNRRPK






AAACAAAGGTTAGTCTCCTA
TGATCTTGACGACGGGGGCT
PRRGRYNRGAAARATRNRGR






CCTCACCTACAAACTCGTCT
ACATTCAGCCCCCAAAAACC
DDRQDPQDRDDQGGPGPVTC






GAATAGACGCCCCCGCGTGG
CACCCACCATCCCCAACGAG
GRPQRYKRAAALRSAFGRDM






GAGCTAACCTTGGTAGCTAG
TGCCGGGGCATTGAAGAGCT
KATVRRIIDGERGDARCEID






CTGCAGTGCCCTGCGACAGC
CCGGCACAATTAGCACTTAG
PKTIEGRFRDELSPPVREGP






GGCCTCGAAGAGCTGCAGAG
CTTATTTATTATTTTTTGTC
ECSLPPWMAEAQAGEHAPSN






CGTCAGCCTGCTTCGACCTC
AACATTTTTGTTTTTTCAAA
DSQPGDAYDGPITALEVEMV






GCTTGTTCTCATTTCAACCT
ITTTTTCACCCCTCACCCCC
LSTLNVGSAPGSDGLSYGFW






ACTCCGTCCTGTGGAATCAA
ACCCTAATAGGTCCCTCGGG
RALDPKGLVLSELFEVCRIE






AAGAGCCCCACTAACAATTA
CTTGGGCCCCTTTTTCGTGC
RRVPGPWKSSRVTLICKDAE






CATTCATAAAATCTAGCAAG
TCGAGAAGCGTCACATCGCC
GDLDDLGNWRPISICQTVYK






AACGAAGAGAAGCGACCAAC
CCACTGACCACGACCTTCCC
IYAAVLARRLQSWALDGGVI






TTTTAATCCATAACTTTTAG
CGACATTGGAGTCCTTGGCG
SRSQKGFMPFEGVYEHVFLL






ATCTTTTTATTTATTAC
TCTCCCAGGTCGAAACAGTC
DSVVADARATRRSLAVCWLD






TGTTTTTAAG
CCAAGTGATAGCACCTAATG
LRNAFGSVDHTTIVEALSRF






CCCTAAGCATATTGCCTTTT
CTCGACTTGTTTCGGCCTGG
GAPAGLVEMISDIYTGGSCR






TTTAGATCTTATAATTATAA
GCCGCCGAGGATTCCCAGAA
IRTRAGFTPDIPVGRGVRQG






AAATAGATTCAAAGTTAACA
CGACCATTCTTCTAAATAAT
CPLSGIIFNLVMEVLLRGVE






CCACCAGGCCGCTACAGAGC
ATTTATATTTCAGAATAAAA
ANNACGYRLSCAGGASVRVL






ATTTTATTTAATCAATTTGT
CTATATATATATCGTTGGCG
AYADDVALVGSSRAEXKIQL






TACCGACCTCCTGCTGCTTC
GGACTTGTCCCGCCTCGATA
GVCERFAAWAGFSFNNKKCA






TTTTTACTTTCTCCAGACTA
CCGAGTGCTGCAGAGCGGCA
AMVLKHQRGGRRLLDSAPLR






CTACCCGGATACAACCCTTG
AAATAAAGAAGAAACCGACG
LCGEEVAILGPDSFYKYLGA






GAAACGAGAGGA
TCGCTCTGCAGCCAAGGACC
HTGYGRQTGGQLVDRVERQV






(SEQ ID NO: 1249)
ACCCAAACTCAAGCCAGCAC
VRLFTSFLTPTQKLSALKRI







CGTCGACAACCAACATCCTC
VLPAMSFHLRVRPCAEGHLR







AAGTCGGCGGTTGCTGGAAC
RLDNTVRRCVKTALRLPKGS







AACTCATAACATCTTCAANA
CRAFFHTSPDAGGLGITSVV







TAAATTATCACCCTGTGCAG
AECDILTVTQAFKMLSSPDH







CAGGAGGCCGTGCTTTTAAA
LVSLVAKGRLGMHAARMGRS







ACTACTCTGTAGTGGCTCAT
ETASACAMADYLSGDSVMGH







GATAATATTTCGCTCCTTTT
XSWKTGYRMPADLWTATRAA







TTGCCCCGTGTAAACTTAGT
SRRLSLRFSPQPQGEFGLES







NGATGCGAATAAAATCAGTT
GTFKIAPRERRSLTRRLHHR







GAATCA
QNLWWRNQWAALPNQGKTVA







(SEQ ID NO: 1372)
AHSAYAASNNWVKGPSSLAP








QALFFGLKARLNQMPTRSVK








ACYSRAPNYDKSCRRCGAEV








ETLPHVLNHCPKSMKSILER








HDSVLAEVLAAIPRGTFASV








DVDRTSREHFRRVGEALRPD








IVARRHDGSVVVADVTCPFE








SCASALDTAAARKIEKYDQL








CANLRQLYRKPVESHALVVG








SLGSWGRTNNTALAALGIRG








AVRSRLAKQLVNLSVEGSHN








IWLRWSGGIPKDLVR








(SEQ ID NO: 1494)





R4
Dong
.

Bomby

GCTAGCTCCCTAAAATCCTA
TAAAAACTAGCATAATTATT
MLRRGRIFLPASTKAGKTRG






x mori

CCTTACGTCCGAGGCGAACA
AACTCATAACTAATGTATAT
RMKWSREVNLFIMRTYYYVT






TCTGTCCACGTGGGGAGCGG
TACTTGGCCAAAAGCCCGTA
KLETDLTIYRKKLHEHFSLK






AAACGCGTACTATCGAAACT
TATACAGTTCCACCGGCTCT
YPNVIISQQRISDQKRAIER






TACGCGGCTAACAAGGTAAA
GTCGACAGACTGAACTGAGA
NKLLSQETLDRLKEEVRKQL






GGTAACCCATTAATATGGAG
AAGGGGAAACATATGGAAAT
EDEQTNNVENEKLNSETYSH






ACAAGACTAAAAAGAAAATT
AATAATAA
EYTTLTPQTILTKKTQQHTN






GAGAGGGCCGCTTCCCGGGG
(SEQ ID NO: 1373)
IISSTQTSHSSTQTESITLL






GCGATCGCCGGGGCACACCT

LENEVDILNTNPTEGATQTQ






GGAGCTGGCGCTGGGTGTTC

EVKDKFETNLTMYSGMDPKA






CAGCATAGGATCCGTCAGCG

RPPLPKLKYSSKLNELIRLF






GCGGAGAGTTGAGCAGGCGG

NNDILVDYISPDTQLSDVHT






GCTCTCGCCGGGGATTTACA

LTYCTAVTISEQLKYKIIAI






ACCCGAAAATGCTACAGCCG

EGNARHKKNFKPPWQQRLEK






CCAACAGAAATCACGATGTA

DIAKLRADIGKLTQYINNNR






GAAAGTAGGAGCAATAGCCC

SKKVVQSVEQIFKNTKIHTS






ATGTGAACCTTACAGCCCGA

HENGNKKSQEFLDTLKQKLA






GTACCGGTTCATACAACCCC

LKAHRLKRYNNSQKRKNENT






TCGGTACAATCATCACCATC

IFLTNEKLFYRNLIKPKTDR






ATCCTCGGGTCATAGAGGCT

DNSNIDIPTAEQLEMYWARL






CACCAACGTCGACTATGG

WENSAKHNDKANWITEEKER






(SEQ ID NO: 1250)

WDTIEEMQFDDVTEEEITTI








TARLHNWKSPGIDKIHNFWF








KKLICLHKTIAKNLTDIISG








NQSIPEFIATGITYMIPKGD








FSIEASQYRPITCLPTIYKI








LTTVITKKINSHIEHNNILA








EEQKGCRRGHMGCKEQLIID








STIMKHATTKNRNLHCTYID








YKKAFDSIPHSWLIQVLEIY








KINPIIISFLRNIMTHWQTT








LKLKNPPNFVTTRQIAIKKG








IYQGDSLSPLWFCLALNPLS








HQLHNDRAGYRIKQQDNTET








IISHLIYMDDIKLYAKNDKE








MKKLIDTTTIFSNDISMQFG








LDKCKTVHIIKGKVQPGDYT








IDDTNTITAMEPSDLYKYLG








FQQLKGLDHITIKQSLTSEY








KKRINAICKTKLSGKHLIKA








LNTYAIPILTYSFGIIKWSK








TDIEQIERITRTTLTKHNNL








HPKSAIERLTIKRQDGGRGM








IDIWHLWRKQIHSLKTFFYI








KSDLSEIHRAIAQNDNNYTP








LNLKQKELIDNTENLRNRNP








QKDMEENWKKKALHGRHPHD








LSQSHIDSKASNMWLKTGSL








FPETEGFLIAIQDQVINTKN








YRKYIIKDPTIRDDKCRKCN








TQPETIQHITGACSTLTQTD








YTHRHNQLANIIHQQLALKH








KLIQNTNTPYYNYKPQTVLE








NDSCKLYYDRAILTDRTIHY








NRPDITLQDKNNKVTYIIDI








AVPNTHNIQKTFTEKMTKYT








ELKEEIVRIWKQKKAYIVPI








IISTTGVVPNHIHNSLKLLD








LKDNIFISLQKAAILNTCRI








VRKFMQLEENQTYYTQ








(SEQ ID NO: 1495)





R4
DongA
AB097

Anopheles

GAAGGCTAACCACAATA
TAACATCCGGTGCAAACTCA
METRSMRKRTTRLPEEGAPT



G
127

gambiae

(SEQ ID NO: 1251)
TTAACATTAAGAAAAGAGAG
GAGPGTGDRASIQRLEDEMV







AGGAGAAATGAGAATGAGAT
QERSFSQRALPVPRTQNRNG







TCATTCACCTTTGGCATTTG
SPINHQGNAASANVAVADRQ







AATAGCCCGGGGTAGGTGAA
QSLILAGGRRQRIMWTREMN







AAGTTCCCAGCATATTGCTG
HYVIRCYYVYTRMETDMPGR







AGAAGTGACAAAATTCGGAT
VKMLGMFNDRFPRFAHQLDL







AATAATAATAATAATAATAA
SKLYIRQRAIILPEELEFIK







TAATAATAATAATAATAATA
LEVRREFGEEEAGWRESSRI







ATAATAATAATATGCATAAT
SARLNTIDQNTSRASEDRDL







AATA
DEPTAPGLSVDIQHQMATAV







(SEQ ID NO: 1374)
TQFHGTDPLSRHRLPKLHYS








YRLKTAVSIINQDVLPQYLD








SVGSIEDLQLIVYSAAVAVV








RTLWLRTYPQGDSEGRPCSK








AEKPAWMRRLENRINATRTK








IGRMQEYQRGNSSMKVVRQI








AEMVKPKELRDLTDANITEV








LDIHLQRLSALAKRLRRYAE








CSKRKEQNRMFNINEREFYN








WIRNDKPNFREGLPDIGDFT








QFWANLCEKPVQHNSEGMRL








AEDERFSDGIEDMPVLVVNA








QDIREATQYTRNGAAPGPDF








VYNFWYKKLITIHEQIAACF








NTVLEDSRKLPKFITGGVTY








FLPKDQNTKNPAKYRPLTCL








SNLNKVLSSVITQKVKDHCD








TNNVMTEEQTGRRKNTQGCK








DQVIIDAVIVGQAAKKQRNL








DMAYIDYKKAYDSVPHSYLL








KVLQLYKVDGNVIKLMQHAM








GMWSTSLHVTDGKVVLRSRS








LNIRRGIFQGDTFSTLWFCL








AMNPLSRTLNQQCNFGYLLK








SEEISTRITHTFFMDDLKLF








AETVQKMHHLLKNVQGFSND








IKMEFGIGKCRSIHLHRGQV








LDADSFRANEQEEIRHMVQG








ETYKFLGFLQLRGIHYAVIK








KELQDKFLHRVSCILKSFLS








VGNKVKAINTFAVALLTYSF








GVMKWSNTDLEALERTIRVV








STKHQMRHPKASVERVILPR








KIGGVGIIDIQALCISQIHQ








LRSYFVESQNRHELYRTVYK








ADHGLSALHLAQQDYQLNCN








IKTVDGKGATWKQKELHGTH








THQLNLEHIDKVSSSTWLVR








CDLFCETEGFMVAIQDRVIA








TWNYRRCILREDVEDRCRKC








NSGGESIEHVIAGCPVLAGS








AYLDRHNDVAKIVHQQLALR








HKLVERFLPCYRYLPDPVQE








NDCIKLYWDREIITDILIRA








NRPDILVYEKRKKRATIDID








IAVTLDHNVQTTFSTKVMKY








HDLAEELKQTWYLEDIRIVP








VIISATGIVPMALLRSLDEL








ELQRELPRIQKAVILRTCST








LRRFLNPYN








(SEQ ID NO: 1496)





R4
R4-
CADV0

Bursaphelenchus

GGGATCCTGGGTTCCTACTA
TAAGAAAAGCATGAAATAAT
MTCNNAVVFPPADGNPAGTA



1_BX
1008175

xylophilus

CCTCGCTCCACCTCCTCGCG
AAGAAATCAGATAAGAATAA
DRNFAIRFPSSEPPGPSGIR






ATGGATCCTGGGGAAGTCTC
CAAGAATACTAATAAGTATA
PSEPLDGRTGIGDVEHAQAG






CGGACTGAGCTAAGAGAGCG
TCATGTAACTATGACAAAAA
NGGFLVDVLEYKEAHRYGSK






TTAAAGTAGAGGGTGACGGC
GAACGCACCAATAAGAACAT
CEFCYVQTKGTVCSKPRTDA






GTAAGTACCTCCAAGTTGCG
GCTTGAGTGGCCAGCTCTGC
WLKCEILFLLHHAYTANQNK






GTGGAGCGGAACATCTACTC
AGGCAAAAGTCGAATTTGGA
SIELAESAFRRAGITRRSKA






TTCGGAGAGAGGGGAAGCTC
ACAGCCGGTAATGGAAGACC
TIAKRWSLIQRGKGTDYKEY






TATGGCGGCGTTAGAAAGGT
TGCAACAAACGTGGGGTAGC
WDEYFEKFRYECNPTPIVRR






TGGACTACGGCAACGCCAGG
AGGCAATATGTAACTATGAC
KRNRLAAGLQSPSSVPNGYE






GAGATGGGGAAGGTTCATCA
AGACCAAAACTCCGAAACTC
FERKRTCETPLDTKASSLPL






GGTGATACTAGTTCGCTACT
TGGTAATGAGCCCGTGCCCC
ICNLLTGIVGVENVEENMSV






GTCATTCGATGTATCCGGAA
CCAAGCATGTGGTCTCGTTC
ECTEPKELSGTANSSVPGLA






CCATACTCGCCAAGTTGTGA
GATGTAGTTAGGAACAGTTC
EGVYERRHNNVNEPAAGCPQ






ACTATGTGAAAGTCTGGATC
TCTTTAACCCGTGATGATTA
DVPVANNLIDSPTTNDRLEA






CAATCCAAGACCACGGGGCG
CGCCCTGTCTTAAATGGCAG
EFKAQLDRAERSYMRRRLPR






CAATTAAAAGGTGTGAAGCA
GTGCCACCAAATACCGAACA
LKNLSPDERMWIGTTVERLR






GCTTGCTGGTGATCACCACG
CTCGTTGAGGTATGGTGGTC
LETVSEPVCEQWRLANAGLY






GTGGTACCTACACCGGCGGG
CGAATGTGAAGCTGGGAGTA
AAIRSIAVMRPLDAAREAHK






GGAACATCTTTAATGCCGAG
CAATTTGGTACGAGAGCACC
TWLLNMKMTERKLRQQIGWV






ATGACCGCGCGAATAATAGG
AGCGCCCCCGATCTAAGTGA
ETTRRTKNEARTERQEIVYR






GAGCCGGCAAAAGCCGAGCG
TGACGCATGCGTCGGAACAA
KVAKLRRERFPEMDLDSVSV






TAAGTGGAAAGGATACAGAA
TGAAGACGGCTGGCAAACAT
HLKRKLELLKGRIQVRTAER






ATTTGCTCAAAAGTATACCA
TCAGGAGTCGCAAA
LRRDTREAAGPYGKTALRGQ






ACCCGCCACTTATAAAAGAC
(SEQ ID NO: 1375)
GFAPNVKDATQYWSGLAQPS






CGAGAAAAGGGTACCACTAG

GQKCSENSAILSDWKELVEC






AGCTCATTATAAAACATCT

NLSSLPDQMEPLVVQGISRA






(SEQ ID NO: 1252)

SPWKSPGPDGIFNYYWRQDF








IVDWLKQLMLDSLRTGHYPW








KLSSGRTVLLYKDGDPTKAE








NYRPITCLNGCFKMINSVVS








EVILKRVENTIALPIEQMAL








RRKVWACVESQIWDQIKQRK








LSDRTQKCKVAWVDFSKAYD








SLNHDAIKFVIGVLKLPTGI








NNYLLDSMQNWSTHLELKSS








GKVVRGPSYPIKRGVLQGDS








LSPTLFVVVTSIIVRHIKTI








ESSDIQMYMDDIKLYGKDQE








TLTRLIKELQTVSNKLGLCM








NLKKCAILGDDLPEEINGIE








HLKESYKYLGVPQREITQVR








ATMAALEKKILTEVDTSLGA








AELSYRQRISRVNSKIAPLV








RFVVQSMLVTPRDVLKVYNR








LGGIDVEIRRRLVKYEIRYK








KSNVARLYLDRKVGGIGFVN








LCRIMVEAVAARAVYCRLAP








SFNEFQDFLAEQNTSPITAA








QTILDKCGINIELSTSTLGD








VKKIVRNHYHELWLTAWKNT








GLYKRWENDHVDIKRSSLWI








NRGNLSANNARIGIGIQDNS








IFCRGFVGNKCDTKYCRLCG








DGIESVSHIVTGCPTHRTNL








YIERHDCVARNVYAYLAIRY








GIPVPHYTQRVKTIEKNGDQ








SVELYWNYKFPCTRALEACR








PDIVLIDKVSKRTHIIEVAV








SWRGRLQEMVDRKVYKYTVN








GEYEADGSSRGWNIVRELND








QYGFPVEVYTLVIGAGGEIL








PCTVKDVERLTGGAATDNLI








ERMERSAVLGSCRIIKRHLA








L








(SEQ ID NO: 1497)





R4
R4-
.

Heterodera

TGGCGATACTCGGAACCTCC
TGAGGACTCANAATTGACAA
MISCDLERETLTQMALFRAR



1_HG


glycines

GGGGAGCCTGGTAGGAGTTG
TACACCTCAGA
SDKTPTHAGIPAPDEVREGG






GCCTACAGGTCGCGAAAGTC
(SEQ ID NO: 1376)
CGQNRTNPAAPRGKAAAIQR






CCTAGGTGCTGCACGGGTTG

QNGITIPIXACAQSGLVRTQ






CGCTAATCCGAGGGCGCTGG

RVQQWSAVEESALKDVVVRN






GTTACCTTCCCATCGGCCAA

TDDRGLINWAKGVLPEWQRL






AAACGTCTGGGCCTTCTTAG

CQLNPTMYMARSSPSLSNKW






CCGCGGGTCCAGTATTTCTG

ASLRRTHVGPGCPSKEGSGP






TTGAGCCTGACAGTTCTTCC

SQDLSDVKIQPARLAHDTVA






CGGATATGGCGAAAGATTAC

ELPQRTVPCGTDGHGVIDSD






AGGGCGGTATTTCGTGAAAC

ETETALAEVSRSSPFGEREP






CTAAAAAWGGTCGGGCCGAA

LDLGATERITRKRLRNAVRD






TGGCACGGACAGACTCACTT

VVPPRKRRVPSTPSRKEQDL






CGGAGTGAGCTCG

VPEVDGPAPTDVLTHPPTES






GGGGCATCCGTGTGTTACCC

EPEPMLDPLSLVQLVRPQLG






CGCTGCACCACGCCGAAGCT

RAMGWAAEEMELGNVVMDVE






GTCATAGCGAGCCCGAAGGG

LKREFNREVRRVGRTPPDQM






GAATGGCCATGGAGACTCCA

YKRGAGPPLPQKREPERVAL






GCCTCACCCTGTAACTCGAA

LEQLIAARVERGINRGLDWF






CCTAAGTCCAGGCCCCTTTC

LELNVAVFAAARVLSRRERV






TGGTGTTGGCTCGCACTGGT

ETLADRLHINDSATLSEVSR






TAGGAACACGACAGTCTCGT

RRAKAERKLRCAREQPWMSR






GTAATCCCACACGCACCGAA

RIRXLGVRVERLKQLADLVR






AGCCCTAGCCCTCGTAGGCG

QRIAGRGNRSSYEGPRRRFR






AAGGCTGCGTTGCTGGTTTC

LRPSLRSVTEAPVNPPLNGN






AGAACTGTGAGCWAGTGGGG

EVYTFWHSLWAQSLRANTDD






TTCGGATGGCCGAGTGTACC

CQLREFKNQLSAARHTDLTS






AACCTGCTTGCTAACAGGTA

VGTSSLVQMFSAALRKMKKG






GCATAGAGTAATATGCTAGT

KAPGPDGIRAAWWGVFRRIA






AAGCAGGGCACGAGAAAGGG

PYVATWVVRVIRGAEPVANW






CCGTAAAGGCTCCGGCTATG

ICNGLTVLLPKSSDNADPSN






AAGGACCTTGCGACCACGCG

YRPITCLNTCYKLFTAVIAQ






TGTGTCTCCCACGTGCGGAT

ITASYVDVLGGLPRQQVALR






TCTTGAAGCCAGAGTCTTGC

KGVWGTSVSLMIDALTVADA






ACTGCGCGCAGGATGGAGCC

RRAKRPLGVCWFDFKKA






TGTGCAACTCCTCCCTCGCT

FDSVPHNLIRWILRVIGLPP






GATCGCAGGAGTGGAGGATC

VILSVIVSVMDQWATRLKIG






ACCACTCTTTTTACCTTGCT

GKVMPKTIPVRTGVFQGDTL






AGCTTGGGGTACCACCTTGA

SPLLFCLSVWPISFALDQFP






GCTGGGGCCGGCCTTGCTAG

QYQFRCANHLQQGFSVGHVF






CTTGGGGTACGACCCTTTGA

YMDDLKCYCPDREVLTAVIQ






GCTGGGGCTGGCCTTACTAG

QVQKSASALGLTIHYKKSAW






CTTGGCGCGCCACCCTTGGA

LDQDGGKSGKAVLGVPXLVG






GCTGGGGTGGCGCAAGATCA

TYKYLGMHERFMIVSKDSLE






CTTGTATACGGTCTAACCAA

SVRGKFMGRLKTLWTSKLTF






TACATTTGAAAAGCGATCAA

GQAMLGTKSXCMPVVRYVLQ






AGCGAA

NLFLPKSEFNQTRLVLREWD






(SEQ ID NO: 1253)

RQIRDLLDECNIRQVFRSKT








ELYVSREEGGWGLPSMEDAL








EEEVVTKLAILVARQETEPL








FRVCEALERKRCPTPLSLGL








QILKDWGVGVELQGRTLLLN








GNTVGPSQATRKLTGELVLR








REAERLSRWRSKVKPGCGMT








GGAWRDVPGIDVHLSNRWLV








KGALSPTVVSNSLAIRANTV








ILRGSGGGYTKGTLLRCRGC








GNTGETRRHIVSACSLGRQK








GAASRRHDNVCRILVRAICH








KLNIEPPNSANFPHVVVLEG








SGAKMWIDFPFVVPHKIRHT








RPDIVVLFEWNGVRRLSVIE








VAVSDVANMQTQHIRKSHRY








GTNSTEPFVAGVTPTYRNDC








LAAQLRAKFKAQQVDVIPII








VGTTGETLDGEFGRIRKGLP








MLTKLQMPRLWSEIQRAVIL








GSYRILVEHLALPKGGA








(SEQ ID NO: 1498)





R4
R4-
.

Parhyale

CCGACCGCCAGCGGGATAAC
TGAGCCTTAGGTCGCGGGAT
MKMSHNRDTPSNGVKGTSVR



1_PH


hawaiensis

TGGCAAACCCTGTCTCGACC
GTGACCCGGCGCCAGAGTGT
LGTSLVRSPVGEAGAVRERG






ACCGGCCCGTGAATCCATCG
AGAGCTGAACATCGCTCAAC
THPSESVSQDSDASVNATGE






GGGCGTATGAGTCTGACACA
CGATCCAATTTGGGTCGTGA
GSVREQAPLSPPGAEEATVP






GGGGGGTGTTTAAGGTGACC
AATCCCCTCGATAATAATAA
TQRRTRHKWSREDRVVLWEC






CTGTTGCGAGGAAATGCGCA
TAATAA
FVASKREGPGYLKRLKQLWD






GCAAAAGCCGGATGAGCCTT
(SEQ ID NO: 1377)
ERGIPGNFPQASLSGQIRQI






AGAACATCGAAGGCCAACGA

CSKNLLSEEERLQIAARMEA






CAACCTGCCGAAAGACTGGC

QVASPSADEPARQVPTRPVT






GACTAATCCAGTCAACCTCC

PPRSPPVEPARRPSIPSEET






TGTAGGTCACCGGCTGGTCA

PDLGAVPSEIDSADPNRSPS






TGCTGAATCTCAGCTTAACC

RGPRHLPAHNMSQSESEDDV






AGGCGTACGAACTTACAGTT

TDPDVSDQQRSDSLEPRDLL






GGAGGGTCGAGCACCCCTGA

RNSSVESTPGHPNQELSDTL






TGGCTGAAAAGGACCATCAA

LSNYVPSEIDSDDPNQSPRR






AGTCGAAGGTAGCCAACGAG

GPRHLPAHDMSLSDSMDEET






AAGACCAACGGCTGATTCAG

EPDLSDQQRSDLLELRDLLR






GCGGAAGAGTCAACTCGTTG

NSSVETTPKGHPSLRHLPEP






AATGCGTTCGACAGCTTGGG

KIRAAAFRVNSVIGKIHTNN






GTAGATGGAACTCCTAAGCC

ITETNALIKAGADLAVRILE






CTGAAAGGCAGTCCATCTTC

VQPRPQRTQRKKDPPWKHRL






GCAGACGCTAAGGTGCCCCG

EKNIAEIRKHLSWISEWRRG






CCGCCTGAGGGTTATCAGGC

NLHDEEKKTLLESRYRCLEV






CCCGCCGCCTGAGGGTTACC

GLTNLEDTLKQRLSAKRSKV






AGGCAACC

RRFEARVAGFHQNQLFNTNQ






(SEQ ID NO: 1254)

KRLYQTLRGEETSSDSPNAE








ESIRFWSDIWSKEVRHNNTA








EWLHDVKEKNVAADPDLTIT








SQQLKKQLSKTKNWKAPGPD








MVQGYWIKTFTSLHSRIAAQ








LNHCLQRGTVPTWMTTGKTV








LIQKDKAKGTEVSNYRPITC








LPLMWKVLTGIIYERVYQHL








DSKKLLPDEQKGCRRNTRGT








KDHLLVDKLLTKDARSKKKN








LSMAWVDYKKAFDMVPHSWI








LECLDIYGIAGNIRNLIATT








MPNWKTQLTSANKHLGEVSI








KRGIFQGDSLSPLLFVLTMI








PLSETLNKAGQGYNYSRTMK








LNHLLYMDDLKLYAKSKDQV








EQLLNIVHQYSQDIKMQFGV








SKCGVLNIERGEVTASEGIT








IEEGTIKDIEEAGYKYLGVM








EYNTILHRTMKDSIRKEYLT








RLRLILKSHLNGGNTIKAIN








TWAVPVVRYSAGIINWTKKD








CTDMDIKTRKLMTIYRALHP








RSCVDRLYINRREGGRGLIS








VEDCVEAEKRALSQHFRESD








DPWARCLVEAKLLKETETAD








QFKERRRLDRTNKWKSMKMS








GQYLEAVQDKIVPDSWNWLL








RGELKRETEGTILAAQEQAL








RTRYIQNKIDKRNVPSTCRI








CRSSDETINHVISECGVLAQ








KEYKRRHDKVARHLHWTLLR








IHNFPVSERWYEHEPAPVVE








NEAVQIYWDKRMETDRVLHA








NRPDIVVKDKQEKSAKLIDI








SIPFDSRIVDKEAEKKEKYR








DLAIELQRLWQMKVDVVPVV








IGALGAMSKNLKTALRELKC








GHLHPGTLQKSALLGTAHII








RKVL








(SEQ ID NO: 1499)





R4
R4-
CADV0

Bursaphelenchus

GAGGATCCTGGGTTCCTACT
TAACAAGTGTAATAAAAACC
MVFNNCKPKHLCPAIRPTGQ



2_BX
10090

xylophilus

ACCCTGCTCCATCTCCTCGC
ACCCATGCGTAGTAAACCGA
QETNGGSEGTAEPTAGPSRP




48

GATGGATCCTTGGGGAAGTC
TCAATTATCTAGCAAAATCG
AVSEDAAQPVPLFEEGEYIR






TCCGGACTGAGCTAAGAGAG
CAGGTCAGAAGACCAAAGAA
AHRDKTCPYCEVLWIGARSS






CGTTAAAGTAGAGGGTGGCG
CCGACCCAGAGGAATAGGAC
KARSDSWPLCQILYLMKRND






GCGTAGTGACTTCCAAGTTG
CAGAGCTGAAACTCTCAGAT
DLRGQRTRYPLLESSLRAAG






CGATGGGGCGGAACATCTAC
ACGCCAACGGTCCTAATAAA
IARTKFAIIKCIRNVLRDRY






TCTTCTGAGAGAGGGAAAGC
ACGTCGTTAAGTAAAGCATC
VPNGPYSEHWKIYRANSGEV






CCTATGGCGGCGGTAGAAAG
GTTAAGTACAAAACAAAAGC
PQGATITKGKRSARVAGLPS






GTTGGGCTACGGCAACACTT
ACTGTAAACGCGAGGCCCCC
PSQSGHHTKRIQAGTGIETE






GCCATGATCAGATTCGATCA
TCTTTGCCAAACTCCGGTAA
TTVTETNTTPEVSHEHRDPC






AAATTAGCCTCTGGGGCTGG
TCCTCGTAGTACGGTGCTTT
GEPETSAANVDKVTELTEDG






CAACCCTACAACGGATTGTA
TTCCCGCTTCACGAATCAGA
SETRGTANVANGGVSVSDPG






AACTGAACTATGCTATGCAA
ACGCTGCCAGATCTTGTCCG
RKRQSSSQNRGNIETTNPEL






AATGAAAATAAAAAATGGGG
ACATGGGCCGTAGGGTTTGG
VGMWEDMFGVQLDGAMRTTE






GCTTTACAATCTAAGGTGTT
GAGTACCAGCGTGGGGCGGA
RPRLPKLKHLSEPERLWIRA






GGCAGATCACGAAAACTGCC
GAGCGTACCTGGGTACACTG
KLEQAWLQCVSYDVEQQWLN






CGTCGATGAGGGTGAGATAA
CATAATCGGGTCTCAGAAAA
ANAVLYAAIRSVAASRPCKE






CATCCTACGGAACAGCCCCT
CCACCTATGGTTTATTATTC
AREAQKTWLDNKKKDEAKLR






GCTGGACCAAACCAAATCAT
TGTCTCCCATCTGCAGGGTA
RLIGRISSVHSMPKGDRTPR






CCACAAATTGGAGGGTTTTC
GCTTTTCGTTGGGCCATAGG
EKKLVKNITKLKNTHYPDMD






TTGGTAGTTTCCCTTGGCAC
AGCCTAGGGGCAAGAGTGCA
WGGLLNHFKVKLSQLKEKIS






GTCTTGTTTCATAAGCCAGA
TGTAGTCTTCAACGGCCATG
VRVAEHKRKVNRNAAGQYGK






ATAAAAACGATACCATACAG
CCAGGGAAACTTGTGAGAGG
SVAGSAGLAPDVVSATAYWS






ACATGAGGCTGGGTACCTGC
TGAGGGATAACTAGCATCAG
GLAQPGPKKFKASSPIFQTW






CAGCCGCGACACGGAAAACC
ATAATATCAGTCATGAAATT
KDDVAKNLNTEPVLLYPIIK






GGTAGGTGCAACCGGAGACA
AGTAACAACCAACGTTCACC
ECIRKPSPWKAPGPDGIYNY






GCTAGGGGAAAGAAAATAGT
GTCGTTGGCAAAACACCGAC
YWQQEFVAQWIQTLVKRTLD






AAAGTGTCGAAAACAAAACA
TAACGATGCTAGTTAGAAAG
IGRFPTALMCGRTVLLFKSG






GGTAGCCCCTGGCTAGAGGG
AGTCGGGTCTTCCCAAAGTT
DKSMPQNYRPITCLNGCFKI






AATGGGACATTGTCCGATTA
AGGTGCTTGCACCGAAGCCG
TNAVLTKVILQRVQDTCALP






GGTTGCCTTGACCCAATGAA
ATCCGCTCTACCCACAGCTC
REQMALKPKVWSCMEAQLRD






AGCCAACCGGGTTTACATTA
TGCCCAGCGTT
QALQSEIGDDCKTAWIDFSK






TCGGTCTACCAAGGGCAATG
(SEQ ID NO: 1378)
AYDSLDHDALRFVIQTIALP






ACCAGAGAAACTGCGACTAT

AGMEEYLLKSLDSWRTQLVL






GCCGTAACCCTTCGCAGATT

SDAGKVVSGKPYPIKRGVLQ






GCCGATGAGAACCATCGATC

GDSLSPALFVLTTSPIVAHL






GTAAGTCGAAGCCAAGCGGA

QRTCPTGRIQLYMDDIKLYG






TTGACCAGTGGAGGGTTATC

KTESDLCMLIKETQRVANKL






CCGACAACAGC

GLNINLKKCALFGKSIKQSI






(SEQ ID NO: 1255)

AGFDPLGDRTYKYLGIPQRD








VADIKQAYDELKAKTVQTIG








ETMACDYLTTRQVINRLNSK








IPPVVRFVTQSALCSAPMTR








GLYNKITELDNVSRAELRKV








LIYKATNVSRFYLATKEGGF








GYASLQQVFVEAVVSRAIYC








LRAPSLCDIREFILSKFDPV








KVARIALARSKIDMDIERMD








MASATRTIRQHYQAKWKTLF








QQSKLYQKWVQHKIDIPNSS








RWLQRGEISPRNCRIAVAVQ








DNTLLCRGFVGSKDPNKQCR








LCNAGIETASHIVTECSTHR








VHMYIERHDSVARNIYAVLA








KNCGFWIPHYSQKIPTVKIT








KSYELYWNYKFPCTQALEAC








RPDIVLIDRAKKRILVVEVA








VSYVTRLEQMTQRKLYKYGV








NGEYQADGETRGWNICRELV








QKYNMRIDLCIVVIGACGEI








LPCMVKEIEKISKVSGRQLL








ERCQRSAVLGTVRTVRRHLA








N








(SEQ ID NO: 1500)





R4
R4-
ABLAO

Heterodera

TGTGGCGATACTCGGAACCT
TAGTCGTAGCCCAGATGTCG
MRKSFLQHIPELSSHIAMSV



2_HG
10003

glycines

CCGGGGAGCCTGGTAGGAGT
ACAGCCCCTACACCGGTAGA
PARNYPKMCSLAQGSSGTLS




89

TGGCCTACAGGTCGCGAAAG
TGGAAGGAGGTATGTATCTA
HNGKGVAMHRCPSDDCAGKD






TCCCTAGGTGCTGCACGGGT
AGCCCACGGCAAGCCACCAG
PPQRGSQKGNLRSVRWTPSE






TGCGCTAATCCGAGCCCCTT
TGGAAACGGTGACCACCTCT
EKAVFEYWSRLEQHAMLNGS






CCGGTGTTGGCTAGCACTGG
GTTCGCGGAAATGCCCCGTA
EARGTCAITRSQFLIHWDGE






AGGGGGGGGGGTCCAAAGAA
GAGTATATGAAGGTCGGAGC
RESRSLSDGVPEYPMRTERA






TACGTGAAGCGGACCCTGTG
ATTGAAGCACAAGTGAGAAC
YYERVRLLRQRGWQWDCANE






TTTTCTCTGACCTCATACGG
CCTGGAAGTATGGTGGTGA
CLVIGQCAEPCRKPNVVAIK






GTATATCCCGATGATATCTA
GCTGGT
ADKGMKRSLVKGKLLSLPHV






TACAGTGTTCATTTTCATTT
AACCTCGAATTCCTCCTATG
MGEINQVSVQVEVPLPSVPA






CTTAACCTGCTTTTTCCTCA
GGTGCTTGCGCCCGTAGGTC
SVPQVEGVESKGFTETEPSN






AGAGAAATATGACCACATTC
ATTTGTGTATGTAGGGATGG
KPSLEGNPAEEGLRKPERVN






GTTTGCCATTTCGAAGGTGC
AAGGAATCTCGATAGCGCAA
VPVHGIISDSERKDLKDRFW






GACTTTTGAGGGAGTTCTCA
ATCGGGATACCGCACTGGCG
SAYKTAKRSVGFRPALKIEP






GGGACATCAAGCTGTTCATG
ATCATGCTGCCAGTGGGCCC
NRVNRAQWEVLDSCVVEVLK






GACGGCTTGACGGCTCGTGG
TGCTGAGTTGGGCGGGTTAG
KRETSNGYRGCVLRHLNVAV






CGGCACGCAGCCGCGCGGGC
CAACCCGTCCAGGGACGCAG
YAAGYVLAEGNKERRQVIRR






CAGTTAGGGCTGGTGCCAAT
ATGCCAAAAGCCTGGTGACA
QSAEWLLRQKSEINNIRRHI






GAGGCAGCGAAGAAGTCTCT
CTCTAAAGGGAGTCTACGGC
GWITDELTRRRTGKNPTSRQ






GAAACGCCAAAAGCAGCGGG
GACGAGAACATCCCCTAAGT
LSNFAWLQRRYQVLGKPVRE






AGAAGAGAGAGAAAGCGGGG
C
TRDLEVQRERLVSRLRLAQD






ACGGGTTAGAGATAATAGCA
(SEQ ID NO: 1379)
RINSSMDREERVRKRMLPLR






TGATTGACGGCGAAGCAATA

RKLEEPLGDSKLDTKQARTF






CTTTGACTTCCAGGTACGGA

WASLIGERKEFGKIPELENW






GGACGGCGGCAAAAAGCAAA

AEEVRSKVTDGQGFASDHVD






AAGGGGACGAAACCGATGGC

QTVWKKILGKARPLKAPGPD






ATTGCCAAAGGGGCCGAAAC

GIPNLLWKRLPSANQALFKW






CATTGAGCTGGACGCTGATG

LMGIKRKQLSVPSWLTKGRV






GGCCGTCTGCCGCGCAAAAG

VLLPKGGDPVDPANYRPIAC






GAGGGGGGAGCCCCTCCGTC

LNTQYKLVTGMVTAWVSEHL






TGGGAAGAAGGTGCGGCGCC

TTYSILPIEQRAMVSGTWGC






ATTCGTCCAACAAACCGGAA

THAMVIDRAITSYAEATGLP






TAGATCTTGATGATCTCAGC

LYVGFVDFAKAFDSVSQPWI






AATTGATCATATTGATCTCT

RYALKVAGVHKRIRCLIGIL






TTTTCCAGTTGTTGCGATAA

MKCWSVRYEVFKSGRVLRSA






ATTATCGTGCATTATTTCTT

PLAVKNGVLQGDTLSPLLFC






CGATTTCTCCAAAGCTTAAC

LSVAVVSSAVGSLFDFEVTI






TCCTTTCCTAATACCTACTC

PGRGVMQQQNHLFYMDDFKG






ATGTATACGTTAGTAGGCAT

FAPSEASLTRMLVTLERTAS






GTTTTATGCAGGTAAAGATA

ALGLKINKRKCALVHPRERE






GACCTGGTGCCCCCGGGCGA

NEETGSDIPVLGLRDTYKYL






CTTGGGATGTGGATGATGGT

GIEERFGIVFEDAWDRVRTK






TGGCGGAGAGTTCTGATGAC

MFERMRTLLCTEHTFGELRA






GCCGTAGTTCCGGAGGAACA

AFASTIAPVARYLFLNVIVG






GTTCCTACTAGTGCCAGCCT

GPSWSETLTKAKDMDLRIRR






GGGCTAGTGGAGCTGGTCTG

LLWERRDNEPGWRFKHCSAD






GCGAGTGCTTTGCTCGTCAG

RLYLRVQYGGLGFVSVEDTL






ATAAGGGGTGGTTGGGGTGG

SESIIYCWAYVQCRPELELA






TACCTCTGGAATGATGTGTC

RELFGTLNRSARSGIKQSIA






GGATAAGCACTCTGCCTTAT

KGARKVFRSYALLSKNSAQR






AAAGCTGTCGTTCTGCCCAG

VSDLDGDASPGFRVGEMIFM






GTTTCTCCTAACCAGGTTGA

EPTRGARAIVKILRKENDSR






ACTTGTAATGAGCCTTTGGG

RLAAWKGRPMGGRVVSLPEL






TATCTGGTCGGGGTCTGCGC

DQVHSYHWLIRARIGRRSFR






GGAGATAGCTCGTGAGGAGC

DCIAAQEGQLKARELMCPHI






TGTTATTACATTATTGGTTG

NAKAKWCRRCGDGRVETEQH






AACGCTTTTGGGTCGTACTG

ILSGCAWSRTGTMLDRHNGV






CACAGATAAAT

VRQVHTALCRKYGLPVSSHV






(SEQ ID NO: 1256)

VPLHAVIENEHAKILYDVAL








HTSPAGVLPREDGSTSYTGL








RSTRPDMVIFDKKARTILIV








EISVPWRENLVKQELIKWRK








YAINSMIEPLELAEAEIPGP








NLKHALGLAYGTSFPTVKVV








PIVVGSCGEVLPNITKRLSE








LGIPKRGIPSLLESIQRAAI








IGSGHVIRAHLSVPRSESET








(SEQ ID NO: 1501)





R4
R4-
CACX0

Strongyloides

CGTCAGAAGAGCAGGTGTTT
TAATTAGATTTATATGTGCC
MQKFSVPKDSSQIFLVDSIL



2_SRa
10020

ratti

TTCAAAGCAAAGACTTATTC
ACTTTACCTCAAAGATATAT
NKHICSTKNKVKDVIKRRSI




06

TACGAAGGGGAAAGATGATC
TAATACTAACTTAATTATAA
IKTLICAAGLTLRKLVCGKL






AAACATGCAGATTTGGCTGC
TTTATTATGAATTATAATAT
GNNKYNSKINQLWKKERKII






AATGAAATAGAATCAAACTA
AATAAGTTTTAAAATAAAA
NCIEDLKHLIETNKRRHNFG






CCATGTGGTGACATCTTGCA
(SEQ ID NO: 1380)
KRLRNAKVSPSEMLKDYYNK






AATACCATTCATACACAAGA

LRYIKNEITACLDEHKKAIL






AGACACGATATGGTTGTGTA

RTKFKLTPSIKIISNIQNHN






CAATATATTAAAAAGACTAA

DEEAELPKEEEFVKYYKELF






AAGAAAAGTATAAGTTAGAT

TNKDGDDKETPHLDNWLKKF






GGTGATTTACAATATGGTAG

SKTLIVDWTINDKEILEALK






ATCAGTATTTAATGGGAAGA

YCGNFKAPGSDMVMKVCYKW






AGGAAAAAATTAGGATCAGA

FKSAQNYLIRWIKSTWYGEY






TCTGGCCATAAGTTTTTGAC

TINKKDTNAVTFMIWKRDGK






TGAACAACCGTTAGTAAACA

PKNDVKSYRPISCLNCDFKL






ATAAACCGGATATTGTTATA

LNKLIANKIYESIEKILPIN






ACGATGAAAGAAGGGAAAAA

QMAVIKNKHGTCEALLLYKS






AAAGATAACGTACATACTAG

LVQSMKFRRTKDVKEIWCSW






AGATGTCGATACCACATATC

IDFSKCYDSISHKCLKKMIQ






TAGAACTTGAAGATGCAAGA

SIKAPPIIHKLILDGIDSWN






AAGAATAAAGTATAAAAAAT

ISICNGKNISKTKIPVKSGI






ATTGTGTGAGCTCCATGGTA

LQGKVASSLYFVLLTGEISY






AAAATAACGAATGATAATGT

ALNKEEQVPIETITPSNTLK






TGATTCGATAGCTCGTGATT

INHISFIDDYQLYATSQKKV






TTAATCTACTAAATTTAATG

EKLTIKLREIAEEMNLKLNP






GAAAGAAAAGAAAGGTGTAA

QKCGIYGTDDLGKRLMLKES






GATAAAGTTTGGATCATTTG

SLNFPYTSEYKYLGLVENSL






TATTTGGCTGTTATGGAGAA

DLKDINIQLFKDKILSKYST






TATGTCTTGACTGAATCTGC

IFESRLTTHQKRKVFNSTIS






TCTAAGAACACAGAAAATCC

PCAAYYLGNLITNKCSIQEL






TGATTGAATTGGGATTCTCC

LNECKKFDQMVRNQLVNQNI






AAAAAAGAGATGGATAGCTT

KKLQVSNSRIYLPKEYNSLG






GATCAAAGAGTGTTCTTACA

LNEIEIEVAANIIRKACYIK






GTTAAATGAATGAGACAGCA

KRETLRGVDKLYIAMSKNGH






AGAATTATTATGAAACATCT

RNTLSDALYITKKYSNFQIN






AGAAGGCGAAAAGAATCAAG

WNIMGMVKDQNNILLDAKKI






ATGAAATCAGCCATTAATAA

IENIKEKRRNLWLEHWKKGN






CTTTTATTTACCCAATTATT

MTYANEAIKKEFHLPDLNID






TATTGCGTTAACTTGATTAT

SKYLMLCYAGSEEQUIYNGH






ATGATATTTATTGTATATTT

VSLVNQSSPSSRLCRKCNKL






ATTGTTTAATTTAGTTTCAT

EETSYHVASVCEFHKKNLHL






AGTACAAATAAAAGAATATA

MRHNSAVYHIITELCRIMKV






CTACGATTCTTTACTTTTAA

KCTLRYPEASGIIKSGNMKI






GTTCTACGAGAACGTTGTTT

AAGVKYTFGTAKIYHNKPDL






TAGAATATTTTAATAATATT

VWYTPEVIYVIEVSISSLKN






TCTACTACAATTAAGTAATT

AKSQMKMKTARYAVNSTKKL






AGAAGAAATCAACGAAAGCA

ENFAALNNLKKGENFVEILS






GCTAAACTTACTCGCAAAAT

HKANFKRVHFMPLVFCTFGE






TCGTTGATCGAGGCTGGAAT

IPKETMKYLEKLNFSNEKIK






GGCACCAACTAATATACTAA

TIASPIARYTGRTLKAHFTN






CCAACAATAGAAAAAAAAAG

(SEQ ID NO: 1502)






AAGAACTTCAGTGGACTTCA








AATAAAATAAAATTATTGGT








AGAATTGTATGATAAAACTG








AGAAAATTTGACTAAAACAA








AAAGATTAGAGCAAATATGT








CATCATTTTCCTAATCATAC








AATTAAAGCGATGATGACGA








AATTGAGAGAATTTGAAAGA








GAAAAAAGGGAAAATTGTGA








AATTAAAATGAGGGAAAATG








AGGAAAAACCTGAGGAAAAA








TTAAATTTTGACAACTATGA








AGAAGCAAAATTGAAAAGAG








GAATTAAGTGTAAAAAAGAA








GTAAAACCAATTGTTATTGA








AAACAAGGATTAGGAATTTT








TGAAAACGGAAAAATTATTT








CTCAAGTTTGTCAACACAAT








CAATTACCAAAGAAAGAAGG








GTAAATCAACCAGA








(SEQ ID NO: 1257)







R4
R4-
CADV0

Bursaphelenchus

GGGTCCTCGGTTCTTACTAC
TGAGACCACCCATGCGCAGA
MISRSQADRPVEGQPVTAMS



3_BX
10088

xylophilus

CGTGCTCCACCTCCTCGCGA
GTATCCGAATCAGTGAAAGT
FHNLEPNNLYPENLRPTGSQ




32

TGGACCCTGGGGTAGGCCTC
CCAAGTTTCAGGACAGAAAC
DANRGVADIAEEVTGPSGLV






CGGGCTGAGCTAAGCAGAGC
GTCAGATAAGTCCAAGAGAA
TNEEAARAPPLFVEGEYKRA






ATTAAAGTAAAGAGTGACGG
ACGAGAAAACAAGTTCAAGT
HCGGGKCHYCRVLWIGARSS






CGCAGTTGCTTCCAAGTTGC
ATGCAAGAGTTAATCAATAA
KARTDSWNLCEILFLINKCM






GGTGGGGCGGAACATCTACT
GAGAGTACCGTAAATGTATG
ELGNVRRIYSPLESSLKEAG






CTTCTGAGAGAGGGGAAGCC
ACCCCCCCCTTTGCCAAGTC
INRTRHAIVKCRLAVMRDRF






CTATGGCGGCGTTAGAAAGG
GACAACTGTCATGCAGGTGT
VDNAPYSEHWRLYNACAENR






TTGGACTGCGGCAACACTAG
CTCTCTTTTCACCCGCCATA
AVVVPMDSATTVKKRTARQA






CCATGATCAGATTCGATCAA
TGGACCAAACGCTATCCAGC
GLESPSQIGVAGKRVHEAET






AATAGCCTCTGGGGCTGGCG
CTCGCTCAGAAGAGCCTTAG
GTDRINAVIETNTTPLEDID






ACCCTATAACGGATTGTAAA
GGCTGGGGAGTACCACATGT
LSPETPEGLAELPSTVEIME






CTGAACTATGCTAACCTGTC
GGCGGAAACTGAATCTGGAT
LTEDGSRSRGTANDADGGVS






AGTAAAGACAGAACGGGGGC
GCGATGCATACCGGGAGCGC
ISDPLRNRPSSSQESRNVPE






TTTGCAATCTAAGGTGTTGG
AGCGAAATCACTTAACGCTG
QVDPDGELVWESLYGAQLRG






CAGACCACTAAAACTGCCCT
GTGCACCTCCTGCTATCGTA
AMRTTDRPRLPKLTKFSAAE






TTGATGAGGGTGAGATAACA
GTACTTCCTAGATAGATGAG
QLWIKSKVEKARLECVSYGI






TCCTACGGAACAGCCCCTGC
TAGGGTGGGCTAAAGGTAGT
EQQWLRASAVLYATIKTVAA






TGATCCAAACAAAATCATCC
CGTCTTCAATGGCAATACCG
CRPYNKAREAHKVWLENKRA






ACAAATCGGAGGGTTTTCTT
AGGGGTCTCGTGACAGGTGA
EEKRVRRIIGRIETVRTMPK






GGTAGTTTCCCTTGGCACGT
GGGATAACTAGTATCAGATA
GKRTDKQIRLARKINRLKRV






CTTGTTTCAAAAGCCAGAAT
ATATCAGTCATGAAATTAGC
SFPEMDWHGFLNHFKAKLDL






ATAAAACAATACCGAAGTAC
AACAACCAACGTCACCGTCG
LKKLISVRVAEHERKISRKI






AGAATGGCTGGAACCTAACA
TTGGCAAAACACCGACTAAC
AGTYGKSVSGQSGFTPDVVA






GCCATGGCACAGAAAACCGG
GATGCTAGTTAGAAAGAGTC
ATTFWSGLAQPGPKKFKKNS






TGGGTGCACACCGGAGACAG
GGGTCTTCCCAAAGTTAGGT
LIFQTWKDSVVENMNTEPVL






CAGGGGAAAGATTGCAAAGT
GCTTGCACCGAAGCCGATCC
LHPLIIECMNKPSPFKATGP






GCCGTAAATAAAGATGGTAG
GCTCTACCCACAGCTCTGCC
DGIFNSYWRQGFIANWVKSL






CTCTCTGACCTGAGGGAATG
CAGCGTT
IQRTIQTGEFPASLMCGRTV






GGACACTGTCCGATTAATGC
(SEQ ID NO: 1381)
LLYKNGDTAKPENYRPITCL






CTTATCCCGAAGACGGTCAA

NGCFKMTNAVITKVIVQRVQ






GGTTCTATCTTATCGGTCTC

DTCALPGEQMALKPKVWACM






CAAAGGGCTCTGGCCATAGA

EAQLRDQALQSEIGNDCKTA






AGCTGCGAGGAAGCCGTAAC

WIDFSKAYDSLDHDAIRFVI






CCTACGCAGATTGCCGCCGT

ETLALPDGMEKYLLKSLESW






GC

KTKLVLSNRGKVATGRPYKI






(SEQ ID NO: 1258)

KRGVLQGDSLSPALFVIATS








PIVSHLKRVCPSGRIQLYMD








DIKLYGKSETELRMLIKEVQ








KVANKLGLQMNLKKCSTYGA








GLTESIAGFDPLGDRAYKYL








GVPQRSVADTNLAFGELEGK








VIRSIEETMACEYLTMRQVV








TRLNSVIGPLVRFVAQSVLT








SQAKVSWIYNKISDLDSKIR








AKLAQTGLRYKKSNVARLYL








SKSKNGIGLVNVQQVLVEAL








VSRAIYCLRAPSLVEIREHI








LTAEFDPVGAARTVLRRSRI








QLEIERVEMASAISAIKTNY








QARWMTKFTQSKLYQKWVHH








DIDLANSNLWLERGEISPQN








ARIAVAAQDNTLLCRGFVGN








RESEKQCRMCNMGIETCSHI








LTECSYHRAHMYIERHDSVA








RNIYAVLAKDHGLWIPHYSQ








PVSSVTKTPTCELYWNYKFP








CTRALEACRPDIVLIDRAKR








TILIVEVAVSYVTRLKQMVS








RKVYKYGVNGEKGADGESRG








WNMIRELSEVYNMKVNLCAV








VIGASGEVLPCTVKAIQSIS








SKTSSRQLLERCQRSAVLGS








TRVVKRHLAEFH








(SEQ ID NO: 1503)





R4
R4-
.

Bursaphelenchus

TGCCAGCGGTGTTGATTAGG
TAATTAGGCAGTGCTCCTAG
MEILWEDLRLKIEDRYGVTL



4_BX


xylophilus

TCCAAGTTCTTTGGCCAAAG
CGGAGTGCCGTGAAGTGGTG
PQRSASSLKNQYPKVILRGL






ATCCGCCCTCGGTTTAGCAG
TCAGTACCCGTCGTGTGGAA
PDSGLPWAGVQVNDTGQVVV






TACCGAACGAGTATACCTTC
AGCCCAGGAGGGTTAGTACC
VDHAEAATLRGSSPAAVDGE






AAGTGGTGGCACTGAATTAG
GACAGTGGGAAACCCGCTGC
AEEPVVPPLPAAEVVESAAD






ACTGAATACTCTGAACTGTA
ACGCAACCTAAAGACATTTG
AAVPDPQSEIVADQGVETRP






GACTTTTGTGCAACTGTGTA
CCCTTCGGGGGAGAGGTATG
VENPPANSRETETEPVEVEP






TGGTGTGGAAGACTTGTTTG
AGACCACACTAGTCATGGTT
YLEGQYKFFVSKILGKSMWR






TACCACTATCAGCTTTATTG
GCTTGCGCAAGCATGACTCA
KPIKYPRRVPETLWRQANEL






GGGCTCGTTACTGTTTCATA
TTCATTGTAAGTTCGTATTA
IERSIRQGEVSIQSLNCMVY






CAGGTAGATGTCCCCTTTAG
TGAGGCCTCGCGGAATGCGA
AAGCAVKSSLDKKDQEAKRR






AGATTTCCCTGCAGTTTGCG
AAGTCCAAGAGCGCCAACTT
ESEWYACRKAEIKALERYLN






CTCCAAGTCGCTAGCCTCTT
GTCTCTTTGGGAGGTTCCTC
FIDLELKRRSASRPLTSRQR






GCGTGTAGTCAAAGGAATAC
CGGGTTCCTTAGGGTTGGCG
QNLGVLITKYGRARVRSGVR






ATTCGCCGTCGGTGACAGGG
TACCATGCTTCGTGTGAGAC
LSELQAMLRDALVGIRKCMA






CTATACCCGGCGACTACGGA
CTAGGCCGCTTGGACCTAGA
KRSADKKRKQGKFVPIQRYL






CTTGTTTATTACGTAGTGCA
GTTAAGCTGTTGCATGTTTG
EPSSAEPRLSPDTVRAYWND






GCCTCGTTTAAGACGAATGT
AGGATGCCTTAGGCGGACGC
IVGSSQQSTSDSTIQDWSSN






GAAAAGAAGGTGTGATTACT
CATG
LSVPSQELNASKIMGWWRAA






AAGCGTTATGAGTCGGGTTA
(SEQ ID NO: 1382)
VSKSKPNKAAGPDGIPGVLW






TCTGGAAACTCCGCCCCGCC

KRFRSASEWVCTWLYRLLQK






GCAATGGCTTCAAGGGTCAA

RRIITPRWLSVGRVVLLPKK






ACTGCTAACATTTTAAACCA

GPLEDPANYRPIACLNTVYK






ATATTTGGCAGCAGCAGACT

LITSVVEMAVREQIQACPGL






ATGATGTCAGAGTGCCGGGG

VPYEQIANRKGVWGCTHASI






GTCTATCGATATAAGACTGA

VDRMITGASREGKGGGFPDL






AAGCGATAGACGTGGAGTGA

RVLFYDCKKAFDSVNRDHMF






AAGGATTCCCTTTGTTAAGA

AVLRVANVNVKVVHLLHTLS






GAATCGGTAGAATTCACTTT

QQWCVRYELRRNNRVERSSP






TTACTTATTCAAGAACTTAA

LRVKRGLLQGDTLSPTWFCL






CAGCAACAAGCACTCGCGAG

CMAPISASIKTLNPGPTLRP






GATTACCGCCCCAGATTCGG

NMGNGRNRGQVAIQVSHVFY






TCGGCGGTACTTCACCTGCT

MDDLKVYCPRVADQRRMEQN






TTCTTCCACTTTCGGAATCT

IPQLFGEIGLSINASKSAAA






GGCATCCTGGCTTTCAGTGT

AAVGRYVESELPVLGTKDEY






GGTGATGGCCGGCTTGAGTT

KYLGIESGFVVNEVAALDRM






TTCTTGAGTCGTGCGAGTGC

QAVLLNRVEAILSVKEHTVG






CTCATCTGGGACGTCCGGAC

QRRDAIRAKAIPGGAYILGH






CGATTGATGGAGTCTGCAGT

IILSDLDPRGAAERMRRLDI






GGACGAGGACTTGATGGACC

EIRRLVKSAGILHDKCSTAR






GTAACCATAGTATATCCCTC

IHLSCEQGGLAWPSMERAYY






ATGCGTCTTCTCGACTCGAG

VAVAYSASYLLTSQDETISR






GGGGTAGCTTGCACTACCCA

ARDYFVSGRLSNKFTVYKHL






CCCTTCTCTTCTCCGATTGG

TSIVDSLGLSVELPDPNGLP






GATTTAGACCTAGCCCTCTG

TGQPSVLARTIARAIDAKLE






GTGTGTCTCGACCCGCGATA

AQWKETLLTYQRAGRVERAD






TCAGATTCCTGAATCGACTG

PTVVDHANSYHWLRKAWINE






TGAGAAATGTCTACGCGCAA

KAYQHAVSVMEGTLLEGVNP






AGATCGACCCATTGCCACCC

HGVLTMCRACKAPSASIAHI






GGCTATGTGGATCGGGCTCT

ITGCAELRKSHMKVRHDGVT






TGACTGCTTATCTCCGGCTT

RWLYNALTEVDGSLPKFHYT






TAATCGCTTGAGGAAAGGGG

QQIPAEMRGERLTVRYDSDI






GGTGTCGCCCGAAAGGGTTA

VTPNKPRHNRPDLVVFDSTR






CGCGATCATCCGATTCCTAC

KVIYIVEVSVTWLSVLQKQY






CGTAAAAACGTAGTTGAAGT

DNKLNRYAVNSNHEFSESIP






AGGATTGAACCTGAGTACCA

YPPGVNLANEIRVLYPQFTG






AGTGAAAAGAGTGGCTATAA

GVKVFPMIISPTGEVHMQFV






TGCTCCATGGTATACCCTAG

PHLAELLENPNIPRILEKIQ






GGATAACCGTGTCGAGCGCA

RSVVLGTDYIIRSYFAM






CTGCTCAATACCCTGATTTG

(SEQ ID NO: 1504)






TTAGTGTAGGTATGTCTAGT








GGCTGCCTAGGCAGATTCGC








CTATTCACTACGTAAAATCT








GGGTGAGTACCATAAGAACC








TCCTGTAGGGCCAGGAGTCA








AACTAGTCCAGGTTCGGTAT








GCTTCGGTATACTCTCTCAC








GGGCTAGTCACCTAAGAGTT








AAGAACCGCCTTTTTCTGCA








CTGTGAATAGAAAAAGAAGG








GCGGGAGAATATACGGGCGA








GGTAAGCACGTCGGAACGGG








GTGTGCCCAACTCGTACCGT








ACGCCCGGACAAGGACCATC








TTTCGTACCCCGTTCACCCG








GCACAAGTCCGATTGTCTCT








CCCGAGAGGCGTCGGCGGAG








GTTGGCTAACGCCGTCTCTC








CAACCATCGGTTTGGGTTTA








AGTAACGCCCCAGTGGCCGG








TAGCCAAACTGATGGAGGCA








GAAATGCCGGTCCGTTGCTA








AATGCGGGAACGGACCAAAA








CGCCGGTGTGGTGGATACAG








GTGGAAGAGTCTTTTGGTCT








ACGCAAGAAAAGACCAGGCT








CAAGTACGAATACGACCGTC








TTCGTGAACGACGCCGTCGA








GACAAAGTTCGAAATCCTAC








GGTCGATAAGGCC








(SEQ ID NO: 1259)







R4
R4_AL
U2944

Ascaris

GGGGCCGGTGGGTTTACTCA
TAGTCGCTAAGGGGTCCGGA
MPCSTNSFFERGTPEPHREP




5

lumbricoides

CTTCTGACCCACCACCAACG
AATGGTCCGGTCCTGCGCTA
ISGTDSSESLGMGTHRSPRL






GAACGAGGGAAAGCAGAGCT
CCCGGTTCTGGTAGCACGTT
NDDEVINGPKGHESDPVHVV






GGGGCCCTCTTCCGATTGGC
CAAGCGCTCAATCGCCTGCC
RAPRTLHPRRLELPIGVNNL






ATGGAACCGACCTCCACGTG
TTGTAGGCAGTCCATCTGTG
GEASQLRQDSAIAEEAQLES






GTGGCCCTGGGCAACGGAAT
GAAGTCGCGCTCTTGATACA
TENHDGRRPPLRGGRKLWSE






TCAAGAGAGGATTTAATCCT
GATGTGGACGGATGGAAGCA
KEIATLRRLCEAYGNRQVCW






CTCTATCATTTGCAAGATGG
GATGATAGAGCCGGTGACGG
KEVQRKFADFHEERTVAALA






ATGAGATCGAGGTATCCGGC
CCCTACTAGCCAAACGC
TKWGALKRPRAPMVGAPPTP






AAACAGGTTCCAAGTGAGCA
(SEQ ID NO: 1383)
DHDPERGPAGEGDGGTTSQE






CCTTTCCCATAGCTGGGAAT

NVPTDDPIPANGPTEGKESD






ATGGGTTAGGCGTCCTCTGA

VRPAVACRCTEPEEQLMESD






CATATAAGAGGAATCAGACT

VRPPAVVRLADPEQHTMKSG






CGTTCGCGCCCGGTCATTAA

VKPVALDGSADLEERPKEKD






CATCGATCAGCGGGAGGGCC

IEQMGVDFEGEPRFRAFRKA






GGACTGAAGTAAATTTCCTG

FYGYFRWAVNSFDREPVKRV






TTGGCCCGAGTGCAGGTGGA

RRDCPKVFYAYADYLIATGS






GCTCGGACCCGAAAAACGAT

SKALGPNQSRIGRLNGLVYA






CCCTAAGAGGACCACAACCC

AARTIHQFWREEVGHRQQGE






GAAGGGATGGACGCAGTCGC

KGWYTKTKATREDLQMLISM






CCCGGCACGTTTGGTGTTGG

MESELARRKEKRKPGAKELE






TTATCCTGGAGTGTTGTGGG

NIHKLVARLGTRSTSGIVRR






ACGAATAGCT

LEMTRQRLKLLEDRISLHEQ






(SEQ ID NO: 1260)

EKRRKRLRKQFAETPSLKLL








TKGAKDRGDTMVTMKSVMDF








WRPIIGRRVTSNPDQLQVLR








DWRDEQKKAYPADLDLEKAD








LEEKYEGAIRRIQPWKAPGP








DGLHAHWWKALPSAKRLLGE








LVVDWLTTGKVTTGWMCRGR








TILIPKKGDRGDPSNYRPIT








CLNTCYKVLTSVMNSVILSH








LSRGEALPMNQRAMRKREWG








CTHAMVLDRAMVMDAMAQKK








HSLSVAWLDYRKAYDSVSHE








YIRWAINSVNIPRSVQLTLK








RLMSDWETRFESTQCRPKLR








SDKMKVLNGIFQGDSLSPTL








FVLCIAPISYALNKGVGQCQ








SSSGWSAGYGFEIGHQFYMD








DLKLYARTPAMLDSQIQVVS








EVSEAMGLHLNLSKCAKAHY








APHGAGGAQEAVEGAEGSRK








GEIPILGLRSTYKYLGVEQR








LLPMEVALKEFEDKFMDRAE








TIFASELTWGQMATAYNTIA








IAGLRYVYSNTNGASPKLLE








ALKRAATLDTRIRDLLRRHK








CRFRNSFVERLYIPRECGGY








GLKSVEDTLRESILATWSYI








ATNPHLAGQQYFFERLAARG








KRTPMADGVKILLDLGVEPQ








VDLKRRTVTVDGIVFEDPTK








LHRYLVGKLLKARTEARIRR








WKEASLAGRLVNDTSIDMRL








SCLWMKKGFVSARNLRDALA








VQEGSLLTRACPALKGKGGQ








EVCRCCHAAPETAEHITSAC








RYWLPSLYVERHDSVARNLY








YVICCRYGITPVHYSNRVSP








LSENSQCRVLWNMDMQTRTP








MKHRKPDIVVFDLKREKILM








FEVSIAHASGLLKQREIKIN








RYTVNSEELPDETITPYPPG








PNLAADLAATYGWQVEFAPV








VVGTCGEHVPAVKEDLQRTL








DLKPHQVEALLERISRSAVI








GTARVVRAHLACS








(SEQ ID NO: 1505)





R4
R4_H
.

Heliconius

ATAAATAATAATAATAATAA
TAACTTATTGTCAGAATTCC
ITYTANMALVTLFMENMENK



mel


melpomene

TAATAATAAGCCCCCTAAAA
TTACTAGTAATAATAATTAT
RYNLRPLPGGRRGASGANAG






TCCAACCATACGTCCGAGTC
CGCTGAAAATCTCCACCCAA
CHSMRTVGDGGLSRRVPLEK






GAACATCTGATTCTCGTGGG
ATATTGCTTGGCTATATGCT
NVTAEQSSSPLTSSSSHSPV






GGGCGGACACGTGAAAATAA
CGCAATTTTTGGTTAACGTA
SSIPSPSSTRTLLNSPNSSP






(SEQ ID NO: 1261)
CCCCAATGATTTGGGAGAAC
TSSHSSLVIRSADVVQEALA







AAAAATGGTAAAACTATAAT
NYPAPTAGSIRARKKWTDIM







AATAATAATTATATTAATAT
NRYIWRTYLIITKCETTLLN







(SEQ ID NO: 1384)
NYLEPLHQEFSSKFPEMQVT








RQRIGDQRRAIIRNKLLSDD








TLAQILIEVKELLQIGDQPL








TQNNIHSTQLSHSNTRIKWS








NELNEEIVKCYFEVTLLEVN








KTSYRKNLYSLFISRNPHLS








HLTEQRIADQRRLIFMNKSV








HNDRIIELKREVEIKLANSN








SLTKNITESNSPSSQTNEIN








DSAYVQSNLQPVEPLDQHCI








NRHNLIEKHYVEQEFNNALI








QFNNTNPETRPYIPRQKSSR








KFSQIVSFLNSEVLPKHLNN








ELDFNALHNIIYTAXYTASL








CNGTKFSFIDNYRPRNSKPS








WQRRLESRIDKYRLQIGRLT








QYISGNRNRKILKTVEEIKT








QYKIHSHHEEPNTELPHFLD








TLKQKLNATSNRLRRYLTCT








KRKQQNNTFVNNEKHFYRTL








SSTNQNTTTQLXEHPTENNL








QQYWANIWETSIEHNADAEW








LNKIPDXEINXMKFKDISIE








TFNQUIQRTHNWKAPGTDNI








HNYWYKKLTCTHSLLLKHIN








QFIQSPCTLPLFITNGITYM








LPKGLDPTNPANYRPITCLQ








TIYKIITACITDIIYKHIDQ








NNILAEQQKGCRKNSQGCKE








QLTIDAIVMKQAHNKNXNTM








YIDYRKAFDSVPHSWLLYIL








KKYKIHPILITFLSSVMLSW








KTRLKLINNNETLITDWIKI








QRGIFQGDALSPLWFCLALN








PLSELLNNTNTGFKLKHNNT








YHIISHLMYMDDIKLYASNN








KELKILADLTQSFSTDIRME








FGIEKCKVHSIKRGKSQQNT








YILNTGEQIESMDENSTYKY








LGFQQAKQIQQKQTKIELTN








KFKFRLNQILRSQLNSRNII








KAINTYAIPILTYSFAIINW








SQTDLSNLQRIINTHMTTHR








KHHPKSCIQRLTISRLDGGR








GLIDIRNLHNNLVTKFRNYF








YAKAEISELHKFIVNIDNKY








TPLNLNDRNIQLNQTLITKQ








QKIEAWSLKSLHGRHLADLS








QTHVDKVASNEWLRRGDLFP








ETEAFMMAIQDQVIDTRNYQ








KHIIKRPNMVNDLCRRCYSS








PETIQHITGACKTIVQTDYK








HRHDQVAAIIHQHLAFKHSL








ITQAQKTPYYKYSPQAILES








TNFKLYWDRTIITDKTVHYN








RPDILLHDKVKXSVYLIDIA








IPNTHNLASTFSNKIDKYTD








LTIELKSQWKVQSVTTVPIV








LSTTGVVPHTLHTSLETLGI








HRLSYILLQKAAILNTCRIV








RKFLSSNN








(SEQ ID NO: 1506)





R4
Rex6


Takifugu

TTCTATGCGCCTTATGCGAC
TAGAGGACCCGAGTCTGAAG
MSGTXTDRVIPARTSPGSTR






rubripes

TGGATAGGCCAGTGGTTTAC
GAAGGAGGCACCGCCCAGGA
SASGVGEPGPPDVKLATGTR






GCCGCTGACTTTGGTGCGGA
GGGCGAGGAAGAGATTTTTT
HSWSRAENVVLMECYYGSNP






AGGTTGTCGGTTCGAATCCA
TTTATATATATATATATATA
SERGYMQRMWEKWVLRNPTS






GGCGAGCCCTTAGGCAAGGC
TATA
SLTKKQLLAQCSNIRNKKLL






TCCTTACGCATATATGCCTA
(SEQ ID NO: 1385)
SQLEIDEARRCASPTVQICY






CACCTCGGT

GKGEPGRQVSXGVISSSPPN






(SEQ ID NO: 1262)

IEIGYKAPMTDGLGTRAADL








RERIMKSWGNSTTSLPRLTH








KVPDQSLLEDMNTALSTIPT








TTITETNQLMYAAATVILQM








LGYKMKSMNSQKEQMAPWRR








RLEAKIMATRREVSLLTELS








RGVNLRTEXPKKYNKLSTTE








ALETAKQRLTALATRLKRYT








REVEARRINKVFSTNPAKVY








SQWQGNKMTTDPPRAETEQY








WKSIWEKEATHNTXAQWLQD








LQTEHSQLPEQDPVVITLAD








IQTRVSKMKSWTAPGPDKIH








AYWLKKLTALHERLAAQMNQ








LLTSGNHPEWLTQGRTVLIM








KDPQKGTIPSNYRPITCLST








TWKLLSGIIAAKISRHMDQY








MSRAQKGIGNNTRGAKHQLL








VDRAIAQDCRTRHTNLCTAW








IDYKKAYDSMPHTWILECLK








LYNINRTLREFIQNSMKLWN








TTLEANSKPIARVSIRCGIY








QGDALSPLLFCIGLNPLSQI








ITKSGYGYQFRSGTTVSHLL








YMDDIKLYAKNERDIDSLIH








LTRIYSKDIGMSFGLDKCGR








MISRRGKVIATDGVELPEGN








ITDVQDSYKYLGIPQANGNH








EEAARRSATAKYLQRLRQVL








KSQLNGKNKIQAINTYALPV








IRYPAGIIPWPLEEIQATDI








KTRKLNGKHKIQAINTYALP








VIRYPAGIIPWPLEEIQATD








IKTRKLLTMHGGFHPKSSVL








RLYTKRKEGGRGLVSVRTTV








QEETTSLREYIKKMAPTDRL








LSECLRQQKPTKEEEPEGLS








WKDKPLHGMYHRQIEEVADI








EKTYQWLEKAGLKDSTEALL








MAAQEQALSTRAIEARVYHT








RQDPRCRLCGDAPETVQHIT








AGCKMLAGKAYMERHNQVAG








IVYRNICTEYGLEVPGSRWE








TPPKVLENKQAKILWDFQIQ








TDKMVVANQPDIVVVDKHQK








TVVVIDVAIPSDSNIRKKEH








EKLEKYQGLKEEMERMWGMK








ATVVPVVIGTLGAVTPKLSR








WLQQIPGTTSEISVQKSAVL








GTAKILRRTLRLPGLW








(SEQ ID NO: 1507)
















TABLE 30







Exemplary monomeric retroviral reverse transcriptases and 


their RT domain signatures















RT


Name
Accession
Organism
Sequence
Signatures





Q4VFZ2_9 
Q4VFZ2
Porcine
MGATGQQQYPWTTRRTVDLGVGRVT
IPR043502,


GAMR-

endogenous
HSFLVIPECPAPLLGRDLLTKMGAQISF
SSF56672,


residues

retrovirus
EQGKPEVSANNKPITVLTLQLDDEYRL
IPR000477,


only


YSPLVKPDQNIQFWLEQFPQAWAETA
PF00078,





GMGLAKQVPPQVIQLKASATPVSVRQ
cd03715





YPLSKEAQEGIRPHVQRLIQQGILVPVQ






SPWNTPLLPVRKPGTNDYRPVQDLRE






VNKRVQDIHPTVPNPYNLLCALPPQRS






WYTVLDLKDAFFCLRLHPTSQPLFAFE






WRDPGTGRTGQLTWTRLPQGFKNSPTI






FDEALHRDLANFRIQHPQVTLLQYVDD






LLLAGATKQDCLEGTKALLLELSDLGY






RASAKKAQICRREVTYLGYSLRDGQR






WLTEARKKTVVQIPAPTTAKQVREFLG






TAGFCRLWIPGFATLAAPLYPLTKEKG






EFSWAPEHQKAFDAIKKALLSAPALAL






PDVTKPFTLYVDERKGVARGVLTQTL






GPWRRPVAYLSKKLDPVASGWPVCLK






AIAAVAILVKDADKLTLGQNITVIAPH






ALENIVRQPPDRWMTNARMTHYQSLL






LTERVTFAPPAALNPATLLPEETDEPVT






HDCHQLLIEETGVRKDLTDIPLTGEVLT






WFTDGSSYVVEGKRMAGAAVVDGTR






TIWASSLPEGTSAQKAELMALTQALRL






AEGKSINIYTDSRYAFATAHVHGAIYK






QRGLLTSAGREIKNKEEILSLLEALHLP






KRLAIIHCPGHQKAKDPISRGNQMADR






VAKQAAQGVNLLPMIETPKAPEPGRQ






YTLEDWQEIKKIDQFSETPEGTCYTSD






GKEILPHKEGLEYVQQIHRLTHLGTKH






LQQLVRTSPYHVLRLPGVADSVVKHC






VPCQLVNANPSRIPPGKRLRGSHPGAH






WEVDFTEVKPAKYGNKYLLVFVDTFS






GWVEAYPTKKETSTVVAKKILEEIFPR






FGIPKVIGSDNGPAFVAQVSQGLAKILG






IDWKLHCAYRPQSSGQVERMNRTIKET






LTKLTAETGVNDWIALLPFVLFRVRNT






PGQFGLTPYELLYGGPPPLVEIASVHSA






DVLLSQPLFSRLKALEWVRQRAWRQL






REAYSGGGDLQIPHRFQVGDSVYVRR






HRAGNLETRWKGPYHVLLTTPTAVKV






EGISTWIHASHVKPAPPPDSGWKAEKT






ENPLKLRLHRVVPYSVNNFSS






(SEQ ID NO: 1559)






POL_SFV1-
P23074
Simian
MDPLQLLQPLEAEIKGTKLKAHWDSG
IPR043502,


residues

foamy
ATITCVPEAFLEDERPIQTMLIKTIHGEK
SSF56672,


only

virus 
QQDVYYLTFKVQGRKVEAEVLASPYD
IPR000477,




type 1
YILLNPSDVPWLMKKPLQLTVLVPLHE
PF00078





YQERLLQQTALPKEQKELLQKLFLKY






DALWQHWENQVGHRRIKPHNIATGTL






APRPQKQYPINPKAKPSIQIVIDDLLKQ






GVLIQQNSTMNTPVYPVPKPDGKWRM






VLDYREVNKTIPLIAAQNQHSAGILSSI






YRGKYKTTLDLTNGFWAHPITPESYW






LTAFTWQGKQYCWTRLPQGFLNSPAL






FTADVVDLLKEIPNVQAYVDDIYISHD






DPQEHLEQLEKIFSILLNAGYVVSLKKS






EIAQREVEFLGFNITKEGRGLTDTFKQK






LLNITPPKDLKQLQSILGLLNFARNFIPN






YSELVKPLYTIVANANGKFISWTEDNS






NQLQHIISVLNQADNLEERNPETRLIIK






VNSSPSAGYIRYYNEGSKRPIMYVNYIF






SKAEAKFTQTEKLLTTMHKGLIKAMD






LAMGQEILVYSPIVSMTKIQRTPLPERK






ALPVRWITWMTYLEDPRIQFHYDKSLP






ELQQIPNVTEDVIAKTKHPSEFAMVFY






TDGSAIKHPDVNKSHSAGMGIAQVQFI






PEYKIVHQWSIPLGDHTAQLAEIAAVE






FACKKALKISGPVLIVTDSFYVAESAN






KELPYWKSNGFLNNKKKPLRHVSKW






KSIAECLQLKPDIIIMHEKGHQQPMTTL






HTEGNNLADKLATQGSYVVHCNTTPS






LDAELDQLLQGHYPPGYPKQYKYTLE






ENKLIVERPNGIRIVPPKADREKIISTAH






NIAHTGRDATFLKVSSKYWWPNLRKD






VVKSIRQCKQCLVTNATNLTSPPILRPV






KPLKPFDKFYIDYIGPLPPSNGYLHVLV






VVDSMTGFVWLYPTKAPSTSATVKAL






NMLTSIAIPKVLHSDQGAAFTSSTFAD






WAKEKGIQLEFSTPYHPQSSGKVERKN






SDIKRLLTKLLIGRPAKWYDLLPVVQL






ALNNSYSPSSKYTPHQLLFGVDSNTPF






ANSDTLDLSREEELSLLQEIRSSLHQPT






SPPASSRSWSPSVGQLVQERVARPASL






RPRWHKPTAILEVVNPRTVIILDHLGN






RRTVSVDNLKLTAYQDNGTSNDSGTM






ALMEEDESSTSST






(SEQ ID NO: 1560)






POL_MPMV-
P07572
Mason-
MGQELSQHERYVEQLKQALKTRGVK
IPR043502,


residues

Pfizer
VKYADLLKFFDFVKDTCPWFPQEGTID
SSF56672,


only

monkey
IKRWRRVGDCFQDYYNTFGPEKVPVT
IPR000477,




virus
AFSYWNLIKELIDKKEVNPQVMAAVA
PF00078,





QTEEILKSNSQTDLTKTSQNPDLDLISL
cd01645,





DSDDEGAKSSSLQDKGLSSTKKPKRFP
PF06817,





VLLTAQTSKDPEDPNPSEVDWDGLED
IPR010661





EAAKYHNPDWPPFLTRPPPYNKATPSA






PTVMAVVNPKEELKEKIAQLEEQIKLE






ELHQALISKLQKLKTGNETVTHPDTAG






GLSRTPHWPGQHIPKGKCCASREKEEQ






IPKDIFPVTETVDGQGQAWRHHNGFDF






AVIKELKTAASQYGATAPYTLAIVESV






ADNWLTPTDWNTLVRAVLSGGDHLL






WKSEFFENCRDTAKRNQQAGNGWDF






DMLTGSGNYSSTDAQMQYDPGLFAQI






QAAATKAWRKLPVKGDPGASLTGVK






QGPDEPFADFVHRLITTAGRIFGSAEAG






VDYVKQLAYENANPACQAAIRPYRKK






TDLTGYIRLCSDIGPSYQQGLAMAAAF






SGQTVKDFLNNKNKEKGGCCFKCGKK






GHFAKNCHEHAHNNAEPKVPGLCPRC






KRGKHWANECKSKTDNQGNPIPPHQG






NRVEGPAPGPETSLWGSQLCSSQQKQP






ISKLTRATPGSAGLDLCSTSHTVLTPEM






GPQALSTGIYGPLPPNTFGLILGRSSITM






KGLQVYPGVIDNDYTGEIKIMAKAVN






NIVTVSQGNRIAQLILLPLIETDNKVQQ






PYRGQGSFGSSDIYWVQPITCQKPSLTL






WLDDKMFTGLIDTGADVTIIKLEDWPP






NWPITDTLTNLRGIGQSNNPKQSSKYL






TWRDKENNSGLIKPFVIPNLPVNLWGR






DLLSQMKIMMCSPNDIVTAQMLAQGY






SPGKGLGKKENGILHPIPNQGQSNKKG






FGNFLTAAIDILAPQQCAEPITWKSDEP






VWVDQWPLTNDKLAAAQQLVQEQLE






AGHITESSSPWNTPIFVIKKKSGKWRLL






QDLRAVNATMVLMGALQPGLPSPVAI






PQGYLKIIIDLKDCFFSIPLHPSDQKRFA






FSLPSTNFKEPMQRFQWKVLPQGMAN






SPTLCQKYVATAIHKVRHAWKQMYII






HYMDDILIAGKDGQQVLQCFDQLKQE






LTAAGLHIAPEKVQLQDPYTYLGFELN






GPKITNQKAVIRKDKLQTLNDFQKLLG






DINWLRPYLKLTTGDLKPLFDTLKGDS






DPNSHRSLSKEALASLEKVETAIAEQF






VTHINYSLPLIFLIFNTALTPTGLFWQD






NPIMWIHLPASPKKVLLPYYDAIADLII






LGRDHSKKYFGIEPSTIIQPYSKSQIDW






LMQNTEMWPIACASFVGILDNHYPPN






KLIQFCKLHTFVFPQIISKTPLNNALLVF






TDGSSTGMAAYTLTDTTIKFQTNLNSA






QLVELQALIAVLSAFPNQPLNIYTDSAY






LAHSIPLLETVAQIKHISETAKLFLQCQ






QLIYNRSIPFYIGHVRAHSGLPGPIAQG






NQRADLATKIVASNINTNLESAQNAHT






LHHLNAQTLRLMFNIPREQARQIVKQC






PICVTYLPVPHLGVNPRGLFPNMIWQM






DVTHYSEFGNLKYIHVSIDTFSGFLLAT






LQTGETTKHVITHLLHCFSIIGLPKQIKT






DNGPGYTSKNFQEFCSTLQIKHITGIPY






NPQGQGIVERAHLSLKTTIEKIKKGEW






YPRKGTPRNILNHALFILNFLNLDDQN






KSAADRFWHNNPKKQFAMVKWKDPL






DNTWHGPDPVLIWGRGSVCVYSQTYD






AARWLPERLVRQVSNNNQSRE






(SEQ ID NO: 1561)






POL_MMTVB-
P03365
Mouse
MGVSGSKGQKLFVSVLQRLLSERGLH
IPR043502,


residues

mammary
VKESSAIEFYQFLIKVSPWFPEEGGLNL
SSF56672,


only

tumor
QDWKRVGREMKRYAAEHGTDSIPKQ
IPR000477,




virus
AYPIWLQLREILTEQSDLVLLSAEAKSV
PF00078,





TEEELEEGLTGLLSTSSQEKTYGTRGT
cd01645,





AYAEIDTEVDKLSEHIYDEPYEEKEKA
PF06817,





DKNEEKDHVRKIKKVVQRKENSEGKR
IPR010661





KEKDSKAFLATDWNDDDLSPEDWDD






LEEQAAHYHDDDELILPVKRKVVKKK






PQALRRKPLPPVGFAGAMAEAREKGD






LTFTFPVVFMGESDEDDTPVWEPLPLK






TLKELQSAVRTMGPSAPYTLQVVDMV






ASQWLTPSDWHQTARATLSPGDYVL






WRTEYEEKSKEMVQKAAGKRKGKVS






LDMLLGTGQFLSPSSQIKLSKDVLKDV






TTNAVLAWRAIPPPGVKKTVLAGLKQ






GNEESYETFISRLEEAVYRMMPRGEGS






DILIKQLAWENANSLCQDLIRPIRKTGT






IQDYIRACLDASPAVVQGMAYAAAMR






GQKYSTFVKQTYGGGKGGQGAEGPV






CFSCGKTGHIRKDCKDEKGSKRAPPGL






CPRCKKGYHWKSECKSKFDKDGNPLP






PLETNAENSKNLVKGQSPSPAQKGDG






VKGSGLNPEAPPFTIHDLPRGTPGSAGL






DLSSQKDLILSLEDGVSLVPTLVKGTLP






EGTTGLIIGRSSNYKKGLEVLPGVIDSD






FQGEIKVMVKAAKNAVIIHKGERIAQL






LLLPYLKLPNPVIKEERGSEGFGSTSHV






HWVQEISDSRPMLHIYLNGRRFLGLLD






TGADKTCIAGRDWPANWPIHQTESSLQ






GLGMACGVARSSQPLRWQHEDKSGII






HPFVIPTLPFTLWGRDIMKDIKVRLMT






DSPDDSQDLMIGAIESNLFADQISWKS






DQPVWLNQWPLKQEKLQALQQLVTE






QLQLGHLEESNSPWNTPVFVIKKKSGK






WRLLQDLRAVNATMHDMGALQPGLP






SPVAVPKGWEIIIIDLQDCFFNIKLHPED






CKRFAFSVPSPNFKRPYQRFQWKVLPQ






GMKNSPTLCQKFVDKAILTVRDKYQD






SYIVHYMDDILLAHPSRSIVDEILTSMI






QALNKHGLVVSTEKIQKYDNLKYLGT






HIQGDSVSYQKLQIRTDKLRTLNDFQK






LLGNINWIRPFLKLTTGELKPLFEILNG






DSNPISTRKLTPEACKALQLMNERLST






ARVKRLDLSQPWSLCILKTEYTPTACL






WQDGVVEWIHLPHISPKVITPYDIFCTQ






LIIKGRHRSKELFSKDPDYIVVPYTKVQ






FDLLLQEKEDWPISLLGFLGEVHFHLP






KDPLLTFTLQTAIIFPHMTSTTPLEKGIV






IFTDGSANGRSVTYIQGREPIIKENTQN






TAQQAEIVAVITAFEEVSQPFNLYTDSK






YVTGLFPEIETATLSPRTKIYTELKHLQ






RLIHKRQEKFYIGHIRGHTGLPGPLAQG






NAYADSLTRILTALESAQESHALHHQN






AAALRFQFHITREQAREIVKLCPNCPD






WGHAPQLGVNPRGLKPRVLWQMDVT






HVSEFGKLKYVHVTVDTYSHFTFATA






RTGEATKDVLQHLAQSFAYMGIPQKIK






TDNAPAYVSRSIQEFLARWKISHVTGIP






YNPQGQAIVERTHQNIKAQLNKLQKA






GKYYTPHHLLAHALFVLNHVNMDNQ






GHTAAERHWGPISADPKPMVMWKDL






LTGSWKGPDVLITAGRGYACVFPQDA






ETPIWVPDRFIRPFTERKEATPTPGTAE






KTPPRDEKDQQESPKNESSPHQREDGL






ATSAGVDLRSGGGP 






(SEQ ID NO: 1562)






POL_MLVMS-
P03355
Moloney
MGQTVTTPLSLTLGHWKDVERIAHNQ
IPR043502,


residues

murine
SVDVKKRRWVTFCSAEWPTFNVGWP
SSF56672,


only

leukemia
RDGTFNRDLITQVKIKVFSPGPHGHPD
IPR000477,




virus
QVPYIVTWEALAFDPPPWVKPFVHPKP
PF00078,





PPPLPPSAPSLPLEPPRSTPPRSSLYPALT
cd03715





PSLGAKPKPQVLSDSGGPLIDLLTEDPP






PYRDPRPPPSDRDGNGGEATPAGEAPD






PSPMASRLRGRREPPVADSTTSQAFPL






RAGGNGQLQYWPFSSSDLYNWKNNN






PSFSEDPGKLTALIESVLITHQPTWDDC






QQLLGTLLTGEEKQRVLLEARKAVRG






DDGRPTQLPNEVDAAFPLERPDWDYT






TQAGRNHLVHYRQLLLAGLQNAGRSP






TNLAKVKGITQGPNESPSAFLERLKEA






YRRYTPYDPEDPGQETNVSMSFIWQSA






PDIGRKLERLEDLKNKTLGDLVREAEK






IFNKRETPEEREERIRRETEEKEERRRTE






DEQKEKERDRRRHREMSKLLATVVSG






QKQDRQGGERRRSQLDRDQCAYCKE






KGHWAKDCPKKPRGPRGPRPQTSLLT






LDDQGGQGQEPPPEPRITLKVGGQPVT






FLVDTGAQHSVLTQNPGPLSDKSAWV






QGATGGKRYRWTTDRKVHLATGKVT






HSFLHVPDCPYPLLGRDLLTKLKAQIH






FEGSGAQVMGPMGQPLQVLTLNIEDE






HRLHETSKEPDVSLGSTWLSDFPQAW






AETGGMGLAVRQAPLIIPLKATSTPVSI






KQYPMSQEARLGIKPHIQRLLDQGILV






PCQSPWNTPLLPVKKPGTNDYRPVQD






LREVNKRVEDIHPTVPNPYNLLSGLPPS






HQWYTVLDLKDAFFCLRLHPTSQPLFA






FEWRDPEMGISGQLTWTRLPQGFKNSP






TLFDEALHRDLADFRIQHPDLILLQYV






DDLLLAATSELDCQQGTRALLQTLGN






LGYRASAKKAQICQKQVKYLGYLLKE






GQRWLTEARKETVMGQPTPKTPRQLR






EFLGTAGFCRLWIPGFAEMAAPLYPLT






KTGTLFNWGPDQQKAYQEIKQALLTA






PALGLPDLTKPFELFVDEKQGYAKGVL






TQKLGPWRRPVAYLSKKLDPVAAGWP






PCLRMVAAIAVLTKDAGKLTMGQPLV






ILAPHAVEALVKQPPDRWLSNARMTH






YQALLLDTDRVQFGPVVALNPATLLPL






PEEGLQHNCLDILAEAHGTRPDLTDQP






LPDADHTWYTDGSSLLQEGQRKAGAA






VTTETEVIWAKALPAGTSAQRAELIAL






TQALKMAEGKKLNVYTDSRYAFATA






HIHGEIYRRRGLLTSEGKEIKNKDEILA






LLKALFLPKRLSIIHCPGHQKGHSAEAR






GNRMADQAARKAAITETPDTSTLLIEN






SSPYTSEHFHYTVTDIKDLTKLGAIYDK






TKKYWVYQGKPVMPDQFTFELLDFLH






QLTHLSFSKMKALLERSHSPYYMLNR






DRTLKNITETCKACAQVNASKSAVKQ






GTRVRGHRPGTHWEIDFTEIKPGLYGY






KYLLVFIDTFSGWIEAFPTKKETAKVV






TKKLLEEIFPRFGMPQVLGTDNGPAFV






SKVSQTVADLLGIDWKLHCAYRPQSS






GQVERMNRTIKETLTKLTLATGSRDW






VLLLPLALYRARNTPGPHGLTPYEILY






GAPPPLVNFPDPDMTRVTNSPSLQAHL






QALYLVQHEVWRPLAAAYQEQLDRP






VVPHPYRVGDTVWVRRHQTKNLEPR






WKGPYTVLLTTPTALKVDGIAAWIHA






AHVKAADPGGGPSSRLTWRVQRSQNP






LKIRLTREAP 






(SEQ ID NO: 1563)






POL_HTL1A-
P03362
Human T-
MGQIFSRSASPIPRPPRGLAAHHWLNFL
IPR043502,


residues

cell
QAAYRLEPGPSSYDFHQLKKFLKIALE
SSF56672,


only

leukemia
TPARICPINYSLLASLLPKGYPGRVNEIL
IPR000477,




virus 1
HILIQTQAQIPSRPAPPPPSSPTHDPPDS
PF00078





DPQIPPPYVEPTAPQVLPVMHPHGAPP






NHRPWQMKDLQAIKQEVSQAAPGSPQ






FMQTIRLAVQQFDPTAKDLQDLLQYL






CSSLVASLHHQQLDSLISEAETRGITGY






NPLAGPLRVQANNPQQQGLRREYQQL






WLAAFAALPGSAKDPSWASILQGLEEP






YHAFVERLNIALDNGLPEGTPKDPILRS






LAYSNANKECQKLLQARGHTNSPLGD






MLRACQTWTPKDKTKVLVVQPKKPPP






NQPCFRCGKAGHWSRDCTQPRPPPGP






CPLCQDPTHWKRDCPRLKPTIPEPEPEE






DALLLDLPADIPHPKNLHRGGGLTSPP






TLQQVLPNQDPASILPVIPLDPARRPVI






KAQVDTQTSHPKTIEALLDTGADMTV






LPIALFSSNTPLKNTSVLGAGGQTQDH






FKLTSLPVLIRLPFRTTPIVLTSCLVDTK






NNWAIIGRDALQQCQGVLYLPEAKRPP






VILPIQAPAVLGLEHLPRPPQISQFPLNP






ERLQALQHLVRKALEAGHIEPYTGPGN






NPVFPVKKANGTWRFIHDLRATNSLTI






DLSSSSPGPPDLSSLPTTLAHLQTIDLRD






AFFQIPLPKQFQPYFAFTVPQQCNYGP






GTRYAWKVLPQGFKNSPTLFEMQLAH






ILQPIRQAFPQCTILQYMDDILLASPSHE






DLLLLSEATMASLISHGLPVSENKTQQ






TPGTIKFLGQIISPNHLTYDAVPTVPIRS






RWALPELQALLGEIQWVSKGTPTLRQP






LHSLYCALQRHTDPRDQIYLNPSQVQS






LVQLRQALSQNCRSRLVQTLPLLGAIM






LTLTGTTTVVFQSKEQWPLVWLHAPL






PHTSQCPWGQLLASAVLLLDKYTLQS






YGLLCQTIHHNISTQTFNQFIQTSDHPS






VPILLHHSHRFKNLGAQTGELWNTFLK






TAAPLAPVKALMPVFTLSPVIINTAPCL






FSDGSTSRAAYILWDKQILSQRSFPLPP






PHKSAQRAELLGLLHGLSSARSWRCL






NIFLDSKYLYHYLRTLALGTFQGRSSQ






APFQALLPRLLSRKVVYLHHVRSHTNL






PDPISRLNALTDALLITPVLQLSPAELHS






FTHCGQTALTLQGATTTEASNILRSCH






ACRGGNPQHQMPRGHIRRGLLPNHIW






QGDITHFKYKNTLYRLHVWVDTFSGAI






SATQKRKETSSEAISSLLQAIAHLGKPS






YINTDNGPAYISQDFLNMCTSLAIRHTT






HVPYNPTSSGLVERSNGILKTLLYKYF






TDKPDLPMDNALSIALWTINHLNVLTN






CHKTRWQLHHSPRLQPIPETRSLSNKQ






THWYYFKLPGLNSRQWKGPQEALQEA






AGAALIPVSASSAQWIPWRLLKRAACP






RPVGGPADPKEKDLQHHG 






(SEQ ID NO: 1564)






POL_FOAMV-
P14350
Human
MNPLQLLQPLPAEIKGTKLLAHWDSG
IPR043502,


residues

spumaretro-
ATITCIPESFLEDEQPIKKTLIKTIHGEK
SSF56672,


only

virus
QQNVYYVTFKVKGRKVEAEVIASPYE
IPR000477,





YILLSPTDVPWLTQQPLQLTILVPLQEY
PF00078





QEKILSKTALPEDQKQQLKTLFVKYDN






LWQHWENQVGHRKIRPHNIATGDYPP






RPQKQYPINPKAKPSIQIVIDDLLKQGV






LTPQNSTMNTPVYPVPKPDGRWRMVL






DYREVNKTIPLTAAQNQHSAGILATIV






RQKYKTTLDLANGFWAHPITPESYWL






TAFTWQGKQYCWTRLPQGFLNSPALF






TADVVDLLKEIPNVQVYVDDIYLSHDD






PKEHVQQLEKVFQILLQAGYVVSLKKS






EIGQKTVEFLGFNITKEGRGLTDTFKTK






LLNITPPKDLKQLQSILGLLNFARNFIPN






FAELVQPLYNLIASAKGKYIEWSEENT






KQLNMVIEALNTASNLEERLPEQRLVI






KVNTSPSAGYVRYYNETGKKPIMYLN






YVFSKAELKFSMLEKLLTTMHKALIKA






MDLAMGQEILVYSPIVSMTKIQKTPLP






ERKALPIRWITWMTYLEDPRIQFHYDK






TLPELKHIPDVYTSSQSPVKHPSQYEGV






FYTDGSAIKSPDPTKSNNAGMGIVHAT






YKPEYQVLNQWSIPLGNHTAQMAEIA






AVEFACKKALKIPGPVLVITDSFYVAES






ANKELPYWKSNGFVNNKKKPLKHISK






WKSIAECLSMKPDITIQHEKGISLQIPVF






ILKGNALADKLATQGSYVVNCNTKKP






NLDAELDQLLQGHYIKGYPKQYTYFL






EDGKVKVSRPEGVKIIPPQSDRQKIVLQ






AHNLAHTGREATLLKIANLYWWPNM






RKDVVKQLGRCQQCLITNASNKASGPI






LRPDRPQKPFDKFFIDYIGPLPPSQGYL






YVLVVVDGMTGFTWLYPTKAPSTSAT






VKSLNVLTSIAIPKVIHSDQGAAFTSST






FAEWAKERGIHLEFSTPYHPQSGSKVE






RKNSDIKRLLTKLLVGRPTKWYDLLPV






VQLALNNTYSPVLKYTPHQLLFGIDSN






TPFANQDTLDLTREEELSLLQEIRTSLY






HPSTPPASSRSWSPVVGQLVQERVARP






ASLRPRWHKPSTVLKVLNPRTVVILDH






LGNNRTVSIDNLKPTSHQNGTTNDTAT






MDHLEKNE






(SEQ ID NO: 1565)






POL_BLVJ-
P03361
Bovine
MGNSPSYNPPAGISPSDWLNLLQSAQR
IPR043502,


residues

leukemia
LNPRPSPSDFTDLKNYIHWFHKTQKKP
SSF56672,


only

virus
WTFTSGGPTSCPPGRFGRVPLVLATLN
IPR000477,





EVLSNEGGAPGASAPEEQPPPYDPPAIL
PF00078





PIISEGNRNRHRAWALRELQDIKKEIEN






KAPGSQVWIQTLRLAILQADPTPADLE






QLCQYIASPVDQTAHMTSLTAAIAAAE






AANTLQGFNPKTGTLTQQSAQPNAGD






LRSQYQNLWLQAGKNLPTRPSAPWSTI






VQGPAESSVEFVNRLQISLADNLPDGV






PKEPIIDSLSYANANRECQQILQGRGPV






AAVGQKLQACAQWAPKNKQPALLVH






TPGPKMPGPRQPAPKRPPPGPCYRCLK






EGHWARDCPTKATGPPPGPCPICKDPS






HWKRDCPTLKSKNKLIEGGLSAPQTIT






PITDSLSEAELECLLSIPLARSRPSVAVY






LSGPWLQPSQNQALMLVDTGAENTVL






PQNWLVRDYPRIPAAVLGAGGVSRNR






YNWLQGPLTLALKPEGPFITIPKILVDT






SDKWQILGRDVPSRLQASISIPEEVRPP






VVGVLDTPPSHIGLEHLPPPPEVPQFPL






NLERLQALQDLVHRSLEAGYISPWDGP






GNNPVFPVRKPNGAWRFVHDLRATNA






LTKPIPALSPGPPDLTAIPTHPPHIICLDL






KDAFFQIPVEDRFRFYLSFTLPSPGGLQ






PHRRFAWRVLPQGFINSPALFERALQE






PLRQVSAAFSQSLLVSYMDDILYASPT






EEQRSQCYQALAARLRDLGFQVASEK






TSQTPSPVPFLGQMVHEQIVTYQSLPTL






QISSPISLHQLQAVLGDLQWVSRGTPTT






RRPLQLLYSSLKRHHDPRAIIQLSPEQL






QGIAELRQALSHNARSRYNEQEPLLAY






VHLTRAGSTLVLFQKGAQFPLAYFQTP






LTDNQASPWGLLLLLGCQYLQTQALS






SYAKPILKYYHNLPKTSLDNWIQSSED






PRVQELLQLWPQISSQGIQPPGPWKTLI






TRAEVFLTPQFSPDPIPAALCLFSDGAT






GRGAYCLWKDHLLDFQAVPAPESAQK






GELAGLLAGLAAAPPEPVNIWVDSKY






LYSLLRTLVLGAWLQPDPVPSYALLYK






SLLRHPAIVVGHVRSHSSASHPIASLNN






YVDQLLPLETPEQWHKLTHCNSRALS






RWPNPRISAWDPRSPATLCETCQKLNP






TGGGKMRTIQRGWAPNHIWQADITHY






KYKQFTYALHVFVDTYSGATHASAKR






GLTTQTTIEGLLEAIVHLGRPKKLNTD






QGANYTSKTFVRFCQQFGVSLSHHVP






YNPTSSGLDERTNGLLKLLLSKYHLDE






PHLPMTQALSRALWTHNQINLLPILKT






RWELHHSPPLAVISEGGETPKGSDKLF






LYLLPGQNNRRWLGPLPALVEASGGA






LLATDPPVWVPWRLLKAFKCLKNDGP






EDAHNRSSDG 






(SEQ ID NO: 1566)






O41894_9RETR-
O41894
Bovine
MPALRPLQVEIKGNHLKGYWDSGAEI
IPR043502,


residues

foamy
TCVPAIYIIEEQPVGKKLITTIHNEKEHD
SSF56672,


only

virus
VYYVEMKIEKRKVQCEVIATALDYVL
IPR000477,





VAPVDIPWYKPGPLELTIKIDVESQKHT
PF00078





LITESTLSPQGQMRLKKLLDQYQALW






QCWENQVGHRRIEPHKIATGALKPRPQ






KQYHINPRAKADIQIVIDDLLRQGVLR






QQNSEMNTPVYPVPKADGRWRMVLD






YREVNKVTPLVATQNCHSASILNTLYR






GPYKSTLDLANGFWAHPIKPEDYWITA






FTWGGKTYCWTVLPQGFLNSPALFTA






DVVDILKDIPNVQVYVDDVYVSSATE






QEHLDILETIFNRLSTAGYIVSLKKSKL






AKETVEFLGFSISQNGRGLTDSYKQKL






MDLQPPTTLRQLQSILGLINFARNFLPN






FAELVAPLYQLIPKAKGQCIPWTMDHT






TQLKTIIQALNSTENLEERRPDVDLIMK






VHISNTAGYIRFYNHGGQKPIAYNNAL






FTSTELKFTPTEKIMATIHKGLLKALDL






SLGKEIHVYSAIASMTKLQKTPLSERK






ALSIRWLKWQTYFEDPRIKFHHDATLP






DLQNLPVPQQDTGKEMTILPLLHYEAI






FYTDGSAIRSPKPNKTHSAGMGIIQAKF






EPDFRIVHLWSFPLGDHTAQYAEIAAF






EFAIRRATGIRGPVLIVTDSNYVAKSYN






EELPYWESNGFVNNKKKTLKHISKWK






AIAECKNLKADIHVIHEPGHQPAEASP






HAQGNALADKQAVSGSYKVFSNELKP






SLDAELEQVLSTGRPNPQGYPNKYEYK






LVNGLCYVDRRGEEGLKIIPPKADRVK






LCQLAHDGPGSAHLGRSALLLKLQQK






YWWPRMHIDASRIVLNCTVCAQTNST






NQKPRPPLVIPHDTKPFQVWYMDYIGP






LPPSNGYQHALVIVDAGTGFTWIYPTK






AQTANATVKALTHLTGTAVPKVLHSD






QGPAFTSSILADWAKDRGIQLEHSAPY






HPQSSGKVERKNSEIKRLLTKLLAGRP






TKWYPLIPIVQLALNNTPNTRQKYTPH






QLMYGADCNLPFENLDTLDLTREEQL






AVLKEVRDGLLDLYPSPSQTTARSWTP






SPGLLVQERVARPAQLRPKWRKPTPIK






KVLNERTVIIDHLGQDKVVSIDNLKPA






AHQKLAQTPDSAEICPSATPCPPNTSL






WYDLDTGTWTCQRCGYQCPDKYHQP






QCTWSCEDRCGHRWKECGNCIPQDGS






SDDASAVAAVEI






(SEQ ID NO: 1567)






POL_MLVBM-
Q7SVK7
Murine
MGQTVTTPLSLTLEHWGDVQRIASNQ
IPR043502,


residues

leukemia
SVGVKKRRWVTFCSAEWPTFGVGWP
SSF56672,


only

virus
QDGTFNLDIILQVKSKVFSPGPHGHPD
IPR000477,





QVPYIVTWEAIAYEPPPWVKPFVSPKL
PF00078,





SLSPTAPILPSGPSTQPPPRSALYPAFTP
cd03715





SIKPRPSKPQVLSDDGGPLIDLLTEDPPP






YGEQGPSSPDGDGDREEATSTSEIPAPS






PMVSRLRGKRDPPAADSTTSRAFPLRL






GGNGQLQYWPFSSSDLYNWKNNNPSF






SEDPGKLTALIESVLTTHQPTWDDCQQ






LLGTLLTGEEKQRVLLEARKAVRGND






GRPTQLPNEVNSAFPLERPDWDYTTPE






GRNHLVLYRQLLLAGLQNAGRSPTNL






AKVKGITQGPNESPSAFLERLKEAYRR






YTPYDPEDPGQETNVSMSFIWQSAPAI






GRKLERLEDLKSKTLGDLVREAEKIFN






KRETPEEREERIRRETEEKEERRRAGDE






QREKERDRRRQREMSKLLATVVTGQR






QDRQGGERRRPQLDKDQCAYCKEKG






HWAKDCPKKPRGPRGPRPQTSLLTLD






DQGGQGQEPPPEPRITLTVGGQPVTFL






VDTGAQHSVLTQNPGPLSDRSAWVQG






ATGGKRYRWTTDRKVHLATGKVTHSF






LHVPDCPYPLLGRDLLTKLKAQIHFEG






SGAQVVGPKGQPLQVLTLGIEDEYRLH






ETSTEPDVSLGSTWLSDFPQAWAETGG






MGLAVRQAPLIIPLKATSTPVSIQQYPM






SHEARLGIKPHIQRLLDQGILVPCQSPW






NTPLLPVKKPGTNDYRPVQDLREVNK






RVEDIHPTVPNPYNLLSGLPPSHQWYT






VLDLKDAFFCLRLHPTSQPLFAFEWRD






PGMGISGQLTWTRLPQGFKNSPTLFDE






ALHRDLADFRIQHPDLILLQYVDDILLA






ATSELDCQQGTRALLQTLGDLGYRAS






AKKAQICQKQVKYLGYLLREGQRWLT






EARKETVMGQPVPKTPRQLREFLGTA






GFCRLWIPGFAEMAAPLYPLTKTGTLF






SWGPDQQKAYQEIKQALLTAPALGLP






DLTKPFELFVDEKQGYAKGVLTQKLG






PWRRPVAYLSKKLDPVAAGWPPCLRM






VAAIAVLTKDAGKLTMGQPLVILAPH






AVEALVKQPPDRWLSNARMTHYQAM






LLDTDRVQFGPVVALNPATLLPLPEEG






APHDCLEILAETHGTRPDLTDQPIPDAD






HTWYTDGSSFLQEGQRKAGAAVTTET






EVIWAGALPAGTSAQRAELIALTQALK






MAEGKRLNVYTDSRYAFATAHIHGEI






YRRRGLLTSEGREIKNKSEILALLKALF






LPKRLSIIHCLGHQKGDSAEARGNRLA






DQAAREAAIKTPPDTSTLLIEDSTPYTP






AYFHYTETDLKKLRDLGATYNQSKGY






WVFQGKPVMPDQFVFELLDSLHRLTH






LGYQKMKALLDRGESPYYMLNRDKT






LQYVADSCTVCAQVNASKAKIGAGVR






VRGHRPGTHWEIDFTEVKPGLYGYKY






LLVFVDTFSGWVEAFPTKRETARVVSK






KLLEEIFPRFGMPQVLGSDNGPAFTSQ






VSQSVADLLGIDWKLHCAYRPQSSGQ






VERINRTIKETLTKLTLAAGTRDWVLL






LPLALYRARNTPGPHGLTPYEILYGAPP






PLVNFHDPDMSELTNSPSLQAHLQALQ






TVQREIWKPLAEAYRDRLDQPVIPHPF






RIGDSVWVRRHQTKNLEPRWKGPYTV






LLTTPTALKVDGISAWIHAAHVKAATT






PPIKPSWRVQRSQNPLKIRLTRGAP






(SEQ ID NO: 2453)
















TABLE 31







Exemplary dimeric retroviral reverse transcriptases and


their RT domain signatures















RT


Name
Accession
Organism
Sequence
Signatures





Q83133_AVIMA
Q83133
Avian
RATVLTVALHLAIPLKWKPN
IPR043502,




myeloblastosis-
HTPVWIDQWPLPEGKLVALT
SSF56672,




associated
QLVEKELQLGHIEPSLSCWN
IPR000477,




virus type
TPVFVIRKASGSYRLLHDLR
PF00078,




1
AVNAKLVPFGAVQQGAPVLS
cd01645,





ALPRGWPLMVLDLKDCFFSI
PF06817,





PLAEQDREAFAFTLPSVNNQ
IPR010661





APARRFQWKVLPQGMTCSPT






ICQLIVGQILEPLRLKMPSL






RMLHYMDDLLLAASSHDGLE






AAGEEVISTLERAGFTISPD






KVQREPGVQYLGYKLGSTYV






APVGLVAEPRIATLWDVQKL






VGSLQSVRPALGIPPRLMGP






FYEQLRGSDPNEAREWNLDM






KMAWREIVQLSTTAALERWD






PALPLEGAVARCEQGAIGVL






GQGLSTHPRPCLWLFSTQPT






KAFTAWLEVLTLLITKLRAS






AVRTFGKEVDILLLPACFRE






DLPLPEGILLALRGFAGKIR






SSDTPSIFDIARPLHVSLKV






RVTDHPVPGPTVFTDASSST






HKGVVVWREGPRWEIKEIAD






LGASVQQLEARAVAMALLLW






PTTPTNVVTDSAFVAKMLLK






MGQEGVPSTAAAFILEDALS






QRSAMAAVLHVRSHSEVPGF






FTEGNDVADSQATFQAYPLR






EAKDLHTALHIGPRALSKAC






NISMQQAREVVQTCPHCNSA






PALEAGVNPRGLGPLQIWQT






DFTLEPRMAPRSWLAVTVDT






ASSAIVVTQHGRVTSVAAQH






HWATAIAVLGRPKAIKTDNG






SCFTSKSTREWLARWGIAHT






TGIPGNSQGQAMVERANRLL






KDKIRVLAEGDGFMKRIPTS






KQGELLAKAMYALNHFERGE






NTKTPIQKHWRPTVLTEGP






PVKIRIETGEWEKGWNVLVW






GRGYAAVKNRDTDKVIWVPS






RKVKPDITQKDEVTKKDEAS






PLFAGISDWAPWEGEQEGLQ






EETASNKQERPGEDTPAANE






S






(SEQ ID NO: 1568)






POL_SIVM1
P05896
Simian
MGARNSVLSGKKADELEKIR
IPR043502,




immuno-
LRPGGKKKYMLKHVVWAANE
SSF56672,




deficiency
LDRFGLAESLLENKEGCQKI
IPR000477,




virus
LSVLAPLVPTGSENLKSLYN
PF00078,





TVCVIWCIHAEEKVKHTEEA
PF06817,





KQIVQRHLVMETGTAETMPK
IPR010661,





TSRPTAPFSGRGGNYPVQQI
PF06815,





GGNYTHLPLSPRTLNAWVKL
IPR010659





IEEKKFGAEVVSGFQALSEG






CLPYDINQMLNCVGDHQAAM






QIIRDIINEEAADWDLQHPQ






QAPQQGQLREPSGSDIAGTT






STVEEQIQWMYRQQNPIPVG






NIYRRWIQLGLQKCVRMYNP






TNILDVKQGPKEPFQSYVDR






FYKSLRAEQTDPAVKNWMTQ






TLL1QNANPDCKLVLKGLGT






NPTLEEMLTACQGVGGPGQK






ARLMAEALKEALAPAPIPFA






AAQQKGPRKPIKCWNCGKEG






HSARQCRAPRRQGCWKCGKM






DHVMAKCPNRQAGFFRPWPL






GKEAPQFPHGSSASGADANC






SPRRTSCGSAKELHALGQAA






ERKQREALQGGDRGFAAPQF






SLWRRPVVTAHIEGQPVEVL






LDTGADDSIVTGIELGPHYT






PKIVGGIGGFINTKEYKNVE






IEVLGKRIKGTIMTGDTPIN






IFGRNLLTALGMSLNLPIAK






VEPVKSPLKPGKDGPKLKQW






PLSKEKIVALREICEKMEKD






GQLEEAPPTNPYNTPTFAIK






KKDKNKWRMLIDFRELNRVT






QDFTEVQLGIPHPAGLAKRK






RITVLDIGDAYFSIPLDEEF






RQYTAFTLPSVNNAEPGKRY






IYKVLPQGWKGSPAIFQYTM






RHVLEPFRKANPDVTLVQYM






DDILIASDRTDLEHDRVVLQ






LKELLNSIGFSSPEEKFQKD






PPFQWMGYELWPTKWKLQKI






ELPQRETWTVNDIQKLVGVL






NWAAQIYPGIKTKHLCRLIR






GKMTLTEEVQWTEMAEAEYE






ENKIILSQeqegcyyqeskp






leatviksQdnqwsYKIHQE






DKILKVGKFAKIKNTHTNGV






RLLAHVIQKIGKEAIVIWGQ






VPKFHLPVEKDVWEQVVWTD






YWQVTWIPEWDFISTPPLVR






LVFNLVKDPIEGEETYYVDG






SCSKQSKEGKAGYITDRGKD






KVKVLEQTTNQQAELEAFLM






ALTDSGPKANIIVDSQYVMG






IITGCPTESESRLVNQIIEE






MIKKTEIYVAWVPAHKGIGG






NQEIDHLVSQGIRQVLFLEK






IEPAQEEHSKYHSNIKELVF






KFGLPRLVAKQIVDTCDKCH






QKGEAIHGQVNSDLGTWQMD






CTHLEGKIVIVAVHVASGFI






EAEVIPQETGRQTALFLLKL






ASRWPITHLHTDNGANFASQ






EVKMVAWWAGIEHTFGVPYN






PQSQGVVEAMNHHLKNQIDR






IREQANSVETIVLMAVHCMN






FKRRGGIGDMTPAERLINMI






TTEQEIQFQQSKNSKFKNFR






VYYREGRDQLWKGPGELLWK






GEGAVILKVGTDIKVVPRRK






AKIIKDYGGGKEMDSSSHME






DTGEAREVA






(SEQ ID NO: 1569)






POL_RSVP
P03354
Rous
MEAVIKVISSACKTYCGKTS
IPR043502,




sarcoma
PSKKEIGAMLSLLQKEGLLM
SSF56672,




virus
SPSDLYSPGSWDPITAALSQ
1PR000477,





RAMILGKSGELKTWGLVLGA
PF00078,





LKAAREEQVTSEQAKFWLGL
cd01645,





GGGRVSPPGPECIEKPATER
PF06817,





RIDKGEEVGETTVQRDAKMA
IPR010661





PEETATPKTVGTSCYHCGTA






IGCNCATASAPPPPYVGSGL






YPSLAGVGEQQGQGGDTPPG






AEQSRAEPGHAGQAPGPALT






DWARVREELASTGPPVVAMP






VVIKTEGPAWTPLEPKLITR






LADTVRTKGLRSPITMAEVE






ALMSSPLLPHDVTNLMRVIL






GPAPYALWMDAWGVQLQTVI






AAATRDPRHPANGQGRGERT






NLNRLKGLADGMVGNPQGQA






ALLRPGELVAITASALQAFR






EVARLAEPAGPWADIMQGPS






ESFVDFANRLIKAVEGSDLP






PSARAPVIIDCFRQKSQPDI






QQLIRTAPSTLTTPGEIIKY






VLDRQKTAPLTDQGIAAAMS






SAIQPLIMAVVNRERDGQTG






SGGRARGLCYTCGSPGHYQA






QCPKKRKSGNSRERCQLCNG






MGHNAKQCRKRDGNQGQRPG






KGLSSGPWPGPEPPAVSLAM






TMEHKDRPLVRVILTNTGSH






PVKQRSVYITALLDSGADIT






IISEEDWPTDWPVMEAANPQ






IHGIGGGIPMRKSRDMIELG






VINRDGSLERPLLLFPAVAM






VRGSILGRDCLQGLGLRLTN






LIGRATVLTVALHLAIPLKW






KPDHTPVWIDQWPLPEGKLV






ALTQLVEKELQLGHIEPSLS






CWNTPVFVIRKASGSYRLLH






DLRAVNAKLVPFGAVQQGAP






VLSALPRGWPLMVLDLKDCF






FSIPLAEQDREAPAFTLPSV






NNQAPARRFQWKVLPQGMTC






SPTICQLVVGQVLEPLRLKH






PSLCMLHYMDDLLLAASSHD






GLEAAGEEVISTLERAGFTI






SPDKVQREPGVQYLGYKLGS






TYVAPVGLVAEPRIATLWDV






QKLVGSLQWLRPALGIPPRL






MGPFYEQLRGSDPNEAREWN






LDMKMAWREIVRLSTTAALE






RWDPALPLEGAVARCEQGAI






GVLGQGLSTHPRPCLWLFST






QPTKAFTAWLEVLTLLITKL






RASAVRTFGKEVDILLLPAC






FREDLPLPEGILLALKGFAG






KIRSSDTPSIFDIARPLHVS






LKVRVTDHPVPGPTVFTDAS






SSTHKGVVVWREGPRWEIKE






IADLGASVQQLEARAVAMAL






LLWPTTPTNVVTDSAFVAKM






LLKMGQEGVPSTAAAFILED






ALSQRSAMAAVLHVRSHSEV






PGFFTEGNDVADSQATFQAY






PLREAKDLHTALHIGPRALS






KACNISMQQAREVVQTCPHC






NSAPALEAGVNPRGLGPLQI






WQTDFTLEPRMAPRSWLA






VTVD






TASSAIVVTQHGRVTSVAVQ






HHWATAIAVLGRPKAIKTDN






GSCFTSKSTREWLARWGIAH






TTGIPGNSQGQAMVERANRL






LKDRIRVLAEGDGFMKRIPT






SKQGELLAKAMYALNHFERG






ENTKTPIQKHWRPTVLTEGP






PVKIRIETGEWEKGWNVLVW






GRGYAAVKNRDTDKVIWVPS






RKVKPDITQKDEVTKKDEAS






PLFAGISDWIPWEDEQEGLQ






GETASNKQERPGEDTLAANE






S






(SEQ ID NO: 1570)






POL_HV2D2
P15833
Human
MGARGSVLSGKKTDELEKVR
IPR043502,




immuno-
LRPGGKKKYMLKHVVWAVNE
SSF56672,




deficiency
LDRFGLAESLLESKEGCQKI
IPR000477,




virus type
LKVLAPLVPTGSENLKSLFN
PF00078,




2
IVCVIFCLHAEEKVKDTEEA
PF06817,





KKIAQRHLAADTEKMPATNK
IP010661,





PTAPPSGGNYPVQQLAGNYV
PF06815,





HLPLSPRTLNAWVKLVEEKK
IPR010659





FGAEVVPGFQALSEGCTPYD






INQMLNCVGEHQAAMQIIRE






IINEEAADWDQQHPSPGPMP






AGQLRDPRGSDIAGTTSTVE






EQIQWMYRAQNPVPVGNIYR






RWIQLGLQKCVRMYNPTNIL






DIKQGPKEPFQSYVDRFYKS






LRAEQTDPAVKNWMTQTLLI






QNANPDCKLVLKGLGMNPTL






EEMLTACQGIGGPGQKARLM






AEALKEALTPAPIPFAAVQQ






KAGKRGTVTCWNCGKQGHTA






RQCRAPRRQGCWKCGKTGHI






MSKCPERQAGFLRVRTLGKE






ASQLPHDPSASGSDTICTPD






EPSRGHDTSGGDTICAPCRS






SSGDAEKLHADGETTEREPR






ETLQGGDRGFAAPQFSLVVR






RPVVKACIEGQSVEVLLDTG






VDDSIVAGIELGSNYTPKIV






GGIGGFINTKEYKDVEIEVV






GKRVRATIMTGDTPINIFGR






NILNTLGMTLNFPVAKVEPV






KVELKPGKDGPKIRQWPLSR






EKILALKEICEKMEKEGQLE






EAPPTNPYNTPTFAIKKKDK






NKWRMLIDFRELNKVTQDFT






EVNWVFPTRQVAEKRRITVI






DVGDAYFSIPLDPNFRQYTA






FTLPSVNNAEPGKRYIYKVL






PQGWKGSQSICQYSMRKVLD






PFRKANSDVIIIQYMDDILI






ASDRSDLEHDRVVSQLKELL






NDMGFSTPEEKFQKDPPFKW






MGYELWPKKWKLQKIQLPEK






EVWTVNAIQKLVGVLNWAAQ






LFPGIKTRHICKLIRGKMTL






TEEVQWTELAEAELQENKII






LEQEQEGSYYKERVPLEATV






QKNLANQWTYKIHQGNKVLK






VGKYAKVKNTHTNGVRLLAH






VVQKIGKEALVIWGEIPVFH






LPVERETWDQWWTDYWQVTW






IPEWDFVSTPPLIRLAYNLV






KDPLEGRETYYTDGSCNRTS






KEGKAGYVTDRGKDKVKVLE






QTTNQQAELEAFALALTDSE






PQVNIIVDSQYVMGIIAAQP






TETESPIVAKIIEEMIKKEA






VYVGWVPAHKGLGGNQEVDH






LVSQGIRQVLFLEKIEPAQE






EHEKYHGNVKELVHKFGIPQ






LVAKQIVNSCDKCQQKGEAI






HGQVNADLGTWQMDCTHLEG






KIIIVAVHVASGFIEAEVIP






QETGRQTALFLLKLASRWPI






THLHTDNGANFTSPSVKMVA






WWVGIEQTFGVPYNPQSQGV






VEAMNHHLKNQIDRLRDQAV






SIETVVLMATHCMNFKRRGG






IGDMTPAERLVNMITTEQEI






QFFQAKNLKFQNFQVYYREG






RDQLWKGPGELLWKGEGAVI






IKVGTEIKVVPRRKAKIIRH






YGGGKGLDCSADMEDTRQAR






EMAQSD






(SEQ ID NO: 1571)






POL_HV1A2
P03369
Human
MGARASVLSGGELDKWEKIR
IPR043502,




immuno-
LRPGGKKKYKLKHIVWASRE
SSF56672,




deficiency
LERFAVNPGLLETSEGCRQI
IPR000477,




virus type
LGQLQPSLQTGSEELRSLYN
PF00078,




1
TVATLYCVHQRIDVKDTKEA
cd01645,





LEKIEEEQNKSKKKAQQAAA
PF06817,





AAGTGNSSQVSQNYPIVQNL
IPR010661,





QGQMVHQAISPRTLNAWVKV
PF06815,





VEEKAFSPEVIPMFSALSEG
IPR010659





ATPQDLNTMLNTVGGHQAAM






QMLKETINEEAAEWDRVHPV






HAGPIAPGQMREPRGSDIAG






TTSTLQEQIGWMTNNPPIPV






GEIYKRWIILGLNKIVRMYS






PTSILDIRQGPKEPFRDYVD






RFYKTLRAEQASQDVKNWMT






ETLLVQNANPDCKTILKALG






PAATLEEMMTACQGVGGPGH






KARVLAEAMSQVTNPANIMM






QRGNFRNQRKTVKCFNCGKE






GHIAKNCRAPRKKGCWRCGR






EGHQMKDCTERQANFLREDL






AFLQGKAREFSSEQTRANSP






TRRELQVWGGENNSLSEAGA






DRQGTVSFNFPQITLWQRPL






VTIRIGGQLKEALLDTGADD






TVL






EEMNLPGKWKPKMIGGIGGF






IKVRQYDQIPVEICGHKAIG






TVLVGPTPVNIIGRNLLTQI






GCTLNFPISPIETVPVKLKP






GMDGPKVKQWPLTEEKIKAL






VEICTEMEKEGKISKIGPEN






PYNTPVFAIKKKDSTKWRKL






VDFRELNKRTQDFWEVQLGI






PHPAGLKKKKSVTVLDVGDA






YFSVPLDKDFRKYTAFTIPS






INNETPGIRYQYNVLPQGWK






GSPAIFQSSMTKILEPFRKQ






NPDIVIYQYMDDLYVGSDLE






IGQHRTKIEELRQHLLRWGF






TTPDKKHQKEPPFLWMGYEL






HPDKWTVQPIMLPEKDSWTV






NDIQKLVGKLNWASQIYAGI






KVKQLCKLLRGTKALTEVIP






LTEEAELELAENREILKEPV






HEVYYDPSKDLVAEIQKQGQ






GQWTYQIYQEPFKNLKTGKY






ARMRGAHTNDVKQLTEAVQK






VSTESIVIWGKIPKFKLPIQ






KETWEAWWMEYWQATWIPEW






EFVNTPPLVKLWYQLEKEPI






VGAETFYVDGAANRETKLGK






AGYVTDRGRQKVVSIADTTN






QKTELQAIHLALQDSGLEVN






IVTDSQYALGIIQAQPDKSE






SELVSQIIEQLIKKEKVYL






AWVPAHKGIGGNEQV






DKLVSAGIRKVLFLNGIDKA






QEEHEKYHSNWRAMASDFNL






PPVVAKEIVASCDKCQLKGE






AMHGQVDCSPGIWQLDCTHL






EGKIILVAVHVASGYIEAEV






IPAETGQETAYFLLKLAGRW






PVKTIHTDNGSNFTSTTVKA






ACWWAGIKQEFGIPYNPQSQ






GVVESMNNELKKIIGQVRDQ






AEHLKTAVQMAVFIHNFKRK






GGIGGYSAGERIVDIIATDI






QTKELQKQITKIQNFRVYYR






DNKDPLWKGPAKLLWKGEGA






VVIQDNSDIKVVPRRKAKII






RDYGKQMAGDDCVASRQDED






(SEQ ID NO: 1572)






POL_FIVPE
P16088
Feline
KEFGKLEGGASCSPSESNAA
IPR043502,




Immuno-
SSNAICTSNGGETIGFVNYN
SSF56672,




deficiency
KVGTTTTLEKRPEILIFVNG
IPR000477,




virus
YPIKFLLDTGADITILNRRD
PF00078,





FQVKNSIENGRQNMIGVGGG
PF06817,





KRGTNYINVHLEIRDENYKT
IPR010661,





QCIFGNVCVLEDNSLIQPLL
PF06815,





GRDNMIKFNIRLVMAQISDK
IPR010659





IPVVKVKMKDPNKGPQIKQW






PLTNEKIEALTEIVERLEKE






GKVKRADSNNPWNTPVFAIK






KKSGKWRMLIDFRELNKLTE






KGAEV0LGLPHPAGLQIKK






QVTVLDIGDAYFTIPLD






PDYAPYTAFTLPRKNNAGPG






RRFVWCSLPQGWILSPLIYQ






STLDNIIQPFIRQNPQLDIY






QYMDDIYIGSNLSKKEHKEK






VEELRKLLLWWGFETPEDKL






QEEPPYTWMGYELHPLTWTI






QQKQLDIPEQPTLNELQKLA






GKINWASQAIPDLSIKALTN






MMRGNQNLNSTRQWTKEARL






EVQKAKKAIEEQVQLGYYDP






SKELYAKLSLVGPHQISYQV






YQKDPEKILWYGKMSRQKKK






AENTCDIALRACYKIREESI






IRIGKEPRYEIPTSREAWES






NLINSPYLKAPPPEVEYIHA






ALNIKRALSMIKDAPIPGAE






TWYIDGGRKLGKAAKAAYWT






DTGKWRVMDLEGSNQKAEIQ






ALLLALKAGSEEMNIITDSQ






YVINIILQQPDMMEGIWQEV






LEELEKKTAIFIDWVPGHKG






IPGNEEVDKLCQTMMIIEGD






GILDKRSEDAGYDLLAAKEI






HLLPGEVKVIPTGVKLMLPK






GYWGLIIGKSSIGSKGLDVL






GGVIDEGYRGEIGVIMINVS






RKSITLMERQKIAQLIILPC






KHEVLEQGKVVMDSERGDNG






YGSTGVFSSWVDRIEEAEIN






FIEKFHSDPQYLRTEFNLPK






MVAEEIRRKCPVCRIIGEQV






GGQLKIGPGIWQMDCTHFDG






KIILVGIHVESGYIWAQIIS






QETADCTVKAVLQLLSAHNV






TELQTDNGPNFKNQKMEGVL






NYMGVKHKFGIPGNPQSQAL






VENVNHTLKVWIQKFLPETT






SLDNALSLAVHSLNFKRRGR






IGGMAPYELLAQQESLRIQD






YFSAIPQKLQAQWIYYKDQK






DKKWKGPMRVEYWGQGSVLL






KDEEKGYFLIPRRHIRRVPE






PCALPEGDE






(SEQ ID NO: 1573)






POL_EIAVY
P03371
Equine
TAWTFLKAMQKCSKKREARG
IPR043502,




infectious
SREAPETNFPDTTEESAQQI
SSF56672,




anemia
CCTRDSSDSKSVPRSERNKK
IPR000477,




virus
GIQCQGEGSSRGSQPGQFVG
PF00078,





VTYNLEKRPTTIVLINDTPL
PF06817,





NVLLDTGADTSVLTTAHYNR
IPR010661,





LKYRGRKYQGTGIIGVGGNV
PF06815,





ETFSTPVTIKKKGRHIKTRM
IPR010659





LVADIPVTILGRDILQDLGA






KLVLAQLSKEIKFRKIELKE






GTMGPKIPQWPLTKEKLEGA






KETVQRLLSEGKISEASDNN






PYNSPIFVIKKRSGKWRLLQ






DLRELNKTVQVGTEISRGLP






HPGGLIKCKHMTVLDIGDAY






FTIPLDPEFRPYTAFTIPSI






NHQEPDKRYVWKCLPQGFVL






SPYIYQKTLQEILQPFRERY






PEVQLYQYMDDLFVGSNGSK






KQHKELIIELRAILQKGFET






PDDKLQEVPPYSWLGYQLCP






ENWKVQKMQLDMVKNPTLND






VQKLMGNITWMSSGVPGLTV






KHIAATTKGCLELNQKVIWT






EEAQKELEENNEKIKNAQGL






QYYNPEEEMLCEVEITKNYE






ATYVIKQSQGILWAGKKIMK






ANKGWSTVKNLMLLLQHVAT






ESITRVGKCPTFKVPFTKEQ






VMWEMQKGWYYSWLPEIVYT






HQVVHDDWRMKLVEEPTSGI






TIYTDGGKQNGEGIAAYVTS






NGRTKQKRLGPVTHQVAERM






AIQMALEDTRDKQVNIVTDS






YYCWKNITEGLGLEGPQNPW






WPIIQNIREKEIVYFAWVPG






HKGIYGNQLADEAAKIKEEI






MLAYQGTQIKEKRDEDAGFD






LCVPYDIMIPVSDTKIIPTD






VKIQVPPNSFGWVTGKSSMA






KQGLLINGGIIDEGYTGEIQ






VICTNIGKSNIKLIEGQKFA






QLIILQHHSNSRQPWDENKI






SQRGDKGFGSTGVFWVENIQ






EAQDEHENWHTSPKILARNY






KIPLTVAKQITQECPHCTKQ






GSGPAGCVMRSPNHWQADCT






HLDNKIILHFVESNSGYIHA






TLLSKENALCTSLAILEWAR






LFSPKSLHTDNGTNFVAEPV






VNLLKFLKIAHTTGIPYHPE






SQGIVERANRTLKEKIQSHR






DNTQTLEAALQLALITCNKG






RESMGGQTPWEVFITNQAQV






IHEKLLLQQAQSSKKFCFYK






IPGEHDWKGPTRVLWKGDGA






VVVNDEGKGIIAVPLTRTKL






LIKPN






(SEQ ID NO: I574)






POL_BIV29
P19560
Bovine
MKRRELEKKLRKVRVTPQQD
IPR043502,




immuno-
KYYTIGNLQWAIRMINLMGI
SSF56672,




deficiency
KCVCDEECSAAEVALIITQF
IPR000477,




virus
SALDLENSPIRGKEEVAIKN
PF00078,





TLKVFWSLLAGYKPESTETA
PF06817,





LGYWEAFTYREREARADKEG
IPR010661





EIKSIYPSLTQNTQNKKQTS






NQTNTQSLPAITTQDGTPRF






DPDLMKQLKIWSDATERNGV






DLHAVNILGVITANLVQEEI






KLLLNSTPKWRLDVQLIESK






VREKENAHRTWKQHHPEAPK






TDEIIGKGLSSAEQATLISV






ECRETFRQWVLQAAMEVAQA






KHATPGPINIHQGPKEPYTD






FINRLVAALEGMAAPETTKE






YLLQHLSIDHANEDCQSILR






PLGPNTPMEKKLEACRVVGS






QKSKMQFLVAAMKEMGIQSP






IPAVLPHTPEAYASQTSGPE






DGRRCYGCGKTGHLKRNCKQ






QKCYHCGKPGHQARNCRSKN






REVLLCPLWAEEPTTEQFSP






EQHEFCDPICTPSYIRLDKQ






PFIKVFIGGRWVKGLVDTGA






DEVVLKNIHWDRIKGYPGTP






IKQIGVNGVNVAKRKTHVEW






RFKDKTGIIDVLFSDTPVNL






FGRSLLRSIVTCFTLLVHTE






KIEPLPVKVRGPGPKVPQWP






LTKEKYQALKEIVKDLLAEG






KISEAAWDNPYNTPVFVIKK






KGTGRWRMLMDFRELNKITV






KGQEFSTGLPYPPGIKECEH






LTAIDIKDAYFTIPLHEDFR






PFTAFSVVPVNREGPIERFQ






WNVLPQGWVCSPAIYQTTTQ






KIIENIKKSHPDVMLYQYMD






DLLIGSNRDDHKQIVQEIRD






KLGSYGFKTPDEKVQEERVK






WIGFELTPKKWRFQPRQLKI






KNPLTVNELQQLVGNCVWVQ






PEVKIPLYPLTDLLRDKTNL






QEKIQLTPEAIKCVEEFNLK






LKDPEWKDRIREGAELVIKI






QMVPRGIVFDLLQDGNPIWG






GVKGLNYDHSNKIKKILRTM






NELNRTVVIMTGREASFLLP






GSSEDWEAALQKEESLTQIF






PVKFYRHSCRWTSICGPVRE






NLTTYYTDGGKKGKTAAAVY






WCEGRTKSKVFPGTNQQAEL






KAICMALLDGPPKMNIITDS






RYAYEGMREEPETWAREGIW






LEIAKILPFKQYVGVGWVPA






HKGIGGNTEADEGVKKALEQ






MAPCSPPEAILLKPGEKQNL






ETGIYMQGLRPQSFLPRADL






PVAITGTMVDSELQLQLLNI






GTEHIRIQKDEVFMTCFLEN






IPSATEDHERWHTSPDILVR






QFHLPKRIAKEIVARCQECK






RTTTSPVRGTNPRGRFLWQM






DNTHWNKTIIWVAVETNSGL






VEAQVIPEETALQVALCILQ






LIQRYTVLHLHSDNGPCFTA






HRIENLCKYLGITKTTGIPY






NPQSQGVVERAHRDLKDRLA






AYQGDCETVEAALSLALVSL






NKKRGGIGGHTPYEIYLESE






HTKYQDQLEQQFSKQKIEKW






CYVRNRRKEWKGPYKVLWDG






DGAAVIEEEGKTALYPHRHM






RFIPPPDSDIQDGSS






(SEQ ID NO: 1575)






A0A142BKH1_ALV
A0A142BKH1
Avian
TVALHLAIPLKWKPDHTPVW
IPR043502,




leukosis
IDQWPLPEGKLVALTQLVEK
SSF56672,




and
ELQLGHIEPSLSCWNTPVFV
IPR000477,




sarcoma
IRKASGSYRLLHDLRAVNAK
PF00078,




virus
LVPFGAVQQGAPVLSALPRG
cd01645,





WPLMVLDLKDCFFSIPLAEQ
PF06817,





DREAFAFTLPSVNNQAPARR
IPR010661





FQWKVLPQGMTCSPTICQLV






VGQVLEPLRLKHPSLRMLHY






MDDLLLAASSHDGLEAAGEE






VISTLERAGFTISPDKIQRE






PGVQYLGYKLGSTYVAPVGL






VAEPRIATLWDVQKLVGSLQ






WLRPALGIPPRLMGPFYEQL






RGSDPNEAREWNLDMKMAWR






EIVQLSTTAALERWDPALPL






EGAVARCEQGAIGVLGQGLS






THPRPCLWLFSTQPTKAFTA






WLEVLTLLITKLRASAVRTF






GKEVDVLLLPACFREDLPLP






EGILLALRGFAGKIRSSDTP






SIFDIARPLHVSLKVRVTDH






PVPGPTVFTDASSSTHKGVV






VWREGPRWEIKEIADLGASV






QQLEARAVAMALLLWPTTPT






NVVTDSAFVAKMLLKMGQEG






VPSTAAAFILEDALSQRSAM






AAVLHVRSHSEVPGFFTEGN






DVADSQATFQAYPLREAKDL






HTALHIGPRALSKACNISMQ






QAREVVQTCPHCNSAPALEA






GVNPRGLGPLQIWQTDFTLE






PRMAPRSWLAVTVATASSAI






VVTQHGRVTSVAARHHWATA






IAVLGRPKAIKTDNGSCFTS






KSTREWLARWGIAHTTGIPG






NSQGQAMVERANRLLKDKIR






VLAEGDGFMKRIPTGKQGEL






LAKAMYALNHFERGENTKTP






IQKHWRPTVLTEGPPVKIRI






ETGEWEKGWNVLVWGRGYAA






VKNRDTDKIIWVPSRKVKPD






ITQKDELTKKDEASPLFAGI






SDWAPWKGEQEGL






(SEQ ID NO: 1576) 
















TABLE 32







InterPro descriptions of signatures present in reverse transcriptases


in Table 30 (monomeric viral RTs) and Table 31 (dimeric viral RTs).










Signature
Database
Short Name
Description





cd01645
CDD
RT_Rtv
RT_Rtv: Reverse transcriptases (RTs) from





retroviruses (Rtvs). RTs catalyze the





conversion of single-stranded RNA into





double-stranded viral DNA for integration into





host chromosomes. Proteins in this subfamily





contain long terminal repeats (LTRs) and are





multifunctional enzymes with RNA-directed





DNA polymerase, DNA directed DNA





polymerase, and ribonuclease hybrid (RNase





H) activities. The viral RNA genome enters the





cytoplasm as part of a nucleoprotein complex,





and the process of reverse transcription





generates in the cytoplasm forming a linear





DNA duplex via an intricate series of steps.





This duplex DNA is colinear with its RNA





template, but contains terminal duplications





known as LTRs that are not present in viral





RNA. It has been proposed that two





specialized template switches, known as





strand-transfer reactions or “jumps”, are





required to generate the LTRs. [PMID:





9831551, PMID: 15107837, PMID: 11080630,





PMID: 10799511, PMID: 7523679, PMID:





7540934, PMID: 8648598, PMID: 1698615]





cd03715
CDD
RT_ZFREV_like
RT_ZFREV_like: A subfamily of reverse





transcriptases (RTs) found in sequences





similar to the intact endogenous retrovirus





ZFERV from zebrafish and to Moloney murine





leukemia virus RT. An RT gene is usually





indicative of a mobile element such as a





retrotransposon or retrovirus. RTs occur in a





variety of mobile elements, including





retrotransposons, retroviruses, group II introns,





bacterial msDNAs, hepadnaviruses, and





caulimoviruses. These elements can be divided





into two major groups. One group contains





retroviruses and DNA viruses whose





propagation involves an RNA intermediate.





They are grouped together with transposable





elements containing long terminal repeats





(LTRs). The other group, also called poly(A)-





type retrotransposons, contain fungal





mitochondrial introns and transposable





elements that lack LTRs. Phylogenetic analysis





suggests that ZFERV belongs to a distinct





group of retroviruses. [PMID: 14694121,





PMID: 2410413, PMID: 9684890, PMID:





10669612, PMID: 1698615, PMID: 8828137]





PF00078
Pfam
RVT_1
A reverse transcriptase gene is usually





indicative of a mobile element such as a





retrotransposon or retrovirus. Reverse





transcriptases occur in a variety of mobile





elements, including retrotransposons,





retroviruses, group II introns, bacterial





msDNAs, hepadnaviruses, and caulimoviruses.





[PMID: 1698615]





IPR000477
InterPro
RT_dom
The use of an RNA template to produce DNA,





for integration into the host genome and





exploitation of a host cell, is a strategy





employed in the replication of retroid





elements, such as the retroviruses and bacterial





retrons. The enzyme catalysing polymerisation





is an RNA-directed DNA-polymerase, or





reverse trancriptase (RT) (2.7.7.49). Reverse





transcriptase occurs in a variety of mobile





elements, including retrotransposons,





retroviruses, group II introns [PMID:





12758069], bacterial msDNAs,





hepadnaviruses, and caulimoviruses.





Retroviral reverse transcriptase is synthesised





as part of the POL polyprotein that contains;





an aspartyl protease, a reverse transcriptase,





RNase H and integrase. POL polyprotein





undergoes specific enzymatic cleavage to yield





the mature proteins. The discovery of





retroelements in the prokaryotes raises





intriguing questions concerning their roles in





bacteria and the origin and evolution of reverse





transcriptases and whether the bacterial reverse





transcriptases are older than eukaryotic reverse





transcriptases [PMID: 8828137], Several





crystal structures of the reverse transcriptase





(RT) domain have been determined [PMID:





1377403].





IPR043502
InterPro
DNA/RNA
This entry represents the DNA/RNA




polymerase
polymerase superfamily, which includes DNA




superfamily
polymerase I, reverse transcriptase, T7 RNA





polymerase, lesion bypass DNA polymerase





(Y-family), RNA-dependent RNA-polymerase





and dsRNA phage RNA-dependent RNA-





polymerase. These enzymes share a similar





protein fold at their active site, which





resembles the palm subdomain of the right-





hand-shaped polymerases. [PMID: 26931141]





SSF56672
Superfamily
DNA/RNA
This superfamily comprises DNA polymerases




polymerases
and RNA polymerases





PF06817
Pfam
RVT_thumb
This domain is known as the thumb domain. It





is composed of a four helix bundle





[PMID: 1377403].





IPR010661
InterPro
RVT thumb
This domain is known as the thumb domain. It





is composed of a four helix bundle. Reverse





transcriptase converts the viral RNA genome





into double-stranded viral DNA. Reverse





transcriptase often occurs in a polyprotein;





with integrase, ribonuclease H and/or protease,





which is cleaved before the enzyme takes





action. The impact of antiretroviral treatment





on the first 400 amino acids of HIV reverse





transcriptase is good. Little is known,





however, of the antiretroviral drug impact on





the C-terminal domains of Pol, which includes





the thumb, connection and RNase H. Evidence





suggests that these might be well conserved





domains. [PMID: 1377403, PMID: 18335052]





PF06815
Pfam
RVT_connect
This domain is known as the connection





domain. This domain lies between the thumb





and palm domains [PMID: 1377403].





IPR010659
InterPro
RVT connect
This domain is known as the connection





domain. This domain lies between the thumb





and palm domains [PMID: 1377403].





cd03715
CDD
RT ZFREV like
RT_ZFREV_like: A subfamily of reverse





transcriptases (RTs) found in sequences





similar to the intact endogenous retrovirus





ZFERV from zebrafish and to Moloney murine





leukemia virus RT. An RT gene is usually





indicative of a mobile element such as a





retrotransposon or retrovirus. RTs occur in a





variety of mobile elements, including





retrotransposons, retroviruses, group II introns,





bacterial msDNAs, hepadnaviruses, and





caulimoviruses. These elements can be divided





into two major groups. One group contains





retroviruses and DNA viruses whose





propagation involves an RNA intermediate.





They are grouped together with transposable





elements containing long terminal repeats





(LTRs). The other group, also called poly(A)-





type retrotransposons, contain fungal





mitochondrial introns and transposable





elements that lack LTRs. Phylogenetic analysis





suggests that ZFERV belongs to a distinct





group of retroviruses. [PMID: 14694121,





PMID: 2410413, PMID: 9684890, PMID:





10669612, PMID: 1698615, PMID: 8828137]









Table 41 provides a listing of retrotransposase proteins and the associated retrotransposon 5′UTRs and 3′UTRs for use in novel Gene Writing systems. Reverse transcriptase domains in the proteins described here were identified using conserved RT signatures, and annotated to indicate the presence and location of RT domains within the polypeptide sequences. In some embodiments, a system or method described herein involves a polypeptide having an amino acid sequence according to Table 41, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or a functional fragment thereof. In some embodiments, a system or method described herein involves a domain (e.g., a reverse transcriptase domain) having an amino acid sequence according to a domain (e.g., a reverse transcriptase domain) of Table 41, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or functional fragment thereof. In some embodiments, a system or method described herein involves a template RNA comprising a sequence according to one or both of a predicted 5′ UTR and a predicted 3′ UTR of Table 41, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or functional fragment thereof.










Lengthy table referenced here




US20240035049A1-20240201-T00002


Please refer to the end of the specification for access instructions.






Table 44 provides Retroviral reverse transcriptase domains for use in Gene Writer polypeptides. Wild-type reverse transcriptase enzymes were collected and prioritized as according to the descriptions herein (see Example 33). The Type column indicates whether the sequence corresponds to a wild-type sequence (“root”) or comprises mutations that may improve the activity of the enzyme (“derivative”).









TABLE 44







Retroviral reverse transcriptase domains for use in Gene Writer polypeptides.


In some embodiments, a system or method described herein involves a reverse


transcriptase domain having an amino acid sequence according to a reverse


transcriptase domain of Table 44, or a sequence having at least 70%, 75%,


80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or functional


fragment thereof.
















SEQ




Virus_
Uniprot_

ID



name
name
ID
type
NO:
peptide





AVIRE_
AVIRE
P03360
root
3136
TAPLEEEYRLFLEAPIQNVTLLEQWKREIPKVWAEINPPGLASTQAPIHV


P03360




QLLSTALPVRVRQYPITLEAKRSLRETIRKFRAAGILRPVHSPWNTPLLP







VRKSGTSEYRMVQDLREVNKRVETIHPTVPNPYTLLSLLPPDRIWYSVLD







LKDAFFCIPLAPESQLIFAFEWADAEEGESGQLTWTRLPQGFKNSPTLFD







EALNRDLQGFRLDHPSVSLLQYVDDLLIAADTQAACLSATRDLLMTLAEL







GYRVSGKKAQLCQEEVTYLGFKIHKGSRSLSNSRTQAILQIPVPKTKRQV







REFLGTIGYCRLWIPGFAELAQPLYAATRGGNDPLVWGEKEEEAFQSLKL







ALTQPPALALPSLDKPFQLFVEETSGAAKGVLTQALGPWKRPVAYLSKRL







DPVAAGWPRCLRAIAAAALLTREASKLTFGQDIEITSSHNLESLLRSPPD







KWLTNARITQYQVLLLDPPRVRFKQTAALNPATLLPETDDTLPIHHCLDT







LDSLTSTRPDLTDQPLAQAEATLFTDGSSYIRDGKRYAGAAVVTLDSVIW







AEPLPIGTSAQKAELIALTKALEWSKDKSVNIYTDSRYAFATLHVHGMIY







RERGLLTAGGKAIKNAPEILALLTAVWLPKRVAVMHCKGHQKDDAPTSTG







NRRADEVAREVAIRPLSTQATIS





AVIRE_
AVIRE
P03360
derivative
3137
TAPLEEEYRLFLEAPIQNVTLLEQWKREIPKVWAEINPPGLASTQAPIHV


P03360_




QLLSTALPVRVRQYPITLEAKRSLRETIRKFRAAGILRPVHSPWNTPLLP


3mut




VRKSGTSEYRMVQDLREVNKRVETIHPTVPNPYTLLSLLPPDRIWYSVLD







LKDAFFCIPLAPESQLIFAFEWADAEEGESGQLTWTRLPQGFKNSPTLFN







EALNRDLQGFRLDHPSVSLLQYVDDLLIAADTQAACLSATRDLLMTLAEL







GYRVSGKKAQLCQEEVTYLGFKIHKGSRSLSNSRTQAILQIPVPK







TKRQVREFLGTIGYCRLWIPGFAELAQPLYAATRPGNDPLVWGEKEEEAF







QSLKLALTQPPALALPSLDKPFQLFVEETSGAAKGVLTQALGPWKRPVAY







LSKRLDPVAAGWPRCLRAIAAAALLTREASKLTFGQDIEITSSHNLESLL







RSPPDKWLTNARITQYQVLLLDPPRVRFKQTAALNPATLLPETDDTLPIH







HCLDTLDSLTSTRPDLTDQPLAQAEATLFTDGSSYIRDGKRYAGAAVVTL







DSVIWAEPLPIGTSAQKAELIALTKALEWSKDKSVNIYTDSRYAFATLHV







HGMIYRERGWLTAGGKAIKNAPEILALLTAVWLPKRVAVMHCKGHQKDDA







PTSTGNRRADEVAREVAIRPLSTQATIS





AVIRE_
AVIRE
P03360
derivative
3138
TAPLEEEYRLFLEAPIQNVTLLEQWKREIPKVWAEINPPGLASTQAPIHV


P03360_




QLLSTALPVRVRQYPITLEAKRSLRETIRKFRAAGILRPVHSPWNTPLLP


3mutA




VRKSGTSEYRMVQDLREVNKRVETIHPTVPNPYTLLSLLPPDRIWYSVLD







LKDAFFCIPLAPESQLIFAFEWADAEEGESGQLTWTRLPQGFKNSPTLFN







EALNRDLQGFRLDHPSVSLLQYVDDLLIAADTQAACLSATRDLLMTLAEL







GYRVSGKKAQLCQEEVTYLGFKIHKGSRSLSNSRTQAILQIPVPKTKRQV







REFLGKIGYCRLFIPGFAELAQPLYAATRPGNDPLVWGEKEEEAFQSLKL







ALTQPPALALPSLDKPFQLFVEETSGAAKGVLTQALGPWKRPVAYLSKRL







DPVAAGWPRCLRAIAAAALLTREASKLTFGQDIEITSSHNLESLLRSPPD







KWLTNARITQYQVLLLDPPRVRFKQTAALNPATLLPETDDTLPIHHCLDT







LDSLTSTRPDLTDQPLAQAEATLFTDGSSYIRDGKRYAGAAVVTLDSVIW







AEPLPIGTSAQKAELIALTKALEWSKDKSVNIYTDSRY







AFATLHVHGMIYRERGWLTAGGKAIKNAPEILALLTAVWLPKRVAVMHCK







GHQKDDAPTSTGNRRADEVAREVAIRPLSTQATIS





BAEVM_
BAEVM
P10272
root
3139
TVSLQDEHRLFDIPVTTSLPDVWLQDFPQAWAETGGLGRAKCQAPIIIDL


P10272




KPTAVPVSIKQYPMSLEAHMGIRQHIIKFLELGVLRPCRSPWNTPLLPVK







KPGTQDYRPVQDLREINKRTVDIHPTVPNPYNLLSTLKPDYSWYTVLDLK







DAFFCLPLAPQSQELFAFEWKDPERGISGQLTWTRLPQGFKNSPTLFDEA







LHRDLTDFRTQHPEVTLLQYVDDLLLAAPTKKACTQGTRHLLQELGEKGY







RASAKKAQICQTKVTYLGYILSEGKRWLTPGRIETVARIPPPRNPREVRE







FLGTAGFCRLWIPGFAELAAPLYALTKESTPFTWQTEHQLAFEALKKALL







SAPALGLPDTSKPFTLFLDERQGIAKGVLTQKLGPWKRPVAYLSKKLDPV







AAGWPPCLRIMAATAMLVKDSAKLTLGQPLTVITPHTLEAIVRQPPDRWI







TNARLTHYQALLLDTDRVQFGPPVTLNPATLLPVPENQPSPHDCRQVLAE







THGTREDLKDQELPDADHTWYTDGSSYLDSGTRRAGAAVVDGHNTIWAQS







LPPGTSAQKAELIALTKALELSKGKKANIYTDSRYAFATAHTHGSIYERR







GLLTSEGKEIKNKAEIIALLKALFLPQEVAIIHCPGHQKGQDPVAVGNRQ







ADRVARQAAMAEVLTLATEPDNTSHIT





BAEVM_
BAEVM
P10272
derivative
3140
TVSLQDEHRLFDIPVTTSLPDVWLQDFPQAWAETGGLGRAKCQAPIIIDL


P10272_




KPTAVPVSIKQYPMSLEAHMGIRQHIIKFLELGVLRPCRSPWNTPLLPVK


3mut




KPGTQDYRPVQDLREINKRTVDIHPTVPNPYNLLSTLKPDYSWYTVLDLK







DAFFCLPLAPQSQELFAFEWKDPERGISGQLTWTRLPQGFKNSPTLFNEA







LHRDLTDFRTQHPEVTLLQYVDDLLLAAPTKKACTQGTRHLLQELGEKGY







RASAKKAQICQTKVTYLGYILSEGKRWLTPGRIETVARIPPPRNPREVRE







FLGTAGFCRLWIPGFAELAAPLYALTKPSTPFTWQTEHQLAFEALKKALL







SAPALGLPDTSKPFTLFLDERQGIAKGVLTQKLGPWKRPVAYLSKKLDPV







AAGWPPCLRIMAATAMLVKDSAKLTLGQPLTVITPHTLEAIVRQPPDRWI







TNARLTHYQALLLDTDRVQFGPPVTLNPATLLPVPENQPSPHDCRQVLAE







THGTREDLKDQELPDADHTWYTDGSSYLDSGTRRAGAAVVDGHNTIWAQS







LPPGTSAQKAELIALTKALELSKGKKANIYTDSRYAFATAHTHGSIYERR







GWLTSEGKEIKNKAEIIALLKALFLPQEVAIIHCPGHQKGQDPVAVGNRQ







ADRVARQAAMAEVLTLATEPDNTSHIT





BAEVM_
BAEVM
P10272
derivative
3141
TVSLQDEHRLFDIPVTTSLPDVWLQDFPQAWAETGGLGRAKCQAPIIIDL


P10272_




KPTAVPVSIKQYPMSLEAHMGIRQHIIKFLELGVLRPCRSPWNTPLLPVK


3mutA




KPGTQDYRPVQDLREINKRTVDIHPTVPNPYNLLSTLKPDYSWYTVLDLK







DAFFCLPLAPQSQELFAFEWKDPERGISGQLTWTRLPQGFKNSPTLFNEA







LHRDLTDFRTQHPEVTLLQYVDDLLLAAPTKKACTQGTRHLLQELGEKGY







RASAKKAQICQTKVTYLGYILSEGKRWLTPGRIETVARIPPPRNPREVRE







FLGKAGFCRLFIPGFAELAAPLYALTKPSTPFTWQTEHQLAFEALKKALL







SAPALGLPDTSKPFTLFLDERQGIAKGVLTQKLGPWKRPVAYLSKKLDPV







AAGWPPCLRIMAATAMLVKDSAKLTLGQPLTVITPHTLEAIVRQPPDRWI







TNARLTHYQALLLDTDRVQFGPPVTLNPATLLPVPENQPSPHDCRQVLAE







THGTREDLKDQELPDADHTWYTDGSSYLDSGTRRAGAAVVDGHNTIWAQS







LPPGTSAQKAELIALTKALELSKGKKANIYTDSRYAFATAHTHGSIYERR







GWLTSEGKEIKNKAEIIALLKALFLPQEVAIIHCPGHQKGQDPVAVGNRQ







ADRVARQAAMAEVLTLATEPDNTSHIT





BLVAU_
BLVA
P25059
root
3142
GVLDAPPSHIGLEHLPPPPEVPQFPLNLERLQALQDLVHRSLEAGYISPW


P25059




DGPGNNPVFPVRKPNGAWRFVHDLRVTNALTKPIPALSPGPPDLTAIPTH







LPHIICLDLKDAFFQIPVEDRFRSYFAFTLPTPGGLQPHRRFAWRVLPQG







FINSPALFERALQEPLRQVSAAFSQSLLVSYMDDILYVSPTEEQRLQCYQ







TMAAHLRDLGFQVASEKTRQTPSPVPFLGQMVHERMVTYQSLPTLQISSP







ISLHQLQTVLGDLQWVSRGTPTTRRPLQLLYSSLKGIDDPRAIIHLSPEQ







QQGIAELRQALSHNARSRYNEQEPLLAYVHLTRAGSTLVLFQKGAQFPLA







YFQTPLTDNQASPWGLLLLLGCQYLQAQALSSYAKTILKYYHNLPKTSLD







NWIQSSEDPRVQELLQLWPQISSQGIQPPGPWKTLVTRAEVFLTPQFSPE







PIPAALCLFSDGAARRGAYCLWKDHLLDFQAVPAPESA







QKGELAGLLAGLAAAPPEPLNIWVDSKYLYSLLRTLVLGAWLQPDPVPSY







ALLYKSLLRHPAIFVGHVRSHSSASHPIASLNNYVDQL





BLVAU_
BLVAU
P25059
derivative
3143
GVLDAPPSHIGLEHLPPPPEVPQFPLNLERLQALQDLVHRSLEAGYISPW


P25059_




DGPGNNPVFPVRKPNGAWRFVHDLRVTNALTKPIPALSPGPPDLTAIPTH


2mut




LPHIICLDLKDAFFQIPVEDRFRSYFAFTLPTPGGLQPHRRFAWRVLPQG







FINSPALFQRALQEPLRQVSAAFSQSLLVSYMDDILYVSPTEEQRLQCYQ







TMAAHLRDLGFQVASEKTRQTPSPVPFLGQMVHERMVTYQSLPTLQISSP







ISLHQLQTVLGDLQWVSRGTPTTRRPLQLLYSSLKPIDDPRAIIHLSPEQ







QQGIAELRQALSHNARSRYNEQEPLLAYVHLTRAGSTLVLFQKGAQFPLA







YFQTPLTDNQASPWGLLLLLGCQYLQAQALSSYAKTILKYYHNLPKTSLD







NWIQSSEDPRVQELLQLWPQISSQGIQPPGPWKTLVTRAEVFLTPQFSPE







PIPAALCLFSDGAARRGAYCLWKDHLLDFQAVPAPESAQKGELAGLLAGL







AAAPPEPLNIWVDSKYLYSLLRTLVLGAWLQPDPVPSYALLYKSLLRHPA







IFVGHVRSHSSASHPIASLNNYVDQL





BLVAU_
BLVAU
P25059
derivative
3144
GVLDAPPSHIGLEHLPPPPEVPQFPLNLERLQALQDLVHRSLEAGYISPW


P25059_




DGPGNNPVFPVRKPNGAWRFVHDLRVTNALTKPIPALSPGPPDLTAPPTH


2mutB




LPHIICLDLKDAFFQIPVEDRFRSYFAFTLPTPGGLQPHRRFAWRVLPQG







FINSPALFQRALQEPLRQVSAAFSQSLLVSYMDDILYVSPTEEQRLQCYQ







TMAAHLRDLGFQVASEKTRQTPSPVPFLGQMVHERMVTYQSLPTLQISSP







ISLHQLQTVLGDLQWVSRGTPTTRRPLQLLYSSLKPIDDPRAIIHLSPEQ







QQGIAELRQALSHNARSRYNEQEPLLAYVHLTRAGSTLVLFQKGAQFPLA







YFQTPLTDNQASPWGLLLLLGCQYLQAQALSSYAKTILKYYHNLPKTSLD







NWIQSSEDPRVQELLQLWPQISSQGIQPPGPWKTLVTRAEVFLTPQFSPE







PIPAALCLFSDGAARRGAYCLWKDHLLDFQAVPAPESAQKGELAGLLAGL







AAAPPEPLNIWVDSKYLYSLLRTLVLGAWLQPDPVPSYALLYKSLLRHPA







IFVGHVRSHSSASHPIASLNNYVDQL





BLVJ_
BLVJ
P03361
root
3145
GVLDTPPSHIGLEHLPPPPEVPQFPLNLERLQALQDLVHRSLEAGYISPW


P03361




DGPGNNPVFPVRKPNGAWRFVHDLRATNALTKPIPALSPGPPDLTAIPTH







PPHIICLDLKDAFFQIPVEDRFRFYLSFTLPSPGGLQPHRRFAWRVLPQG







FINSPALFERALQEPLRQVSAAFSQSLLVSYMDDILYASPTEEQRSQCYQ







ALAARLRDLGFQVASEKTSQTPSPVPFLGQMVHEQIVTYQSLPTLQISSP







ISLHQLQAVLGDLQWVSRGTPTTRRPLQLLYSSLKRHHDPRAIIQLSPEQ







LQGIAELRQALSHNARSRYNEQEPLLAYVHLTRAGSTLVLFQKGAQFPLA







YFQTPLTDNQASPWGLLLLLGCQYLQTQALSSYAKPILKYYHNLPKTSLD







NWIQSSEDPRVQELLQLWPQISSQGIQPPGPWKTLITRAEVFLTPQFSPD







PIPAALCLFSDGATGRGAYCLWKDHLLDFQAVPAPESAQKGELAGLLAGL







AAAPPEPVNIWVDSKYLYSLLRTLVLGAWLQPDPVPSYALLYKSLLRHPA







IVVGHVRSHSSASHPIASLNNYVDQL





BLVJ_
BLVJ
P03361
derivative
3146
GVLDTPPSHIGLEHLPPPPEVPQFPLNLERLQALQDLVHRSLEAGYISPW


P03361_




DGPGNNPVFPVRKPNGAWRFVHDLRATNALTKPIPALSPGPPDLTAIPTH


2mut




PPHIICLDLKDAFFQIPVEDRFRFYLSFTLPSPGGLQPHRRFAWRVLPQG







FINSPALFNRALQEPLRQVSAAFSQSLLVSYMDDILYASPTEEQRSQCPE







QLQGIAELRQALSHNARSRYNEQEPLLAYVHLTRAGSTLVLFQKGAQFPL







AYFQTPLTDNQASPWGLLLLLGCQYLQTQALSSYAKPILKYYHNLPKTSL







DNWIQSSEDPRVQELLQLWPQISSQGIQPPGPWKTLITRAEVFLTPQFSP







DPIPAALCLFSDGATGRGAYCLWKDHLLDFQAVPAPESAQKGELAGLLAG







LAAAPPEPVNIWVDSKYLYSLLRTWVLGAWLQPDPVPSYALLYKSLLRHP







AIVVGHVRSHSSASHPIASLNNYVDQL





BLVJ_
BLVJ
P03361
derivative
3147
GVLDTPPSHIGLEHLPPPPEVPQFPLNLERLQALQDLVHRSLEAGYISPW


P03361_




DGPGNNPVFPVRKPNGAWRFVHDLRATNALTKPIPALSPGPPDLTAPP


2mutB




THPPHIICLDLKDAFFQIPVEDRFRFYLSFTLPSPGGLQPHRRFAWRVLP







QGFINSPALFQRALQEPLRQVSAAFSQSLLVSYMDDILYASPTEEQRSQC







YQALAARLRDLGFQVASEKTSQTPSPVPFLGQMVHEQIVTYQSLPTLQIS







SPISLHQLQAVLGDLQWVSRGTPTTRRPLQLLYSSLKRHHDPRAIIQLSP







EQLQGIAELRQALSHNARSRYNEQEPLLAYVHLTRAGSTLVLFQKGAQFP







LAYFQTPLTDNQASPWGLLLLLGCQYLQTQALSSYAKPILKYYHNLPKTS







LDNWIQSSEDPRVQELLQLWPQISSQGIQPPGPWKTLITRAEVFLTPQFS







PDPIPAALCLFSDGATGRGAYCLWKDHLLDFQAVPAPESAQKGELAGLLA







GLAAAPPEPVNIWVDSKYLYSLLRTWVLGAWLQPDPVPSYALLYKSLLRH







PAIVVGHVRSHSSASHPIASLNNYVDQL





FFV_
FFV
O93209
root
3148
MDLLKPLTVERKGVKIKGYWNSQADITCVPKDLLQGEEPVRQQNVTTIHG


O93209




TQEGDVYYVNLKIDGRRINTEVIGTTLDYANITPGDVPWILKKPLELTIK







LDLEEQQGTLLNNSILSKKGKEELKQLFEKYSALWQSWENQVGHRRIRPH







KIATGTVKPTPQKQYHINPKAKPDIQIVINDLLKQGVLIQKESTMNTPVY







PVPKPNGRWRMVLDYRAVNKVTPLIAVQNQHSYGILGSLFKGRYKTTIDL







SNGFWAHPIVPEDYWITAFTWQGKQYCWTVLPQGFLNSPGLFTGDVVDLL







QGIPNVEVYVDDVYISHDSEKEHLEYLDILFNRLKEAGYIISLKKSNIAN







SIVDFLGFQITNEGRGLTDTFKEKLENITAPTTLKQLQSILGLLNFARNF







IPDFTELIAPLYALIPKSTKNYVPWQIEHSTTLETLITKLNGAEYLQGRK







GDKTLIMKVNASYTTGYIRYYNEGEKKPISYVSIVFSKTELKFTELEKLL







TTVHKGLLKALDLSMGQNIHVYSPIVSMQNIQKTPQTAKKALASRWLSWL







SYLEDPRIRFFYDPQMPALKDLPAVDTGKDNKKHPSNFQHIFYTDGSAIT







SPTKEGHLNAGMGIVYFINKDGNLQKQQEWSISLGNHTAQFAEIAAFEFA







LKKCLPLGGNILVVTDSNYVAKAYNEELDVWASNGFVNNRKKPLKHISKW







KSVADLKRLRPDVVVTHEPGHQKLDSSPHAYGNNLADQLATQASFKVH





FFV_
FFV
O93209
derivative
3149
VPWILKKPLELTIKLDLEEQQGTLLNNSILSKKGKEELKQLFEKYSALWQ


O93209-




SWENQVGHRRIRPHKIATGTVKPTPQKQYHINPKAKPDIQIVINDLLKQG


Pro




VLIQKESTMNTPVYPVPKPNGRWRMVLDYRAVNKVTPLIAVQNQHSYGIL







GSLFKGRYKTTIDLSNGFWAHPIVPEDYWITAFTWQGKQYCWTVLPQGFL







NSPGLFTGDVVDLLQGIPNVEVYVDDVYISHDSEKEHLEYLDILFNRLKE







AGYIISLKKSNIANSIVDFLGFQITNEGRGLTDTFKEKLENITAPTTLKQ







LQSILGLLNFARNFIPDFTELIAPLYALIPKSTKNYVPWQIEHSTTLETL







ITKLNGAEYLQGRKGDKTLIMKVNASYTTGYIRYYNEGEKKPISYVSIVF







SKTELKFTELEKLLTTVHKGLLKALDLSMGQNIHVYSPIVSMQNIQKTPQ







TAKKALASRWLSWLSYLEDPRIRFFYDPQMPALKDLPAVDTGKDNKKHPS







NFQHIFYTDGSAITSPTKEGHLNAGMGIVYFINKDGNLQKQQEWSISLGN







HTAQFAEIAAFEFALKKCLPLGGNILVVTDSNYVAKAYNEELDVWASNGF







VNNRKKPLKHISKWKSVADLKRLRPDVVVTHEPGHQKLDSSPHAYGNNLA







DQLATQASFKVH





FFV_
FFV
O93209
derivative
3150
VPWILKKPLELTIKLDLEEQQGTLLNNSILSKKGKEELKQLFEKYSALWQ


O93209-




SWENQVGHRRIRPHKIATGTVKPTPQKQYHINPKAKPDIQIVINDLLKQG


Pro_2




VLIQKESTMNTPVYPVPKPNGRWRMVLDYRAVNKVTPLIAVQNQHSYGIL


mut




GSLFKGRYKTTIDLSNGFWAHPIVPEDYWITAFTWQGKQYCWTVLPQGFL







NSPGLFNGDVVDLLQGIPNVEVYVDDVYISHDSEKEHLEYLDILFNRLKE







AGYIISLKKSNIANSIVDFLGFQITNEGRGLTDTFKEKLENITAPTTLKQ







LQSILGLLNFARNFIPDFTELIAPLYALIPKSPKNYVPWQIEHSTTLETL







ITKLNGAEYLQGRKGDKTLIMKVNASYTTGYIRYYNEGEKKPISYVSIVF







SKTELKFTELEKLLTTVHKGLLKALDLSMGQNIHVYSPIVSMQNIQKTPQ







TAKKALASRWLSWLSYLEDPRIRFFYDPQMPALKDLPAVDTGKDNKKHPS







NFQHIFYTDGSAITSPTKEGHLNAGMGIVYFINKDGNLQKQQEWSISLGN







HTAQFAEIAAFEFALKKCLPLGGNILVVTDSNYVAKAYNEELDVWASNGF







VNNRKKPLKHISKWKSVADLKRLRPDVVVTHEPGHQKLDSSPHAYGNNLA







DQLATQASFKVH





FFV_
FFV
O93209
derivative
3151
VPWILKKPLELTIKLDLEEQQGTLLNNSILSKKGKEELKQLFEKYSALWQ


O93209-




SWENQVGHRRIRPHKIATGTVKPTPQKQYHINPKAKPDIQIVINDLLKQG


Pro_




VLIQKESTMNTPVYPVPKPNGRWRMVLDYRAVNKVTPLIAVQNQHSYGIL


2mutA




GSLFKGRYKTTIDLSNGFWAHPIVPEDYWITAFTWQGKQYCWTVLPQGFL







NSPGLFNGDVVDLLQGIPNVEVYVDDVYISHDSEKEHLEYLDILFNRLKE







AGYIISLKKSNIANSIVDFLGFQITNEGRGLTDTFKEKLENITAPTTLKQ







LQSILGKLNFARNFIPDFTELIAPLYALIPKSPKNYVPWQIEHSTTLETL







ITKLNGAEYLQGRKGDKTLIMKVNASYTTGYIRYYNEGEKKPTGKDNKKH







PSNFQHIFYTDGSAITSPTKEGHLNAGMGIVYFINKDGNLQKQQEWSISL







GNHTAQFAEIAAFEFALKKCLPLGGNILVVTDSNYVAKAYNEELDVWASN







GFVNNRKKPLKHISKWKSVADLKRLRPDVVVTHEPGHQKLDSSPHAYGNN







LADQLATQASFKVH





FFV_
FFV
O93209
derivative
3152
MDLLKPLTVERKGVKIKGYWNSQADITCVPKDLLQGEEPVRQQNVTTIHG


O93209_2




TQEGDVYYVNLKIDGRRINTEVIGTTLDYAIITPGDVPWILKKPLELTIK


mut




LDLEEQQGTLLNNSILSKKGKEELKQLFEKYSALWQSWENQVGHRRIRPH







KIATGTVKPTPQKQYHINPKAKPDIQIVINDLLKQGVLIQKESTMNTPVY







PVPKPNGRWRMVLDYRAVNKVTPLIAVQNQHSYGILGSLFKGRYKTTIDL







SNGFWAHPIVPEDYWITAFTWQGKQYCWTVLPQGFLNSPG







LFNGDVVDLLQGIPNVEVYVDDVYISHDSEKEHLEYLDILFNRLKEAGYI







ISLKKSNIANSIVDFLGFQITNEGRGLTDTFKEKLENITAPTTLKQLQSI







LGLLNFARNFIPDFTELIAPLYALIPKSPKNYVPWQIEHSTTLETLITKL







NGAEYLQGRKGDKTLIMKVNASYTTGYIRYYNEGEKKPISYVSIVFSKTE







LKFTELEKLLTTVHKGLLKALDLSMGQNIHVYSPIVSMQNIQKTPQTAKK







ALASRWLSWLSYLEDPRIRFFYDPQMPALKDLPAVDTGKDNKKHPSNFQH







IFYTDGSAITSPTKEGHLNAGMGIVYFINKDGNLQKQQEWSISLGNHTAQ







FAEIAAFEFALKKCLPLGGNILVVTDSNYVAKAYNEELDVWASNGFVNNR







KKPLKHISKWKSVADLKRLRPDVVVTHEPGHQKLDSSPHAYGNNLADQLA







TQASFKVH





FFV_
FFV
O93209
derivative
3153
MDLLKPLTVERKGVKIKGYWNSQADITCVPKDLLQGEEPVRQQNVTTIHG


O93209_




TQEGDVYYVNLKIDGRRINTEVIGTTLDYAIITPGDVPWILKKPLELTIK


2mutA




LDLEEQQGTLLNNSILSKKGKEELKQLFEKYSALWQSWENQVGHRRIRPH







KIATGTVKPTPQKQYHINPKAKPDIQIVINDLLKQGVLIQKESTMNTPVY







PVPKPNGRWRMVLDYRAVNKVTPLIAVQNQHSYGILGSLFKGRYKTTIDL







SNGFWAHPIVPEDYWITAFTWQGKQYCWTVLPQGFLNSPGLFNGDVVDLL







QGIPNVEVYVDDVYISHDSEKEHLEYLDILFNRLKEAGYIISLKKSNIAN







SIVDFLGFQITNEGRGLTDTFKEKLENITAPTTLKQLQSILGKLNFARNF







IPDFTELIAPLYALIPKSPKNYVPWQIEHSTTLETLITKLNGAEYLQGRK







GDKTLIMKVNASYTTGYIRYYNEGEKKPISYVSIVFSKTELKFTELEKLL







TTVHKGLLKALDLSMGQNIHVYSPIVSMQNIQKTPQTAKKALASRWLSWL







SYLEDPRIRFFYDPQMPALKDLPAVDTGKDNKKHPSNFQHIFYTDGSAIT







SPTKEGHLNAGMGIVYFINKDGNLQKQQEWSISLGNHTAQFAEIAAFEFA







LKKCLPLGGNILVVTDSNYVAKAYNEELDVWASNGFVNNRKKPLKHISKW







KSVADLKRLRPDVVVTHEPGHQKLDSSPHAYGNNLADQLATQASFKVH





FLV_
FIV
P10273
root
3154
TLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGTAHCQAPVLIQ


P10273




LKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPV







KKPGTEDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDL







KDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFDE







ALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKG







YRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNSRQVR







EFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFEDIKKAL







LSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDT







VASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKW







LSNARMTHYQAMLLDAERVHFGPTVSLNPATLL







PLPSGGNHHDCLQILAETHGTRPDLTDQPLPDADLTWYTDGSSFIRNGER







EAGAAVTTESEVIWAAPLPPGTSAQRAELIALTQALKMAEGKKLTVYTDS







RYAFATTHVHGEIYRRRGLLTSEGKEIKNKNEILALLEALFLPKRLSIIH







CPGHQKGDSPQAKGNRLADDTAKKAATETHSSLTVLP





FLV_
FLV
P10273
derivative
3155
TLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGTAHCQAPVLIQ


P10273_




LKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPV


3mut




KKPGTEDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDL







KDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFNE







ALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKG







YRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNSRQVR







EFLGTAGYCRLWIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFEDIKKAL







LSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDT







VASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKW







LSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGGNHHDCLQILAE







THGTRPDLTDQPLPDADLTWYTDGSSFIRNGEREAGAAVTTESEVIWAAP







LPPGTSAQRAELIALTQALKMAEGKKLTVYTDSRYAFATTHVHGEIYRRR







GWLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRL







ADDTAKKAATETHSSLTVLP





FLV_
FLV
P10273
derivative
3156
TLQLEEEYRLFEPESTQKQEMDIWLKNFPQAWAETGGMGTAHCQAPVLIQ


P10273_




LKATATPISIRQYPMPHEAYQGIKPHIRRMLDQGILKPCQSPWNTPLLPV


3mutA




KKPGTEDYRPVQDLREVNKRVEDIHPTVPNPYNLLSTLPPSHPWYTVLDL







KDAFFCLRLHSESQLLFAFEWRDPEIGLSGQLTWTRLPQGFKNSPTLFNE







ALHSDLADFRVRYPALVLLQYVDDLLLAAATRTECLEGTKALLETLGNKG







YRASAKKAQICLQEVTYLGYSLKDGQRWLTKARKEAILSIPVPKNSRQVR







EFLGKAGYCRLFIPGFAELAAPLYPLTRPGTLFQWGTEQQLAFEDIKKAL







LSSPALGLPDITKPFELFIDENSGFAKGVLVQKLGPWKRPVAYLSKKLDT







VASGWPPCLRMVAAIAILVKDAGKLTLGQPLTILTSHPVEALVRQPPNKW







LSNARMTHYQAMLLDAERVHFGPTVSLNPATLLPLPSGGNHHDCLQILAE







THGTRPDLTDQPLPDADLTWYTDGSSFIRNGEREAGAAVTTESEVIWAAP







LPPGTSAQRAELIALTQALKMAEGKKLTVYTDSRYAFATTHVHGEIYRRR







GWLTSEGKEIKNKNEILALLEALFLPKRLSIIHCPGHQKGDSPQAKGNRL







ADDTAKKAATETHSSLTVLP





FOAMV_
FOAMV
P14350
root
3157
MNPLQLLQPLPAEIKGTKLLAHWNSGATITCIPESFLEDEQPIKKTLIKT


P14350




IHGEKQQNVYYVTFKVKGRKVEAEVIASPYEYILLSPTDVPWLTQQPLQL







TILVPLQEYQEKILSKTALPEDQKQQLKTLFVKYDNLWQHWENQVGHRKI







RPHNIATGDYPPRPQKQYPINPKAKPSIQIVIDDLLKQGVLTPQNSTMNT







PVYPVPKPDGRWRMVLDYREVNKTIPLTAAQNQHSAGILATIVRQKYKTT







LDLANGFWAHPITPESYWLTAFTWQGKQYCWTRLPQGFLNSPALFTADVV







DLLKEIPNVQVYVDDIYLSHDDPKEHVQQLEKVFQILLQAGYVVSLKKSE







IGQKTVEFLGFNITKEGRGLTDTFKTKLLNITPPKDLKQLQSILGLLNFA







RNFIPNFAELVQPLYNLIASAKGKYIEWSEENTKQLNMVIEALNTASNLE







ERLPEQRLVIKVNTSPSAGYVRYYNETGKKPIMYLNYVFSKAELKFSMLE







KLLTTMHKALIKAMDLAMGQEILVYSPIVSMTKIQKTPLPERKALPIRWI







TWMTYLEDPRIQFHYDKTLPELKHIPDVYTSSQSPVKHPSQYEGVFYTDG







SAIKSPDPTKSNNAGMGIVHATYKPEYQVLNQWSIPLGNHTAQMAEIAAV







EFACKKALKIPGPVLVITDSFYVAESANKELPYWKSNGFVNNKKKPLKHI







SKWKSIAECLSMKPDITIQHEKGISLQIPVFILKGNALADKLATQGSYVV







N





FOAMV_
FOAMV
P14350
derivative
3158
VPWLTQQPLQLTILVPLQEYQEKILSKTALPEDQKQQLKTLFVKYDNLWQ


P14350-




HWENQVGHRKIRPHNIATGDYPPRPQKQYPINPKAKPSIQIVIDDLLKQG


Pro




VLTPQNSTMNTPVYPVPKPDGRWRMVLDYREVNKTIPLTAAQNQHSAGIL







ATIVRQKYKTTLDLANGFWAHPITPESYWLTAFTWQGKQYCWTRLPQGFL







NSPALFTADVVDLLKEIPNVQVYVDDIYLSHDDPKEHVQQLEKVFQILLQ







AGYVVSLKKSEIGQKTVEFLGFNITKEGRGLTDTFKTK







LLNITPPKDLKQLQSILGLLNFARNFIPNFAELVQPLYNLIASAKGKYIE







WSEENTKQLNMVIEALNTASNLEERLPEQRLVIKVNTSPSAGYVRYYNE 







TGKKPIMYLNYVFSKAELKFSMLEKLLTTMHKALIKAMDLAMGQEILVYS







PIVSMTKIQKTPLPERKALPIRWITWMTYLEDPRIQFHYDKTLPELK HI







PDVYTSSQSPVKHPSQYEGVFYTDGSAIKSPDPTKSNNAGMGIVHATYKP







EYQVLNQWSIPLGNHTAQMAEIAAVEFACKKALKIPGPVLVITDS FYVA







ESANKELPYWKSNGFVNNKKKPLKHISKWKSIAECLSMKPDITIQHEKGI







SLQIPVFILKGNALADKLATQGSYVVN





FOAMV_
FOAMV
P14350
derivative
3159
VPWLTQQPLQLTILVPLQEYQEKILSKTALPEDQKQQLKTLFVKYDNLWQ


P14350-




HWENQVGHRKIRPHNIATGDYPPRPQKQYPINPKAKPSIQIVIDDLLKQG


Pro_2




VLTPQNSTMNTPVYPVPKPDGRWRMVLDYREVNKTIPLTAAQNQHSAGIL


mut




ATIVRQKYKTTLDLANGFWAHPITPESYWLTAFTWQGKQYCWTRLPQGFL







NSPALFNADVVDLLKEIPNVQVYVDDIYLSHDDPKEHVQQLEKVFQILLQ







AGYVVSLKKSEIGQKTVEFLGFNITKEGRGLTDTFKTKLLNITPPKDLKQ







LQSILGLLNFARNFIPNFAELVQPLYNLIAPAKGKYIEWSEENTKQLNMV







IEALNTASNLEERLPEQRLVIKVNTSPSAGYVRYYNETGKKPIMYLNYVF







SKAELKFSMLEKLLTTMHKALIKAMDLAMGQEILVYSPIVSMTKIQKTPL







PERKALPIRWITWMTYLEDPRIQFHYDKTLPELKHIPDVYTSSQSPVKHP







SQYEGVFYTDGSAIKSPDPTKSNNAGMGIVHATYKPEYQVLNQWSIPLGN







HTAQMAEIAAVEFACKKALKIPGPVLVITDSFYVAESANKELPYWKSNGF







VNNKKKPLKHISKWKSIAECLSMKPDITIQHEKGISLQIPVFILKGNALA







DKLATQGSYVVN





FOAMV_
FOAMV
P14350
derivative
3160
VPWLTQQPLQLTILVPLQEYQEKILSKTALPEDQKQQLKTLFVKYDNLWQ


P14350-




HWENQVGHRKIRPHNIATGDYPPRPQKQYPINPKAKPSIQIVIDDLLKQG


Pro_2




VLTPQNSTMNTPVYPVPKPDGRWRMVLDYREVNKTIPLTAAQNQHSAGIL


mutA




ATIVRQKYKTTLDLANGFWAHPITPESYWLTAFTWQGKQYCWTRLPQGFL







NSPALFNADVVDLLKEIPNVQVYVDDIYLSHDDPKEHVQQLEKVFQILLQ







AGYVVSLKKSEIGQKTVEFLGFNITKEGRGLTDTFKTKLLNITPPKDLKQ







LQSILGKLNFARNFIPNFAELVQPLYNLIAPAKGKYIEWSEENTKQLNMV







IEALNTASNLEERLPEQRLVIKVNTSPSAGYVRYYNETGKKPIMYLNYVF







SKAELKFSMLEKLLTTMHKALIKAMDLAMGQEILVYSPIVSMTKIQKTPL







PERKALPIRWITWMTYLEDPRIQFHYDKTLPELKHIPDVYTSSQSPVKHP







SQYEGVFYTDGSAIKSPDPTKSNNAGMGIVHATYKPEYQVLNQWSIPLGN







HTAQMAEIAAVEFACKKALKIPGPVLVITDSFYVAESANKELPYWKSNGF







VNNKKKPLKHISKWKSIAECLSMKPDITIQHEKGISLQIPVFILKGNALA







DKLATQGSYVVN





FOAMV_
FOAMV
P14350
derivative
3161
MNPLQLLQPLPAEIKGTKLLAHWNSGATITCIPESFLEDEQPIKKTLIKT


P14350_




IHGEKQQNVYYVTFKVKGRKVEAEVIASPYEYILLSPTDVPWLTQQPLQL


2mut




TILVPLQEYQEKILSKTALPEDQKQQLKTLFVKYDNLWQHWENQVGHRKI







RPHNIATGDYPPRPQKQYPINPKAKPSIQIVIDDLLKQGVLTPQNSTMNT







PVYPVPKPDGRWRMVLDYREVNKTIPLTAAQNQHSAGILATIVRQKYKTT







LDLANGFWAHPITPESYWLTAFTWQGKQYCWTRLPQGFLNSPALFNADVV







DLLKEIPNVQVYVDDIYLSHDDPKEHVQQLEKVFQILLQAGYVVSLKKSE







IGQKTVEFLGFNITKEGRGLTDTFKTKLLNITPPKDLKQLQSILGLLNFA







RNFIPNFAELVQPLYNLIAPAKGKYIEWSEENTKQLNMVIEALNTASNLE







ERLPEQRLVIKVNTSPSAGYVRYYNETGKKPIMYLNYVFSKAELKFSMLE







KLLTTMHKALIKAMDLAMGQEILVYSPIVSMTKIQKTPLPERKALPIRWI







TWMTYLEDPRIQFHYDKTLPELKHIPDVYTSSQSPVKHPSQYEGVFYTDG







SAIKSPDPTKSNNAGMGIVHATYKPEYQVLNQWSIPLGNHTAQMAEIAAV







EFACKKALKIPGPVLVITDSFYVAESANKEL







PYWKSNGFVNNKKKPLKHISKWKSIAECLSMKPDITIQHEKGISLQIPVF







ILKGNALADKLATQGSYVVN





FOAMV_
FOAMV
P14350
derivative
3162
MNPLQLLQPLPAEIKGTKLLAHWNSGATITCIPESFLEDEQPIKKTLIKT


P14350_




IHGEKQQNVYYVTFKVKGRKVEAEVIASPYEYILLSPTDVPWLTQQPLQL


2mutA




TILVPLQEYQEKILSKTALPEDQKQQLKTLFVKYDNLWQHWENQVGHRKI







RPHNIATGDYPPRPQKQYPINPKAKPSIQIVIDDLLKQGVLTPQNSTMNT







PVYPVPKPDGRWRMVLDYREVNKTIPLTAAQNQHSAGILATIVRQKYKTT







LDLANGFWAHPITPESYWLTAFTWQGKQYCWTRLPQGFLNSPALFNADVV







DLLKEIPNVQVYVDDIYLSHDDPKEHVQQLEKVFQILLQAGYVVSLKKSE







IGQKTVEFLGFNITKEGRGLTDTFKTKLLNITPPKDLKQLQSILGKLNFA







RNFIPNFAELVQPLYNLIAPAKGKYIEWSEENTKQLNMVIEALNTASNLE







ERLPEQRLVIKVNTSPSAGYVRYYNETGKKPIMYLNYVFSKAELKFSMLE







KLLTTMHKALIKAMDLAMGQEILVYSPIVSMTKIQKTPLPERKALPIRWI







TWMTYLEDPRIQFHYDKTLPELKHIPDVYTSSQSPVKHPSQYEGVFYTDG







SAIKSPDPTKSNNAGMGIVHATYKPEYQVLNQWSIPLGNHTAQMAEIAAV







EFACKKALKIPGPVLVITDSFYVAESANKELPYWKSNGFVNNKKKPLKHI







SKWKSIAECLSMKPDITIQHEKGISLQIPVFILKGNALADKLATQGSYVV







N





GALV_
GALV
P21414
root
3163
VLNLEEEYRLHEKPVPSSIDPSWLQLFPTVWAERAGMGLANQVPPVVVEL


P21414




RSGASPVAVRQYPMSKEAREGIRPHIQKFLDLGVLVPCRSPWNTPLLPVK







KPGTNDYRPVQDLREINKRVQDIHPTVPNPYNLLSSLPPSYTWYSVLDLK







DAFFCLRLHPNSQPLFAFEWKDPEKGNTGQLTWTRLPQGFKNSPTLFDEA







LHRDLAPFRALNPQVVLLQYVDDLLVAAPTYEDCKKGTQKLLQELSKLGY







RVSAKKAQLCQREVTYLGYLLKEGKRWLTPARKATVMKIPVPTTPRQVRE







FLGTAGFCRLWIPGFASLAAPLYPLTKESIPFIWTEEHQQAFDHIKKALL







SAPALALPDLTKPFTLYIDERAGVARGVLTQTLGPWRRPVAYLSKKLDPV







ASGWPTCLKAVAAVALLLKDADKLTLGQNVTVIASHSLESIVRQPPDRWM







TNARMTHYQSLLLNERVSFAPPAVLNPATLLPVESEATPVHRCSEILAEE







TGTRRDLEDQPLPGVPTWYTDGSSFITEGKRRAGAPIVDGKRTVWASSLP







EGTSAQKAELVALTQALRLAEGKNINIYTDSRYAFATAHIHGAIYKQRGL







LTSAGKDIKNKEEILALLEAIHLPRRVAIIHCPGHQRGSNPVATGNRRAD







EAAKQAALSTRVLAGTTKP





GALV_
GALV
P21414
derivative
3164
VLNLEEEYRLHEKPVPSSIDPSWLQLFPTVWAERAGMGLANQVPPVVVEL


P21414_




RSGASPVAVRQYPMSKEAREGIRPHIQKFLDLGVLVPCRSPWNTPLLPVK


3mut




KPGTNDYRPVQDLREINKRVQDIHPTVPNPYNLLSSLPPSYTWYSVLDLK







DAFFCLRLHPNSQPLFAFEWKDPEKGNTGQLTWTRLPQGFKNSPTLFNEA







LHRDLAPFRALNPQVVLLQYVDDLLVAAPTYEDCKKGTQKLLQELSKLGY







RVSAKKAQLCQREVTYLGYLLKEGKRWLTPARKATVMKIPVPTTPRQVRE







FLGTAGFCRLWIPGFASLAAPLYPLTKPSIPFIWTEEHQQAFDHIKKALL







SAPALALPDLTKPFTLYIDERAGVARGVLTQTLGPWRRPVAYLSKKLDPV







ASGWPTCLKAVAAVALLLKDADKLTLGQNVTVIASHSLESIVRQPPDRWM







TNARMTHYQSLLLNERVSFAPPAVLNPATLLPVESEATPVHRCSEILAEE







TGTRRDLEDQPLPGVPTWYTDGSSFITEGKRRAGAPIVDGKRTVWASSLP







EGTSAQKAELVALTQALRLAEGKNINIYTDSRYAFATAHIHGAIYKQRGW







LTSAGKDIKNKEEILALLEAIHLPRRVAIIHCPGHQRGSNPVATGNRRAD







EAAKQAALSTRVLAGTTKP





GALV_
GALV
P21414
derivative
3165
VLNLEEEYRLHEKPVPSSIDPSWLQLFPTVWAERAGMGLANQVPPVVVEL


P21414_




RSGASPVAVRQYPMSKEAREGIRPHIQKFLDLGVLVPCRSPWNTPLLPVK


3mutA




KPGTNDYRPVQDLREINKRVQDIHPTVPNPYNLLSSLPPSYTWYSVLDLK







DAFFCLRLHPNSQPLFAFEWKDPEKGNTGQLTWTRLPQGFKNSPTLFNEA







LHRDLAPFRALNPQVVLLQYVDDLLVAAPTYEDCKKGTQKLLQELSKLGY







RVSAKKAQLCQREVTYLGYLLKEGKRWLTPARKATVMKIPVPTTPRQVRE







FLGKAGFCRLFIPGFASLAAPLYPLTKPSIPFIWTEEHQQAFDHIKKALL







SAPALALPDLTKPFTLYIDERAGVARGVLTQTLGP







wRRPVAYLSKKLDPVASGWPTCLKAVAAVALLLKDADKLTLGQNVTVIAS







HSLESIVRQPPDRWMTNARMTHYQSLLLNERVSFAPPAVLNPATL







LPVESEATPVHRCSEILAEETGTRRDLEDQPLPGVPTWYTDGSSFITEGK







RRAGAPIVDGKRTVWASSLPEGTSAQKAELVALTQALRLAEGKNINIYTD







SRYAFATAHIHGAIYKQRGWLTSAGKDIKNKEEILALLEAIHLPRRVAII







HCPGHQRGSNPVATGNRRADEAAKQAALSTRVLAGTTKP





HTL1A_
HTL1A
P03362
root
3166
AVLGLEHLPRPPQISQFPLNPERLQALQHLVRKALEAGHIEPYTGPGNNP


P03362




VFPVKKANGTWRFIHDLRATNSLTIDLSSSSPGPPDLSSLPTTLAHLQTI







DLRDAFFQIPLPKQFQPYFAFTVPQQCNYGPGTRYAWKVLPQGFKNSPTL







FEMQLAHILQPIRQAFPQCTILQYMDDILLASPSHEDLLLLSEATMASLI







SHGLPVSENKTQQTPGTIKFLGQIISPNHLTYDAVPTVPIRSRWALPELQ







ALLGEIQWVSKGTPTLRQPLHSLYCALQRHTDPRDQIYLNPSQVQSLVQL







RQALSQNCRSRLVQTLPLLGAIMLTLTGTTTVVFQSKEQWPLVWLHAPLP







HTSQCPWGQLLASAVLLLDKYTLQSYGLLCQTIHHNISTQTFNQFIQTSD







HPSVPILLHHSHRFKNLGAQTGELWNTFLKTAAPLAPVKALMPVFTLSPV







IINTAPCLFSDGSTSRAAYILWDKQILSQRSFPLPPPHKSAQRAELLGLL







HGLSSARSWRCLNIFLDSKYLYHYLRTLALGTFQGRSSQAPFQALLPRLL







SRKVVYLHHVRSHTNLPDPISRLNALTDALLITPVLQL





HTL1A_
HTL1A
P03362
derivative
3167
AVLGLEHLPRPPQISQFPLNPERLQALQHLVRKALEAGHIEPYTGPGNNP


P03362_




VFPVKKANGTWRFIHDLRATNSLTIDLSSSSPGPPDLSSLPTTLAHLQTI


2mut




DLRDAFFQIPLPKQFQPYFAFTVPQQCNYGPGTRYAWKVLPQGFKNSPTL







FQMQLAHILQPIRQAFPQCTILQYMDDILLASPSHEDLLLLSEATMASLI







SHGLPVSENKTQQTPGTIKFLGQIISPNHLTYDAVPTVPIRSRWALPELQ







ALLGEIQWVSKGTPTLRQPLHSLYCALQPHTDPRDQIYLNPSQVQSLVQL







RQALSQNCRSRLVQTLPLLGAIMLTLTGTTTVVFQSKEQWPLVWLHAPLP







HTSQCPWGQLLASAVLLLDKYTLQSYGLLCQTIHHNISTQTFNQFIQTSD







HPSVPILLHHSHRFKNLGAQTGELWNTFLKTAAPLAPVKALMPVFTLSPV







IINTAPCLFSDGSTSRAAYILWDKQILSQRSFPLPPPHKSAQRAELLGLL







HGLSSARSWRCLNIFLDSKYLYHYLRTLALGTFQGRSSQAPFQALLPRLL







SRKVVYLHHVRSHTNLPDPISRLNALTDALLITPVLQL





HTL1A_
HTL1A
P03362
derivative
3168
AVLGLEHLPRPPQISQFPLNPERLQALQHLVRKALEAGHIEPYTGPGNNP


P03362_




VFPVKKANGTWRFIHDLRATNSLTIDLSSSSPGPPDLSSPPTTLAHLQTI


2mutB




DLRDAFFQIPLPKQFQPYFAFTVPQQCNYGPGTRYAWKVLPQGFKNSPTL







FQMQLAHILQPIRQAFPQCTILQYMDDILLASPSHEDLLLLSEATMASVQ







LRQALSQNCRSRLVQTLPLLGAIMLTLTGTTTVVFQSKEQWPLVWLHAPL







PHTSQCPWGQLLASAVLLLDKYTLQSYGLLCQTIHHNISTQTFNQFIQTS







DHPSVPILLHHSHRFKNLGAQTGELWNTFLKTAAPLAPVKALMPVFTLSP







VIINTAPCLFSDGSTSRAAYILWDKQILSQRSFPLPPPHKSAQRAELLGL







LHGLSSARSWRCLNIFLDSKYLYHYLRTLALGTFQGRSSQAPFQALLPRL







LSRKVVYLHHVRSHTNLPDPISRLNALTDALLITPVLQL





HTLIC_
HTL1C
P14078
root
3169
AVLGLEHLPRPPEISQFPLNPERLQALQHLVRKALEAGHIEPYTGPGNNP


P14078




VFPVKKANGTWRFIHDLRATNSLTIDLSSSSPGPPDLSSLPTTLAHLQTI







DLKDAFFQIPLPKQFQPYFAFTVPQQCNYGPGTRYAWRVLPQGFKNSPTL







FEMQLAHILQPIRQAFPQCTILQYMDDILLASPSHADLQLLSEATMASVQ







LRQALSQNCRSRLVQTLPLLGAIMLTLTGTTTVVFQSKQQWPLVWLHAPL







PHTSQCPWGQLLASAVLLLDKYTLQSYGLLCQTIHHNISTQTFNQFIQTS







DHPSVPILLHHSHRFKNLGAQTGELWNTFLKTTAPLAPVKALMPVFTLSP







VIINTAPCLFSDGSTSQAAYILWDKHILSQRSFPLPPPHKSAQRAELLGL







LHGLSSARSWRCLNIFLDSKYLYHYLRTLALGTFQGRSSQAPFQALLPRL







LSRKVVYLHHVRSHTNLPDPISRLNALTDALLITPVLQL





HTLIC_
HTL1C
P14078
derivative
3170
AVLGLEHLPRPPEISQFPLNPERLQALQHLVRKALEAGHIEPYTGPGNNP


P14078_2




VFPVKKANGTWRFIHDLRATNSLTIDLSSSSPGPPDLSSLPTTLAHLQTI


mut




DLKDAFFQIPLPKQFQPYFAFTVPQQCNYGPGTRYAWRVLPQGFKNSPTL







FQMQLAHILQPIRQAFPQCTILQYMDDILLASPSHADLQLLSEATMA







SLISHGLPVSENKTQQTPGTIKFLGQIISPNHLTYDAVPKVPIRSRWALP







ELQALLGEIQWVSKGTPTLRQPLHSLYCALQPHTDPRDQIYLNPSQVQSL







VQLRQALSQNCRSRLVQTLPLLGAIMLTLTGTTTVVFQSKQQWPLVWLHA







PLPHTSQCPWGQLLASAVLLLDKYTLQSYGLLCQTIHHNISTQTFNQFIQ







TSDHPSVPILLHHSHRFKNLGAQTGELWNTFLKTTAPLAPVKALMPVFTL







SPVIINTAPCLFSDGSTSQAAYILWDKHILSQRSFPLPPPHKSAQRAELL







GLLHGLSSARSWRCLNIFLDSKYLYHYLRTLALGTFQGRSSQAPFQALLP







RLLSRKVVYLHHVRSHTNLPDPISRLNALTDALLITPVLQL





HTLIC_
HTL1C
P14078
derivative
3171
AVLGLEHLPRPPEISQFPLNPERLQALQHLVRKALEAGHIEPYTGPGNNP


P14078_2




VFPVKKANGTWRFIHDLRATNSLTIDLSSSSPGPPDLSSPPTTLAHLQTI


mutB




DLKDAFFQIPLPKQFQPYFAFTVPQQCNYGPGTRYAWRVLPQGFKNSPTL







FQMQLAHILQPIRQAFPQCTILQYMDDILLASPSHADLQLLSEATMASLI







SHGLPVSENKTQQTPGTIKFLGQIISPNHLTYDAVPKVPIRSRWALPELQ







ALLGEIQWVSKGTPTLRQPLHSLYCALQPHTDPRDQIYLNPSQVQSLVQL







RQALSQNCRSRLVQTLPLLGAIMLTLTGTTTVVFQSKQQWPLVWLHAPLP







HTSQCPWGQLLASAVLLLDKYTLQSYGLLCQTIHHNISTQTFNQFIQTSD







HPSVPILLHHSHRFKNLGAQTGELWNTFLKTTAPLAPVKALMPVFTLSPV







IINTAPCLFSDGSTSQAAYILWDKHILSQRSFPLPPPHKSAQRAELLGLL







HGLSSARSWRCLNIFLDSKYLYHYLRTLALGTFQGRSSQAPFQALLPRLL







SRKVVYLHHVRSHTNLPDPISRLNALTDALLITPVLQL





HTLIL_
HTL1L
POC211
root
3172
GLEHLPRPPEISQFPLNPERLQALQHLVRKALEAGHIEPYTGPGNNPVFP


P0C211




VKKANGTWRFIHDLRATNSLTVDLSSSSPGPPDLSSLPTTLAHLQTIDLK







DAFFQIPLPKQFQPYFAFTVPQQCNYGPGTRYAWKVLPQGFKNSPTLFEM







QLASILQPIRQAFPQCVILQYMDDILLASPSPEDLQQLSEATMASLISHG







LPVSQDKTQQTPGTIKFLGQUISPNHITYDAVPTVPIRSRWALPELQALL







GEIQWVSKGTPTLRQPLHSLYCALQGHTDPRDQIYLNPSQVQSLMQLQQA







LSQNCRSRLAQTLPLLGAIMLTLTGTTTVVFQSKQQWPLVWLHAPLPHTS







QCPWGQLLASAVLLLDKYTLQSYGLLCQTIHHNISIQTFNQFIQTSDHPS







VPILLHHSHRFKNLGAQTGELWNTFLKTAAPLAPVKALTPVFTLSPIIIN







TAPCLFSDGSTSQAAYILWDKHILSQRSFPLPPPHKSAQQAELLGLLHGL







SSARSWHCLNIFLDSKYLYHYLRTLALGTFQGKSSQAPFQALLPRLLAHK







VIYLHHVRSHTNLPDPISKLNALTDALLITPIL





HTLIL_
HTL1L
POC211
derivative
3173
GLEHLPRPPEISQFPLNPERLQALQHLVRKALEAGHIEPYTGPGNNPVFP


P0C211_2




VKKANGTWRFIHDLRATNSLTVDLSSSSPGPPDLSSLPTTLAHLQTIDLK


mut




DAFFQIPLPKQFQPYFAFTVPQQCNYGPGTRYAWKVLPQGFKNSPTLFQM







QLASILQPIRQAFPQCVILQYMDDILLASPSPEDLQQLSEATMASLISHG







LPVSQDKTQQTPGTIKFLGQIISPNHITYDAVPTVPIRSRWALPELQALL







GEIQWVSKGTPTLRQPLHSLYCALQGHTDPRDQIYLNPSQVQSLMQLQQA







LSQNCRSRLAQTLPLLGAIMLTLTGTTTVVFQSKQQWPLVWLHAPLPHTS







QCPWGQLLASAVLLLDKYTLQSYGLLCQTIHHNISIQTFNQFIQTSDHPS







VPILLHHSHRFKNLGAQTGELWNTFLKTAAPLAPVKALTPVFTLSPIIIN







TAPCLFSDGSTSQAAYILWDKHILSQRSFPLPPPHKSAQQAELLGLLHGL







SSARSWHCLNIFLDSKYLYHYLRTLAWGTFQGKSSQAPFQALLPRLLAHK







VIYLHHVRSHTNLPDPISKLNALTDALLITPIL





HTLIL_
HTL1L
POC211
derivative
3174
GLEHLPRPPEISQFPLNPERLQALQHLVRKALEAGHIEPYTGPGNNPVFP


P0C211_2




VKKANGTWRFIHDLRATNSLTVDLSSSSPGPPDLSSPPTTLAHLQTIDLK


mutB




DAFFQIPLPKQFQPYFAFTVPQQCNYGPGTRYAWKVLPQGFKNSPTLFQM







QLASILQPIRQAFPQCVILQYMDDILLASPSPEDLQQLSEATMASLISHG







LPVSQDKTQQTPGTIKFLGQUISPNHITYDAVPTVPIRSRWALPELQALL







GEIQWVSKGTPTLRQPLHSLYCALQGHTDPRDQIYLNPSQVQSLMQLQQA







LSQNCRSRLAQTLPLLGAIMLTLTGTTTVVFQSKQQWPLVWLHAPLPHTS







QCPWGQLLASAVLLLDKYTLQSYGLLCQTIHHNISIQTFNQFIQTSDHPS







VPILLHHSHRFKNLGAQTGELWNTFLKTAAPLAPVKALTPVFTLSPIIIN







TAPCLFSDGSTSQAAYILWDKHILSQRSFPLPPPHKSAQQAE







LLGLLHGLSSARSWHCLNIFLDSKYLYHYLRTLAWGTFQGKSSQAPFQAL







LPRLLAHKVIYLHHVRSHTNLPDPISKLNALTDALLITPIL





HTL32_
HTL32
Q0R5R2
root
3175
GLEHLPPPPEVSQFPLNPERLQALTDLVSRALEAKHIEPYQGPGNNPIFP


QOR5R2




VKKPNGKWRFIHDLRATNSVTRDLASPSPGPPDLTSLPQGLPHLRTIDLT







DAFFQIPLPTIFQPYFAFTLPQPNNYGPGTRYSWRVLPQGFKNSPTLFEQ







QLSHILTPVRKTFPNSLIIQYMDDILLASPAPGELAALTDKVTNALTKEG







LPLSPEKTQATPGPIHFLGQVISQDCITYETLPSINVKSTWSLAELQSML







GELQWVSKGTPVLRSSLHQLYLALRGHRDPRDTIKLTSIQVQALRTIQKA







LTLNCRSRLVNQLPILALIMLRPTGTTAVLFQTKQKWPLVWLHTPHPATS







LRPWGQLLANAVIILDKYSLQHYGQVCKSFHHNISNQALTYYLHTSDQSS







VAILLQHSHRFHNLGAQPSGPWRSLLQMPQIFQNIDVLRPPFTISPVVIN







HAPCLFSDGSASKAAFIIWDRQVIHQQVLSLPSTCSAQAGELFGLLAGLQ







KSQPWVALNIFLDSKFLIGHLRRMALGAFPGPSTQCELHTQLLPLLQGKT







VYVHHVRSHTLLQDPISRLNEATDALMLAPLLPL





HTL32_
HTL32
Q0R5R2
derivative
3176
GLEHLPPPPEVSQFPLNPERLQALTDLVSRALEAKHIEPYQGPGNNPIFP


QOR5R2_2




VKKPNGKWRFIHDLRATNSVTRDLASPSPGPPDLTSLPQGLPHLRTIDLT


mut




DAFFQIPLPTIFQPYFAFTLPQPNNYGPGTRYSWRVLPQGFKNSPTLFQQ







QLSHILTPVRKTFPNSLIIQYMDDILLASPAPGELAALTDKVTNALTKEG







LPLSPEKTQATPGPIHFLGQVISQDCITYETLPSINVKSTWSLAELQSML







GELQWVSKGTPVLRSSLHQLYLALRGHRDPRDTIKLTSIQVQALRTIQKA







LTLNCRSRLVNQLPILALIMLRPTGTTAVLFQTKQKWPLVWLHTPHPATS







LRPWGQLLANAVIILDKYSLQHYGQVCKSFHHNISNQALTYYLHTSDQSS







VAILLQHSHRFHNLGAQPSGPWRSLLQMPQIFQNIDVLRPPFTISPVVIN







HAPCLFSDGSASKAAFIIWDRQVIHQQVLSLPSTCSAQAGELFGLLAGLQ







KSQPWVALNIFLDSKFLIGHLRRMAWGAFPGPSTQCELHTQLLPLLQGKT







VYVHHVRSHTLLQDPISRLNEATDALMLAPLLPL





HTL32_
HTL32
Q0R5R2
derivative
3177
GLEHLPPPPEVSQFPLNPERLQALTDLVSRALEAKHIEPYQGPGNNPIFP


QOR5R2_2




VKKPNGKWRFIHDLRATNSVTRDLASPSPGPPDLTSPPQGLPHLRTIDLT


mutB




DAFFQIPLPTIFQPYFAFTLPQPNNYGPGTRYSWRVLPQGFKNSPTLFQQ







QLSHILTPVRKTFPNSLIIQYMDDILLASPAPGELAALTDKVTNALTKEG







LPLSPEKTQATPGPIHFLGQVISQDCITYETLPSINVKSTWSLAELQSML







GELQWVSKGTPVLRSSLHQLYLALRGHRDPRDTIKLTSIQVQALRTIQKA







LTLNCRSRLVNQLPILALIMLRPTGTTAVLFQTKQKWPLVWLHTPHPATS







LRPWGQLLANAVIILDKYSLQHYGQVCKSFHHNISNQALTYYLHTSDQSS







VAILLQHSHRFHNLGAQPSGPWRSLLQMPQIFQNIDVLRPPFTISPVVIN







HAPCLFSDGSASKAAFIIWDRQVIHQQVLSLPSTCSAQAGELFGLLAGLQ







KSQPWVALNIFLDSKFLIGHLRRMAWGAFPGPSTQCELHTQLLPLLQGKT







VYVHHVRSHTLLQDPISRLNEATDALMLAPLLPL





HTL3P_
HTL3P
Q4U0X6
root
3178
GLEHLPPPPEVSQFPLNPERLQALTDLVSRALEAKHIEPYQGPGNNPIFP


Q4U0X6




VKKPNGKWRFIHDLRATNSLTRDLASPSPGPPDLTSLPQDLPHLRTIDLT







DAFFQIPLPAVFQPYFAFTLPQPNNHGPGTRYSWRVLPQGFKNSPTLFEQ







QLSHILAPVRKAFPNSLIIQYMDDILLASPALRELTALTDKVTNALTKEG







LPMSLEKTQATPGSIHFLGQVISPDCITYETLPSIHVKSIWSLAELQSML







GELQWVSKGTPVLRSSLHQLYLALRGHRDPRDTIELTSTQVQALKTIQKA







LALNCRSRLVSQLPILALIILRPTGTTAVLFQTKQKWPLVWLHTPHPATS







LRPWGQLLANAIITLDKYSLQHYGQICKSFHHNISNQALTYYLHTSDQSS







VAILLQHSHRFHNLGAQPSGPWRSLLQVPQIFQNIDVLRPPFIISPVVID







HAPCLFSDGATSKAAFILWDKQVIHQQVLPLPSTCSAQAGELFGLLAGLQ







KSKPWPALNIFLDSKFLIGHLRRMALGAFLGPSTQCDLHARLFPLLQGKT







VYVHHVRSHTLLQDPISRLNEATDALMLAPLLPL





HTL3P_
HTL3P
Q4U0X6
derivative
3179
GLEHLPPPPEVSQFPLNPERLQALTDLVSRALEAKHIEPYQGPGNNPIFP


Q4U0X6_2




VKKPNGKWRFIHDLRATNSLTRDLASPSPGPPDLTSLPQDLPHLRTIDLT


mut




DAFFQIPLPAVFQPYFAFTLPQPNNHGPGTRYSWRVLPQGFKNSPTLFQQ







QLSHILAPVRKAFPNSLIIQYMDDILLASPALRELTALTDKVTNALTKE







GLPMSLEKTQATPGSIHFLGQVISPDCITYETLPSIHVKSIWSLAELQSM







LGELQWVSKGTPVLRSSLHQLYLALRGHRDPRDTIELTSTQVQALKTIQK







ALALNCRSRLVSQLPILALIILRPTGTTAVLFQTKQKWPLVWLHTPHPAT







SLRPWGQLLANAIITLDKYSLQHYGQICKSFHHNISNQALTYYLHTSDQS







SVAILLQHSHRFHNLGAQPSGPWRSLLQVPQIFQNIDVLRPPFIISPVVI







DHAPCLFSDGATSKAAFILWDKQVIHQQVLPLPSTCSAQAGELFGLLAGL







QKSKPWPALNIFLDSKFLIGHLRRMAWGAFLGPSTQCDLHARLFPLLQGK







TVYVHHVRSHTLLQDPISRLNEATDALMLAPLLPL





HTL3P_
HTL3P
Q4UOX6
derivative
3180
GLEHLPPPPEVSQFPLNPERLQALTDLVSRALEAKHIEPYQGPGNNPIFP


Q4U0X6_




VKKPNGKWRFIHDLRATNSLTRDLASPSPGPPDLTSPPQDLPHLRTIDLT


2mutB




DAFFQIPLPAVFQPYFAFTLPQPNNHGPGTRYSWRVLPQGFKNSPTLFQQ







QLSHILAPVRKAFPNSLIIQYMDDILLASPALRELTALTDKVTNALTKEG







LPMSLEKTQATPGSIHFLGQVISPDCITYETLPSIHVKSIWSLAELQSML







GELQWVSKGTPVLRSSLHQLYLALRGHRDPRDTIELTSTQVQALKTIQKA







LALNCRSRLVSQLPILALIILRPTGTTAVLFQTKQKWPLVWLHTPHPATS







LRPWGQLLANAIITLDKYSLQHYGQICKSFHHNISNQALTYYLHTSDQSS







VAILLQHSHRFHNLGAQPSGPWRSLLQVPQIFQNIDVLRPPFIISPVVID







HAPCLFSDGATSKAAFILWDKQVIHQQVLPLPSTCSAQAGELFGLLAGLQ







KSKPWPALNIFLDSKFLIGHLRRMAWGAFLGPSTQCDLHARLFPLLQGKT







VYVHHVRSHTLLQDPISRLNEATDALMLAPLLPL





HTLV2_
HTLV2
P03363
root
3181
HLPPPPQVDQFPLNLPERLQALNDLVSKALEAGHIEPYSGPGNNPVFPVK


P03363




KPNGKWRFIHDLRATNAITTTLTSPSPGPPDLTSLPTALPHLQTIDLTDA







FFQIPLPKQYQPYFAFTIPQPCNYGPGTRYAWTVLPQGFKNSPTLFEQQL







AAVLNPMRKMFPTSTIVQYMDDILLASPTNEELQQLSQLTLQALTTHGLP







ISQEKTQQTPGQIRFLGQVISPNHITYESTPTIPIKSQWTLTELQVILGE







IQWVSKGTPILRKHLQSLYSALHGYRDPRACITLTPQQLHALHAIQQALO







HNCRGRLNPALPLLGLISLSTSGTTSVIFQPKQNWPLAWLHTPHPPTSLC







PWGHLLACTILTLDKYTLQHYGQLCQSFHHNMSKQALCDFLRNSPHPSVG







ILIHHMGRFHNLGSQPSGPWKTLLHLPTLLQEPRLLRPIFTLSPVVLDTA







PCLFSDGSPQKAAYVLWDQTILQQDITPLPSHETHSAQKGELLALICGLR







AAKPWPSLNIFLDSKYLIKYLHSLAIGAFLGTSAHQTLQAALPPLLQGKT







IYLHHVRSHTNLPDPISTFNEYTDSLILAPLVPL





HTLV2_
HTLV2
P03363
derivative
3182
HLPPPPQVDQFPLNLPERLQALNDLVSKALEAGHIEPYSGPGNNPVFPVK


P03363_




KPNGKWRFIHDLRATNAITTTLTSPSPGPPDLTSLPTALPHLQTIDLTDA


2mut




FFQIPLPKQYQPYFAFTIPQPCNYGPGTRYAWTVLPQGFKNSPTLFQQQL







AAVLNPMRKMFPTSTIVQYMDDILLASPTNEELQQLSQLTLQALTTHGLP







ISQEKTQQTPGQIRFLGQVISPNHITYESTPTIPIKSQWTLTELQVILGE







IQWVSKGTPILRKHLQSLYSALHPYRDPRACITLTPQQLHALHAIQQALQ







HNCRGRLNPALPLLGLISLSTSGTTSVIFQPKQNWPLAWLHTPHPPTSLC







PWGHLLACTILTLDKYTLQHYGQLCQSFHHNMSKQALCDFLRNSPHPSVG







ILIHHMGRFHNLGSQPSGPWKTLLHLPTLLQEPRLLRPIFTLSPVVLDTA







PCLFSDGSPQKAAYVLWDQTILQQDITPLPSHETHSAQKGELLALICGLR







AAKPWPSLNIFLDSKYLIKYLHSLAIGAFLGTSAHQTLQAALPPLLQGKT







IYLHHVRSHTNLPDPISTFNEYTDSLILAPLVPL





HTLV2_
HTLV2
P03363
derivative
3183
HLPPPPQVDQFPLNLPERLQALNDLVSKALEAGHIEPYSGPGNNPVFPVK


P03363_




KPNGKWRFIHDLRATNAITTTLTSPSPGPPDLTSPPTALPHLQTIDLTDA


2mutB




FFQIPLPKQYQPYFAFTIPQPCNYGPGTRYAWTVLPQGFKNSPTLFQQQL







AAVLNPMRKMFPTSTIVQYMDDILLASPTNEELQQLSQLTLQALTTHGLP







ISQEKTQQTPGQIRFLGQVISPNHITYESTPTIPIKSQWTLTELQVILGE







IQWVSKGTPILRKHLQSLYSALHPYRDPRACITLTPQQLHALHAIQQALQ







HNCRGRLNPALPLLGLISLSTSGTTSVIFQPKQNWPLAWLHTPHPPTSLC







PWGHLLACTILTLDKYTLQHYGQLCQSFHHNMSKQALCDFLRNSPHPSVG







ILIHHMGRFHNLGSQPSGPWKTLLHLPTLLQEPRLLRPIFTLSPVVLDTA







PCLFSDGSPQKAAYVLWDQTILQQDITPLPSHETHSAQKGELL







ALICGLRAAKPWPSLNIFLDSKYLIKYLHSLAIGAFLGTSAHQTLQAALP







PLLQGKTIYLHHVRSHTNLPDPISTFNEYTDSLILAPLVPL





JSRV_
JSRV
P31623
root
3184
PLGTSDSPVTHADPIDWKSEEPVWVDQWPLTQEKLSAAQQLVQEQLRLGH


P31623




IEPSTSAWNSPIFVIKKKSGKWRLLQDLRKVNETMMHMGALQPGLPTPSA







IPDKSYIIVIDLKDCFYTIPLAPQDCKRFAFSLPSVNFKEPMQRYQWRVL







PQGMTNSPTLCQKFVATAIAPVRQRFPQLYLVHYMDDILLAHTDEHLLYQ







AFSILKQHLSLNGLVIADEKIQTHFPYNYLGFSLYPRVYNTQLVKLQTDH







LKTLNDFQKLLGDINWIRPYLKLPTYTLQPLFDILKGDSDPASPRTLSLE







GRTALQSIEEAIRQQQITYCDYQRSWGLYILPTPRAPTGVLYQDKPLRWI







YLSATPTKHLLPYYELVAKIIAKGRHEAIQYFGMEPPFICVPYALEQQDW







LFQFSDNWSIAFANYPGQITHHYPSDKLLQFASSHAFIFPKIVRRQPIPE







ATLIFTDGSSNGTAALIINHQTYYAQTSFSSAQVVELFAVHQALLTVPTS







FNLFTDSSYVVGALQMIETVPIIGTTSPEVLNLFTLIQQVLHCRQHPCFF







GHIRAHSTLPGALVQGNHTADVLTKQVFFQS





JSRV_
JSRV
P31623
derivative
3185
PLGTSDSPVTHADPIDWKSEEPVWVDQWPLTQEKLSAAQQLVQEQLRLGH


P31623_




IEPSTSAWNSPIFVIKKKSGKWRLLQDLRKVNETMMHMGALQPGLPTPSP


2mutB




IPDKSYIIVIDLKDCFYTIPLAPQDCKRFAFSLPSVNFKEPMQRYQWRVL







PQGMTNSPTLCQKFVATAIAPVRQRFPQLYLVHYMDDILLAHTDEHLLYQ







AFSILKQHLSLNGLVIADEKIQTHFPYNYLGFSLYPRVYNTQLVKLQTDH







LKTLNDFQKLLGDINWIRPYLKLPTYTLQPLFDILKGDSDPASPRTLSLE







GRTALQSIEEAIRQQQITYCDYQRSWGLYILPTPRAPTGVLYQDKPLRWI







YLSATPTKHLLPYYELVAKIIAKGRHEAIQYFGMEPPFICVPYALEQQDW







LFQFSDNWSIAFANYPGQITHHYPSDKLLQFASSHAFIFPKIVRRQPIPE







ATLIFTDGSSNGTAALIINHQTYYAQTSFSSAQVVELFAVHQALLTVPTS







FNLFTDSSYVVGALQMIETVPIIGTTSPEVLNLFTLIQQVLHCRQHPCFF







GHIRAHSTLPGALVQGNHTADVLTKQVFFQS





KORV_
KORV
101160
root
3186
TLGDQGSRGSDPLPEPRVTLTVEGIPTEFLVNTGAEHSVLTKPMGKMGSK


Q9TTC1




RTVVAGATGSKVYPWTTKRLLKIGQKQVTHSFLVIPECPAPLLGRDLLTK







LKAQIQFSTEGPQVTWEDRPAMCLVLNLEEEYRLHEKPVPPSIDPSWLQL







FPMVWAEKAGMGLANQVPPVVVELKSDASPVAVRQYPMSKEAREGIRPHI







QRFLDLGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVQDIHPT







VPNPYNLLSSLPPSHTWYSVLDLKDAFFCLKLHPNSQPLFAFEWRDPEKG







NTGQLTWTRLPQGFKNSPTLFDEALHRDLASFRALNPQVVMLQYVDDLLV







AAPTYRDCKEGTRRLLQELSKLGYRVSAKKAQLCREEVTYLGYLLKGGKR







WLTPARKATVMKIPTPTTPRQVREFLGTAGFCRLWIPGFASLAAPLYPLT







REKVPFTWTEAHQEAFGRIKEALLSAPALALPDLTKPFALYVDEKEGVAR







GVLTQTLGPWRRPVAYLSKKLDPVASGWPTCLKAIAAVALLLKDADKLTL







GQNVLVIAPHNLESIVRQPPDRWMTNARMTHYQSLLLNERVSFAPPAILN







PATLLPVESDDTPIHICSEILAEETGTRPDLRDQPLPGVPAWYTDGSSFI







MDGRRQAGAAIVDNKRTVWASNLPEGTSAQKAELIALTQALRLAEGKSIN







IYTDSRYAFATAHVHGAIYKQRGLLTSAGKDIKNKEEILALLEAIHLPKR







VAIIHCPGHQRGTDPVATGNRKADEAAKQAAQSTRILTETTKN





KORV_
KORV
Q9C1
derivative
3187
LLGRDLLTKLKAQIQFSTEGPQVTWEDRPAMCLVLNLEEEYRLHEKPVPP


Q9TTC1-




SIDPSWLQLFPMVWAEKAGMGLANQVPPVVVELKSDASPVAVRQYPMSKE


Pro




AREGIRPHIQRFLDLGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVN







KRVQDIHPTVPNPYNLLSSLPPSHTWYSVLDLKDAFFCLKLHPNSQPLFA







FEWRDPEKGNTGQLTWTRLPQGFKNSPTLFDEALHRDLASFRALNPQVVM







LQYVDDLLVAAPTYRDCKEGTRRLLQELSKLGYRVSAKKAQLCREEVTYL







GYLLKGGKRWLTPARKATVMKIPTPTTPRQVREFLGTAGFCRLWIPGFAS







LAAPLYPLTREKVPFTWTEAHQEAFGRIKEALLSAPALALPDLTKPFALY







VDEKEGVARGVLTQTLGPWRRPVAYLSKKLDPVASGWPTCLKAIAAVALL







LKDADKLTLGQNVLVIAPHNLESIVRQPPD







RWMTNARMTHYQSLLLNERVSFAPPAILNPATLLPVESDDTPIHICSEIL







AEETGTRPDLRDQPLPGVPAWYTDGSSFIMDGRRQAGAAIVDNKRTVWAS







NLPEGTSAQKAELIALTQALRLAEGKSINIYTDSRYAFATAHVHGAIYKQ







RGLLTSAGKDIKNKEEILALLEAIHLPKRVAIIHCPGHQRGTDPVATGNR







KADEAAKQAAQSTRILTETTKN





KORV_
KORV
Q9TTC1
derivative
3188
PMSKEAREGIRPHIQRFLDLGILVPCQSPWNTPLLPVKKPGTNDYRPVQD


Q9TTC1-




LREVNKRVQDIHPTVPNPYNLLSSLPPSHTWYSVLDLKDAFFCLKLHPNS


Pro3




QPLFAFEWRDPEKGNTGQLTWTRLPQGFKNSPTLFNEALHRDLASFRALN


mut




PQVVMLQYVDDLLVAAPTYRDCKEGTRRLLQELSKLGYRVSAKKAQLCRE







EVTYLGYLLKGGKRWLTPARKATVMKIPTPTTPRQVREFLGTAGFCRLWI







PGFASLAAPLYPLTRPKVPFTWTEAHQEAFGRIKEALLSAPALALPDLTK







PFALYVDEKEGVARGVLTQTLGPWRRPVAYLSKKLDPVASGWPTCLKAIA







AVALLLKDADKLTLGQNVLVIAPHNLESIVRQPPDRWMTNARMTHYQSLL







LNERVSFAPPAILNPATLLPVESDDTPIHICSEILAEETGTRPDLRDQPL







PGVPAWYTDGSSFIMDGRRQAGAAIVDNKRTVWASNLPEGTSAQKAELIA







LTQALRLAEGKSINIYTDSRYAFATAHVHGAIYKQRGWLTSAGKDIKNKE







EILALLEAIHLPKRVAIIHCPGHQRGTDPVATGNRKADEAAKQAAQSTRI







LTETTKN





KORV_
KORV
Q9TTC1
derivative
3189
LLGRDLLTKLKAQIQFSTEGPQVTWEDRPAMCLVLNLEEEYRLHEKPVPP


Q9TTC1-




SIDPSWLQLFPMVWAEKAGMGLANQVPPVVVELKSDASPVAVRQYPMSKE


Pro3




AREGIRPHIQRFLDLGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVN


mutA




KRVQDIHPTVPNPYNLLSSLPPSHTWYSVLDLKDAFFCLKLHPNSQPLFA







FEWRDPEKGNTGQLTWTRLPQGFKNSPTLFNEALHRDLASFRALNPQVVM







LQYVDDLLVAAPTYRDCKEGTRRLLQELSKLGYRVSAKKAQLCREEVTYL







GYLLKGGKRWLTPARKATVMKIPTPTTPRQVREFLGKAGFCRLFIPGFAS







LAAPLYPLTRPKVPFTWTEAHQEAFGRIKEALLSAPALALPDLTKPFALY







VDEKEGVARGVLTQTLGPWRRPVAYLSKKLDPVASGWPTCLKAIAAVALL







LKDADKLTLGQNVLVIAPHNLESIVRQPPDRWMTNARMTHYQSLLLNERV







SFAPPAILNPATLLPVESDDTPIHICSEILAEETGTRPDLRDQPLPGVPA







WYTDGSSFIMDGRRQAGAAIVDNKRTVWASNLPEGTSAQKAELIALTQAL







RLAEGKSINIYTDSRYAFATAHVHGAIYKQRGWLTSAGKDIKNKEEILAL







LEAIHLPKRVAIIHCPGHQRGTDPVATGNRKADEAAKQAAQSTRILTETT







KN





KORV_
KORV
Q9TTC1
derivative
3190
TLGDQGSRGSDPLPEPRVTLTVEGIPTEFLVNTGAEHSVLTKPMGKMGSK


Q9TTC1_




RTVVAGATGSKVYPWTTKRLLKIGQKQVTHSFLVIPECPAPLLGRDLLTK


3mut




LKAQIQFSTEGPQVTWEDRPAMCLVLNLEEEYRLHEKPVPPSIDPSWLQL







FPMVWAEKAGMGLANQVPPVVVELKSDASPVAVRQYPMSKEAREGIRPHI







QRFLDLGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVQDIHPT







VPNPYNLLSSLPPSHTWYSVLDLKDAFFCLKLHPNSQPLFAFEWRDPEKG







NTGQLTWTRLPQGFKNSPTLFNEALHRDLASFRALNPQVVMLQYVDDLLV







AAPTYRDCKEGTRRLLQELSKLGYRVSAKKAQLCREEVTYLGYLLKGGKR







WLTPARKATVMKIPTPTTPRQVREFLGTAGFCRLWIPGFASLAAPLYPLT







RPKVPFTWTEAHQEAFGRIKEALLSAPALALPDLTKPFALYVDEKEGVAR







GVLTQTLGPWRRPVAYLSKKLDPVASGWPTCLKAIAAVALLLKDADKLTL







GQNVLVIAPHNLESIVRQPPDRWMTNARMTHYQSLLLNERVSFAPPAILN







PATLLPVESDDTPIHICSEILAEETGTRPDLRDQPLPGVPAWYTDGSSFI







MDGRRQAGAAIVDNKRTVWASNLPEGTSAQKAELIALTQALRLAEGKSIN







IYTDSRYAFATAHVHGAIYKQRGWLTSAGKDIKNKEEILALLEAIHLPKR







VAIIHCPGHQRGTDPVATGNRKADEAAKQAAQSTRILTETTKN





KORV_
KORV
Q9TTC1
derivative
3191
TLGDQGSRGSDPLPEPRVTLTVEGIPTEFLVNTGAEHSVLTKPMGKMGSK


Q9TTC1_ 
mutA



RTVVAGATGSKVYPWTTKRLLKIGQKQVTHSFLVIPECPAPLLGRDL


3




REGIRPHIQRFLDLGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNK







RVQDIHPTVPNPYNLLSSLPPSHTWYSVLDLKDAFFCLKLHPNSQPLFAF







EWRDPEKGNTGQLTWTRLPQGFKNSPTLFNEALHRDLASFRALNPQVVML







QYVDDLLVAAPTYRDCKEGTRRLLQELSKLGYRVSAKKAQLCREEVTYLG







YLLKGGKRWLTPARKATVMKIPTPTTPRQVREFLGKAGFCRLFIPGFASL







AAPLYPLTRPKVPFTWTEAHQEAFGRIKEALLSAPALALPDLTKPFALYV







DEKEGVARGVLTQTLGPWRRPVAYLSKKLDPVASGWPTCLKAIAAVALLL







KDADKLTLGQNVLVIAPHNLESIVRQPPDRWMTNARMTHYQSLLLNERVS







FAPPAILNPATLLPVESDDTPIHICSEILAEETGTRPDLRDQPLPGVPAW







YTDGSSFIMDGRRQAGAAIVDNKRTVWASNLPEGTSAQKAELIALTQALR







LAEGKSINIYTDSRYAFATAHVHGAIYKQRGWLTSAGKDIKNKEEILALL







EAIHLPKRVAIIHCPGHQRGTDPVATGNRKADEAAKQAAQSTRILTETTK







N





MLVAV_
MLVAV
P03356
root
3192
TLNLEDEYRLYETSAEPEVSPGSTWLSDFPQAWAETGGMGLAVRQAPLII


P03356




PLKATSTPVSIKQYPMSQEAKLGIKPHIQRLLDQGILVPCQSPWNTPLLP







VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHRWYTVLD







LKDAFFCLRLHPTSQPLFAFEWRDPGMGISGQLTWTRLPQGFKNSPTLFD







EALHRDLADFRIQHPDLILLQYVDDILLAATSELDCQQGTRALLLTLGNL







GYRASAKKAQLCQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQL







REFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA







LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD







PVAAGWPPCLRMVAAIAVLRKDAGKLTMGQPLVILAPHAVEALVKQPPDR







WLSNARMTHYQAMLLDTDRVQFGPVVALNPATLLPLPEEGAPHDCLEILA







ETHGTRPDLTDQPIPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWAR







ALPAGTSAQRAELIALTQALKMAEGKRLNVYTDSRYAFATAHIHGEIYRR







RGLLTSEGREIKNKSEILALLKALFLPKRLSIIHCLGHQKGDSAEARGNR







LADQAAREAAIKTPPDTSTLL





MLVAV_
MLVAV
P03356
derivative
3193
TLNLEDEYRLYETSAEPEVSPGSTWLSDFPQAWAETGGMGLAVRQAPLII


P03356_




PLKATSTPVSIKQYPMSQEAKLGIKPHIQRLLDQGILVPCQSPWNTPLLP


3mut




VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHRWYTVLD







LKDAFFCLRLHPTSQPLFAFEWRDPGMGISGQLTWTRLPQGFKNSPTLFN







EALHRDLADFRIQHPDLILLQYVDDILLAATSELDCQQGTRALLLTLGNL







GYRASAKKAQLCQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQL







REFLGTAGFCRLWIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQA







LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD







PVAAGWPPCLRMVAAIAVLRKDAGKLTMGQPLVILAPHAVEALVKQPPDR







WLSNARMTHYQAMLLDTDRVQFGPVVALNPATLLPLPEEGAPHDCLEILA







ETHGTRPDLTDQPIPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWAR







ALPAGTSAQRAELIALTQALKMAEGKRLNVYTDSRYAFATAHIHGEIYRR







RGWLTSEGREIKNKSEILALLKALFLPKRLSIIHCLGHQKGDSAEARGNR







LADQAAREAAIKTPPDTSTLL





MLVAV_
MLVAV
P0335
derivative
3194
TLNLEDEYRLYETSAEPEVSPGSTWLSDFPQAWAETGGMGLAVRQAPLII


P03356_




PLKATSTPVSIKQYPMSQEAKLGIKPHIQRLLDQGILVPCQSPWNTPLLP


3mutA




VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHRWYTVLD







LKDAFFCLRLHPTSQPLFAFEWRDPGMGISGQLTWTRLPQGFKNSPTLFN







EALHRDLADFRIQHPDLILLQYVDDILLAATSELDCQQGTRALLLTLGNL







GYRASAKKAQLCQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQL







REFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQA







LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD







PVAAGWPPCLRMVAAIAVLRKDAGKLTMGQPLVILAPHAVEALVKQPPDR







WLSNARMTHYQAMLLDTDRVQFGPVVALNPATLLPLPEEGAPHDCLEILA







ETHGTRPDLTDQPIPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWAR







ALPAGTSAQRAELIALTQALKMAEG







KRLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGREIKNKSEILALLKALF







LPKRLSIIHCLGHQKGDSAEARGNRLADQAAREAAIKTPPDTSTLL





MLVBM_
MLVBM
Q7SVK7
root
3195
LGIEDEYRLHETSTEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIP


Q7SVK7_3




LKATSTPVSIQQYPMSHEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPV


mutAWS




KKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDL







KDAFFCLRLHPTSQPLFAFEWRDPGMGISGQLTWTRLPQGFKNSPTLFNE







ALHRDLADFRIQHPDLILLQYVDDILLAATSELDCQQGTRALLQTLGDLG







YRASAKKAQICQKQVKYLGYLLREGQRWLTEARKETVMGQ







PVPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFSWGPDQQ







KAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRP







VAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVE







ALVKQPPDRWLSNARMTHYQAMLLDTDRVQFGPVVALNPATLLPLPEEGA







PHDCLEILAETHGTRPDLTDQPIPDADHTWYTDGSSFLQEGQRKAGAAVT







TETEVIWAGALPAGTSAQRAELIALTQALKMAEGKRLNVYTDSRYAFATA







HIHGEIYRRRGWLTSEGREIKNKSEILALLKALFLPKRLSIIHCLGHQKG







DSAEARGNRLADQAAREAAIKTPPDTSTLLI





MLVBM_
MLVBM
Q7SVK7
derivative
3196
TLGIEDEYRLHETSTEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLII


Q7SVK7




PLKATSTPVSIQQYPMSHEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP







VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLD







LKDAFFCLRLHPTSQPLFAFEWRDPGMGISGQLTWTRLPQGFKNSPTLFD







EALHRDLADFRIQHPDLILLQYVDDILLAATSELDCQQGTRALLQTLGDL







GYRASAKKAQICQKQVKYLGYLLREGQRWLTEARKETVMGQPVPKTPRQL







REFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFSWGPDQQKAYQEIKQA







LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD







PVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDR







WLSNARMTHYQAMLLDTDRVQFGPVVALNPATLLPLPEEGAPHDCLEILA







ETHGTRPDLTDQPIPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWAG







ALPAGTSAQRAELIALTQALKMAEGKRLNVYTDSRYAFATAHIHGEIYRR







RGLLTSEGREIKNKSEILALLKALFLPKRLSIIHCLGHQKGDSAEARGNR







LADQAAREAAIKTPPDTSTLL





MLVBM_
MLVBM
Q7SVK7
derivative
3197
TLGIEDEYRLHETSTEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLII


Q7SVK7_




PLKATSTPVSIQQYPMSHEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


3mut




VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLD







LKDAFFCLRLHPTSQPLFAFEWRDPGMGISGQLTWTRLPQGFKNSPTLFN







EALHRDLADFRIQHPDLILLQYVDDILLAATSELDCQQGTRALLQTLGDL







GYRASAKKAQICQKQVKYLGYLLREGQRWLTEARKETVMGQPVPKTPRQL







REFLGTAGFCRLWIPGFAEMAAPLYPLTKPGTLFSWGPDQQKAYQEIKQA







LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD







PVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDR







WLSNARMTHYQAMLLDTDRVQFGPVVALNPATLLPLPEEGAPHDCLEILA







ETHGTRPDLTDQPIPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWAG







ALPAGTSAQRAELIALTQALKMAEGKRLNVYTDSRYAFATAHIHGEIYRR







RGWLTSEGREIKNKSEILALLKALFLPKRLSIIHCLGHQKGDSAEARGNR







LADQAAREAAIKTPPDTSTLL





MLVBM_
MLVBM
Q7SVK7
root
3195
LGIEDEYRLHETSTEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIP


Q7SVK7_3




LKATSTPVSIQQYPMSHEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPV


mutAWS




KKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDL







KDAFFCLRLHPTSQPLFAFEWRDPGMGISGQLTWTRLPQGFKNSPTLFNE







ALHRDLADFRIQHPDLILLQYVDDILLAATSELDCQQGTRALLQTLGDLG







YRASAKKAQICQKQVKYLGYLLREGQRWLTEARKETVMGQPVPKTPRQLR







EFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFSWGPDQQKAYQEIKQAL







LTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDP







VAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRW







LSNARMTHYQAMLLDTDRVQFGPVVALNP







ATLLPLPEEGAPHDCLEILAETHGTRPDLTDQPIPDADHTWYTDGSSFLQ







EGQRKAGAAVTTETEVIWAGALPAGTSAQRAELIALTQALKMAEGKRLNV







YTDSRYAFATAHIHGEIYRRRGWLTSEGREIKNKSEILALLKALFLPKRL







SIIHCLGHQKGDSAEARGNRLADQAAREAAIKTPPDTSTLLI





MLVBM_
MLVBM
Q7SVK7
derivative
3196
TLGIEDEYRLHETSTEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLII


Q7SVK7




PLKATSTPVSIQQYPMSHEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP







VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLD







LKDAFFCLRLHPTSQPLFAFEWRDPGMGISGQLTWTRLPQGFKNSPTLFD







EALHRDLADFRIQHPDLILLQYVDDILLAATSELDCQQGTRALLQTLGDL







GYRASAKKAQICQKQVKYLGYLLREGQRWLTEARKETVMGQPVPKTPRQL







REFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFSWGPDQQKAYQEIKQA







LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD







PVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDR







WLSNARMTHYQAMLLDTDRVQFGPVVALNPATLLPLPEEGAPHDCLEILA







ETHGTRPDLTDQPIPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWAG







ALPAGTSAQRAELIALTQALKMAEGKRLNVYTDSRYAFATAHIHGEIYRR







RGLLTSEGREIKNKSEILALLKALFLPKRLSIIHCLGHQKGDSAEARGNR







LADQAAREAAIKTPPDTSTLL





MLVBM_
MLVBM
Q7SVK7
derivative
3197
TLGIEDEYRLHETSTEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLII


Q7SVK7_




PLKATSTPVSIQQYPMSHEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


3mut




VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLD







LKDAFFCLRLHPTSQPLFAFEWRDPGMGISGQLTWTRLPQGFKNSPTLFN







EALHRDLADFRIQHPDLILLQYVDDILLAATSELDCQQGTRALLQTLGDL







GYRASAKKAQICQKQVKYLGYLLREGQRWLTEARKETVMGQPVPKTPRQL







REFLGTAGFCRLWIPGFAEMAAPLYPLTKPGTLFSWGPDQQKAYQEIKQA







LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPATLLPLPEEGAPH







DCLEILAETHGTRPDLTDQPIPDADHTWYTDGSSFLQEGQRKAGAAVTTE







TEVIWAGALPAGTSAQRAELIALTQALKMAEGKRLNVYTDSRYAFATAHI







HGEIYRRRGWLTSEGREIKNKSEILALLKALFLPKRLSIIHCLGHQKGDS







AEARGNRLADQAAREAAIKTPPDTSTLL





MLVCB_
MLVCB
P08361
root
3198
TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLII


P08361




PLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP







VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLD







LKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFD







EALHRDLAGFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGDL







GYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPIPKTPRQL







REFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAFQEIKQA







LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD







PVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDR







WLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHDCLDILA







EAHGTRSDLMDQPLPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWAR







ALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRR







RGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGNSAEARGNR







MADQAAREVATRETPETSTLL





MLVCB_
MLVCB
P08361
derivative
3199
TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLII


P08361_3




PLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


mut




VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLD







LKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFN







EALHRDLAGFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGDL







GYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPIPKTPRQL







REFLGTAGFCRLWIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAFQEIKQA







LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLG







PATLLPLPEEGLQHDCLDILAEAHGTRSDLMDQPLPDADHTWYTDGSSFL







QEGQRKAGAAVTTETEVIWARALPAGTSAQRAELIALTQALKMAEGKKLN







VYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKR







LSIIHCPGHQKGNSAEARGNRMADQAAREVATRETPETSTLL





MLVCB_
MLVCB


3200
TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLII


P08361_3




PLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


mutA




VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLD







LKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFN







EALHRDLAGFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGDL







GYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPIPKTPRQL







REFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAFQEIKQA







LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPATLLPLPEEGLQH







DCLDILAEAHGTRSDLMDQPLPDADHTWYTDGSSFLQEGQRKAGAAVTTE







TEVIWARALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHI







HGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGNS







AEARGNRMADQAAREVATRETPETSTLL





MLVF5_
MLVF5
P26810
root
3201
TLNIEDEYRLHETSKGPDVPLGSTWLSDFPQAWAETGGMGLAFRQAPLII


P26810




SLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP







VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLD







LKDAFFCLRLHPTSQSLFAFEWKDPEMGISGQLTWTRLPQGFKNSPTLFD







EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGDL







GYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQL







REFLGTAGLCRLWIPGFAEMAAPLYPLTKTGTLFKWGPDQQKAYQEIKQA







LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD







PVAAGWPPCLRMVAAIAVLTKDVGKLTMGQPLVILAPHAVEALVKQPPDR







WLSNARMTHYQALLLDTDRVQFGPIVALNPATLLPLPEEGLQHDCLDILA







EAHGTRPDLTDQPLPDADHTWYTDGSSFLQEGQRRAGAAVTTETEVIWAK







ALPAGTSAQRAELIALTQALKMAAGKKLNVYTDSRYAFATAHIHGEIYRR







RGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGNHAEARGNR







MADQAAREVATRETPETSTLL





MLVF5_
MLVF5
P26810
derivative
3202
TLNIEDEYRLHETSKGPDVPLGSTWLSDFPQAWAETGGMGLAFRQAPLII


P26810_




SLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


3mut




VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLD







LKDAFFCLRLHPTSQSLFAFEWKDPEMGISGQLTWTRLPQGFKNSPTLFN







EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGDL







GYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQL







REFLGTAGLCRLWIPGFAEMAAPLYPLTKPGTLFKWGPDQQKAYQEIKQA







LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD







PVAAGWPPCLRMVAAIAVLTKDVGKLTMGQPLVILAPHAVEALVKQPPDR







WLSNARMTHYQALLLDTDRVQFGPIVALNPATLLPLPEEGLQHDCLDILA







EAHGTRPDLTDQPLPDADHTWYTDGSSFLQEGQRRAGAAVTTETEVIWAK







ALPAGTSAQRAELIALTQALKMAAGKKLNVYTDSRYAFATAHIHGEIYRR







RGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGNHAEARGNR







MADQAAREVATRETPETSTLL





MLVF5_
MLVF5
P26810
derivative
3203
TLNIEDEYRLHETSKGPDVPLGSTWLSDFPQAWAETGGMGLAFRQAPLII


P26810_3




SLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


mutA




VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLD







LKDAFFCLRLHPTSQSLFAFEWKDPEMGISGQLTWTRLPQGFKNSPTLFN







EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGDL







GYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQL







REFLGKAGLCRLFIPGFAEMAAPLYPLTKPGTLFKWGPDQQKAYQEIKQA







LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD







PVAAGWPPCLRMVAAIAVLTKDVGKLTMGQPLVILAPHAVEALVKQPPDR







WLSNARMTHYQALLLDTDRVQFGPIVALNP







ATLLPLPEEGLQHDCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSFLQ







EGQRRAGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAAGKKLNV







YTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRL







SIIHCPGHQKGNHAEARGNRMADQAAREVATRETPETSTLL





MLVFF_
MLVFF
P26809
root
3204
TLNIEDEYRLHETSKGPDVPLGSTWLSDFPQAWAETGGMGLAVRQAPLII


P26809




PLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP







VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLD







LKDAFFCLRLHPTSQSLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFD







EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGDL







GYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQL







REFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFEWGPDQQKAYQEIKQA







LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD







PVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDR







WLSNARMTHYQALLLDTDRVQFGPIVALNPATLLPLPEEGLQHDCLDILA







EAHGTRPDLTDQPLPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVVWAK







ALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRR







RGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGNRAEARGNR







MADQAAREVATRETPETSTLL





MLVFF_
MLVFF
P26809
derivative
3205
TLNIEDEYRLHETSKGPDVPLGSTWLSDFPQAWAETGGMGLAVRQAPLII


P26809_




PLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


3mut




VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLD







LKDAFFCLRLHPTSQSLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFN







EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGDL







GYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQL







REFLGTAGFCRLWIPGFAEMAAPLYPLTKPGTLFEWGPDQQKAYQEIKQA







LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD







PVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDR







WLSNARMTHYQALLLDTDRVQFGPIVALNPATLLPLPEEGLQHDCLDILA







EAHGTRPDLTDQPLPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVVWAK







ALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRR







RGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGNRAEARGNR







MADQAAREVATRETPETSTLL





MLVFF_
MLVEF
P26809
derivative
3206
TLNIEDEYRLHETSKGPDVPLGSTWLSDFPQAWAETGGMGLAVRQAPLII


P26809_




PLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


3mutA




VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLD







LKDAFFCLRLHPTSQSLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFN







EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGDL







GYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQL







REFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFEWGPDQQKAYQEIKQA







LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD







PVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDR







WLSNARMTHYQALLLDTDRVQFGPIVALN







PATLLPLPEEGLQHDCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSFL







QEGQRKAGAAVTTETEVVWAKALPAGTSAQRAELIALTQALKMAEGKKLN







VYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKR







LSIIHCPGHQKGNRAEARGNRMADQAAREVATRETPETSTLL





MLVMS_
MLVMS
P03355
root
3207
TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLII


P03355_




PLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


PLV919




VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLD







LKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFN







EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNL







GYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQL







REFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQA







LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKL







GPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVIL







APHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALNPATLLP







LPEEGLQHNCLDILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRK







AGAAVTTETEVIWAKALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSR







YAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHC







PGHQKGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTA







DGSEFE





MLVMS_
MLVMS
P03355
derivative
1548
TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLII


P03355




PLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP







VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLD







LKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFD







EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNL







GYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQL







REFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA







LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD







PVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDR







WLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILA







EAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAK







ALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRR







RGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNR







MADQAARKAAITETPDTSTLL





MLVMS_
MLVMS
P03355
derivative
3208
TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLII


P03355_3




PLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


mut




VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLD







LKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFN







EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNL







GYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQL







REFLGTAGFCRLWIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQA







LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD







PVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDR







WLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILA







EAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAK







ALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRR







RGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNR







MADQAARKAAITETPDTSTLL





MLVMS_
MLVMS
P03355
derivative
3209
TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLII


P03355_




PLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


3mutA_




VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLD


WS




LKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFN







EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNL







GYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQL







REFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQA







LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD







PVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDR







WLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILA







EAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAK







ALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRR







RGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNR







MADQAARKAAITETPDTSTLL





MLVRD_
MLVRD
P11227
root
3210
TLNIEDEYRLHEISTEPDVSPGSTWLSDFPQAWAETGGMGLAVRQAPLII


P11227




PLKATSTPVSIKQYPMSQEAKLGIKPHIQRLLDQGILVPCQSPWNTPLL







PVKKPGTNDYRPVQGLREVNKRVEDIHPTVPNPYNLLSGLPTSHRWYTVL







DLKDAFFCLRLHPTSQPLFASEWRDPGMGISGQLTWTRLPQGFKNSPTLF







DEALHRGLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLKTLGN







LGYRASAKKAQICQKQVKYLGYLLREGQRWLTEARKETVMGQPTPKTPRQ







LREFLGTAGFCRLWIPRFAEMAAPLYPLTKTGTLFNWGPDQQKAYHEIKQ







ALLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKL







DPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPD







RWLSNARMTHYQAMLLDTDRVQFGPVVALNPATLLPLPEEGAPHDCLEIL







AETHGTEPDLTDQPIPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWA







RALPAGTSAQRAELIALTQALKMAEGKRLNVYTDSRYAFATAHIHGEIYK







RRGLLTSEGREIKNKSEILALLKALFLPKRLSIIHCLGHQKGDSAEARGN







RLADQAAREAAIKTPPDTSTLL





MLVRD_
MLVRD
P11227
derivative
3211
TLNIEDEYRLHEISTEPDVSPGSTWLSDFPQAWAETGGMGLAVRQAPLII


P11227_




PLKATSTPVSIKQYPMSQEAKLGIKPHIQRLLDQGILVPCQSPWNTPLLP


3mut




VKKPGTNDYRPVQGLREVNKRVEDIHPTVPNPYNLLSGLPTSHRWYTVLD







LKDAFFCLRLHPTSQPLFASEWRDPGMGISGQLTWTRLPQGFKNSPTLFN







EALHRGLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLKTLGNL







GYRASAKKAQICQKQVKYLGYLLREGQRWLTEARKETVMGQPTPKTPRQL







REFLGTAGFCRLWIPRFAEMAAPLYPLTKPGTLFNWGPDQQKAYHEIKQA







LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD







PVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDR







WLSNARMTHYQAMLLDTDRVQFGPVVALNPATLLPLPEEGAPHDCLEILA







ETHGTEPDLTDQPIPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWAR







ALPAGTSAQRAELIALTQALKMAEGKRLNVYTDSRYAFATAHIHGEIYKR







RGWLTSEGREIKNKSEILALLKALFLPKRLSIIHCLGHQKGDSAEARGNR







LADQAAREAAIKTPPDTSTLL





MLVRD_
MLVRD
P11227
derivative
3212
TLNIEDEYRLHEISTEPDVSPGSTWLSDFPQAWAETGGMGLAVRQAPLII


P11227_




PLKATSTPVSIKQYPMSQEAKLGIKPHIQRLLDQGILVPCQSPWNTPLLP


3mutA




VKKPGTNDYRPVQGLREVNKRVEDIHPTVPNPYNLLSGLPTSHRWYTVLD







LKDAFFCLRLHPTSQPLFASEWRDPGMGISGQLTWTRLPQGFKNSPTLFN







EALHRGLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLKTLGNL







GYRASAKKAQICQKQVKYLGYLLREGQRWLTEARKETVMGQPTPKTPRQL







REFLGKAGFCRLFIPRFAEMAAPLYPLTKPGTLFNWGPDQQKAYHEIKQA







LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD







PVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDR







WLSNARMTHYQAMLLDTDRVQFGPVVALNPATLLPLPEEGAPHDCLEILA







ETHGTEPDLTDQPIPDADHTWYTDGSSFLQEGQRKAGAAVTTETEVIWAR







ALPAGTSAQRAELIALTQALKMAEGKRLNVYTDSRYAFATAHIHGEIYKR







RGWLTSEGREIKNKSEILALLKALFLPKRLSIIHCLGHQKGDSAEARGNR







LADQAAREAAIKTPPDTSTLL





MMTVB_
MMTVB
P03365
root
3213
VQEISDSRPMLHIYLNGRRFLGLLDTGADKTCIAGRDWPANWPIHQTESS


P03365_WS




LQGLGMACGVARSSQPLRWQHEDKSGIIHPFVIPTLPFTLWGRDIMKDIK







VRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQWPLKQEKLQA







LQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLLQDLRAVNATMHD







MGALQPGLPSPVAVPKGWEIIIIDLQDCFFNIKLHPEDCKRFAFSVPSPN







FKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVRDKYQDSYIVHYMDD







ILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLKYLGTHIQGD







SVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTTGELKPLFEILNG







DSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSLCILKTEYTP







TACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIKGRHRSKELFSKDPD







YIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPLLTFTLQTAI







IFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKENTQNTAQQAE







IVAVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTKIYTELKHLQ







RLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILTA





MMTVB_
MMTVB
P03365
derivative
3214
WVQEISDSRPMLHIYLNGRRFLGLLNTGADKTCIAGRDWPANWPIHQTES


P03365




SLQGLGMACGVARSSQPLRWQHEDKSGIIHPFVIPTLPFTLWGRDIMKDI







KVRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQWPLKQEKLQ







ALQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLLQDLRAVNATMH







DMGALQPGLPSPVAVPKGWEIIIIDLQDCFFNIKLHPEDCKRFAFSVPSP







NFKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVRDKYQDSYIVHYMD







DILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLKYLGTHIQG







DSVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTTGELKPLFEILN







GDSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSLCILKTEYT







PTACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIKGRHRSKELFSKDP







DYIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPLLTFTLQTA







IIFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKENTQNTAQQA







EIVAVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTKIYTELKHL







QRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILT





MMTVB_
MMTVB
P03365
derivative
3215
GRDIMKDIKVRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQW


P03365-




PLKQEKLQALQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLLQDL


Pro




RAVNATMHDMGALQPGLPSPVAVPKGWEIIIIDLQDCFFNIKLHPEDCKR







FAFSVPSPNFKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVRDKYQD







SYIVHYMDDILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLK







YLGTHIQGDSVSYQKLQIRTDKLRTLNDFQKLLGNINWIR







PFLKLTTGELKPLFEILNGDSNPISTRKLTPEACKALQLMNERLSTARVK







RLDLSQPWSLCILKTEYTPTACLWQDGVVEWIHLPHISPKVITPYDIFCT







QLIIKGRHRSKELFSKDPDYIVVPYTKVQFDLLLQEKEDWPISLLGFLGE







VHFHLPKDPLLTFTLQTAIIFPHMTSTTPLEKGIVIFTDGSANGRSVTYI







QGREPIIKENTQNTAQQAEIVAVITAFEEVSQPFNLYTDSKYVTGLFPEI







ETATLSPRTKIYTELKHLQRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAY







ADSLTRILT





MMTVB_
MMTVB
P03365
derivative
3216
GRDIMKDIKVRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQW


P03365-Pro_




PLKQEKLQALQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLLQDL


2mut




RAVNATMHDMGALQPGLPSPVAVPKGWEIIIIDLQDCFFNIKLHPEDCKR







FAFSVPSPNFKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVRDKYQD







SYIVHYMDDILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLK







YLGTHIQGDSVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTTGEL







KPLFEILNPDSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSL







CILKTEYTPTACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIKGRHRS







KELFSKDPDYIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPL







LTFTLQTAIIFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKEN







TQNTAQQAEIVAVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTK







IYTELKHLQRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILT





MMTVB_
MMTVB
P03365
derivative
3217
GRDIMKDIKVRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQW


P03365-




PLKQEKLQALQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLLQDL


Pro_




RAVNATMHDMGALQPGLPSPVAPPKGWEIIIIDLQDCFFNIKLHPEDCKR


2mutB




FAFSVPSPNFKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVRDKYQD







SYIVHYMDDILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLK







YLGTHIQGDSVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTTGEL







KPLFEILNPDSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSL







CILKTEYTPTACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIKGRHRS







KELFSKDPDYIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPL







LTFTLQTAIIFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKEN







TQNTAQQAEIVAVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTK







IYTELKHLQRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILT





MMTVB_
MMTVB
P03365
derivative
3218
WVQEISDSRPMLHIYLNGRRFLGLLNTGADKTCIAGRDWPANWPIHQTES


P03365_




SLQGLGMACGVARSSQPLRWQHEDKSGIIHPFVIPTLPFTLWGRDIMRAV


2mut




NATMHDMGALQPGLPSPVAVPKGWEIIIIDLQDCFFNIKLHPEDCKRFAF







SVPSPNFKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVRDKYQDSYI







VHYMDDILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLKYLG







THIQGDSVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTTGELKPL







FEILNPDSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSLCIL







KTEYTPTACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIKGRHRSKEL







FSKDPDYIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPLLTF







TLQTAIIFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKENTQN







TAQQAEIVAVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTKIYT







ELKHLQRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILT





MMTVB_
MMTVB
P03365
derivative
3219
WVQEISDSRPMLHIYLNGRRFLGLLNTGADKTCIAGRDWPANWPIHQTES


P03365_




SLQGLGMACGVARSSQPLRWQHEDKSGIIHPFVIPTLPFTLWGRDIMKDI


2mutB




KVRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQWPLKQEKLQ







ALQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLLQDLRAVNATMH







DMGALQPGLPSPVAPPKGWEIIIIDLQDCFFNIKLHPEDCKRFAFSVPSP







NFKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVRDKYQDSYIVHYMD







DILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLKYLGTHIQG







DSVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTTGELKPLFEILN







PDSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSLCILKTEYT







PTACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIKGRHRSKELFSKDP







DYIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPLLTFTLQTA







IIFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKENTQNTAQQA







EIVAVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTKIYTELKHL







QRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILT





MMTVB_
MMTVB
P03365
derivative
3220
VQEISDSRPMLHIYLNGRRFLGLLDTGADKTCIAGRDWPANWPIHQTESS


P03365_




LQGLGMACGVARSSQPLRWQHEDKSGIIHPFVIPTLPFTLWGRDIMKDIK


2mutB_




VRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQWPLKQEKLQA


WS




LQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLLQDLRAVNATMHD







MGALQPGLPSPPAVPKGWEIIIIDLQDCFFNIKLHPEDCKRFAFSVPSPN







FKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVRDKYQDSYIVHYMDD







ILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLKYLGTHIQGD







SVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTTGELKPLFEILNP







DSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSLCILKTEYTP







TACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIKGRHRSKELFSKDPD







YIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPLLTFTLQTAI







IFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKENTQNTAQQAE







IVAVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTKIYTELKHLQ







RLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILTA





MMTVB_
MMTVB
P03365
derivative
3221
VQEISDSRPMLHIYLNGRRFLGLLDTGADKTCIAGRDWPANWPIHQTESS


P03365_




LQGLGMACGVARSSQPLRWQHEDKSGIIHPFVIPTLPFTLWGRDIMKDIK


2mut_




VRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQWPLKQEKLQA


WS




LQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLLQDLRAVNATMHD







MGALQPGLPSPVAVPKGWEIIIIDLQDCFFNIKLHPEDCKRFAFSVPSPN







FKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVRDKYQDSYIVHYMDD







ILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLKYLGTHIQGD







SVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTTGELKPLFEILNP







DSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSLCILKTEYTP







TACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIKGRHRSKELFSKDPD







YIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPLLTFTLQTAI







IFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREP







IIKENTQNTAQQAEIVAVITAFEEVSQPFNLYTDSKYVTGLFPEIETATL







SPRTKIYTELKHLQRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLT 







RILTA





MMTVB_
MMTVB
P03365
root
3213
VQEISDSRPMLHIYLNGRRFLGLLDTGADKTCIAGRDWPANWPIHQTESS


P03365_




LQGLGMACGVARSSQPLRWQHEDKSGIIHPFVIPTLPFTLWGRDIMKDIK


WS




VRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQWPLKQEKLQA







LQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLLQDLRAVNATMHD







MGALQPGLPSPVAVPKGWEIIIIDLQDCFFNIKLHPEDCKRFAFSVPSPN







FKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVRDKYQDSYIVHYMDD







ILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLKYLGTHIQGD







SVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTTGELKPLFEILNG







DSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSLCILKTEYTP







TACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIKGRHRSKELFSKDPD







YIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPLLTFTLQTAI







IFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKENTQNTAQQAE







IVAVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTKIYTELKHLQ







RLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILTA





MMTVB_
MMTVB
P03365
derivative
3214
WVQEISDSRPMLHIYLNGRRFLGLLNTGADKTCIAGRDWPANWPIHQTES


P03365




SLQGLGMACGVARSSQPLRWQHEDKSGIIHPFVIPTLPFTLWGRDIMKDI







KVRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQWPLKQEKLQ







ALQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLLQDLRAVNATMH







DMGALQPGLPSPVAVPKGWEIIIIDLQDCFFNIKLHPEDCKRFAFSVPSP







NFKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVRDKYQDSYIVHYMD







DILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLKYLGTHIQG







DSVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTTGELKPLFEILN







GDSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSLCILKTEYT







PTACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIKGRHRSKELFSKDP







DYIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPLLTFTLQTA







IIFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKENTQNTAQQA







EIVAVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTKIYTELKHL







QRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILT





MMTVB_
MMTVB
P03365
derivative
3215
GRDIMKDIKVRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQW


P03365-




PLKQEKLQALQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLLQDL


Pro




RAVNATMHDMGALQPGLPSPVAVPKGWEIIIIDLQDCFFNIKLHPEDCKR







FAFSVPSPNFKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVRDKYQD







SYIVHYMDDILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLK







YLGTHIQGDSVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTTGEL







KPLFEILNGDSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSL







CILKTEYTPTACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIKGRHRS







KELFSKDPDYIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPL







LTFTLQTAIIFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKEN







TQNTAQQAEIVAVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTK







IYTELKHLQRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILT





MMTVB_
MMTVB
P03365
derivative
3216
GRDIMKDIKVRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQW


P03365-




PLKQEKLQALQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLLQDL


Pro_




RAVNATMHDMGALQPGLPSPVAVPKGWEIIIIDLQDCFFNIKLHPEDCKR


2mut




FAFSVPSPNFKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVRDKYQD







SYIVHYMDDILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLK







YLGTHIQGDSVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTTGEL







KPLFEILNPDSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSL







CILKTEYTPTACLWQDGVVEWIHLPHISPKVITPYDIFCT







QLIIKGRHRSKELFSKDPDYIVVPYTKVQFDLLLQEKEDWPISLLGFLGE







VHFHLPKDPLLTFTLQTAIIFPHMTSTTPLEKGIVIFTDGSANGRSVTYI







QGREPIIKENTQNTAQQAEIVAVITAFEEVSQPFNLYTDSKYVTGLFPEI







ETATLSPRTKIYTELKHLQRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAY







ADSLTRILT





MMTVB_
MMTVB
P03365
derivative
3217
GRDIMKDIKVRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQW


P03365-




PLKQEKLQALQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLLQDL


Pro_




RAVNATMHDMGALQPGLPSPVAPPKGWEIIIIDLQDCFFNIKLHPEDCKR


2mutB




FAFSVPSPNFKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVRDKYQD







SYIVHYMDDILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLK







YLGTHIQGDSVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTTGEL







KPLFEILNPDSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSL







CILKTEYTPTACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIKGRHRS







KELFSKDPDYIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPL







LTFTLQTAIIFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKEN







TQNTAQQAEIVAVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTK







IYTELKHLQRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILT





MMTVB_
MMTVB
P03365
derivative
3218
WVQEISDSRPMLHIYLNGRRFLGLLNTGADKTCIAGRDWPANWPIHQTES


P03365_




SLQGLGMACGVARSSQPLRWQHEDKSGIIHPFVIPTLPFTLWGRDIMKDI


2mut




KVRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQWPLKQEKLQ







ALQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLLQDLRAVNATMH







DMGALQPGLPSPVAVPKGWEIIIIDLQDCFFNIKLHPEDCKRFAFSVPSP







NFKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVRDKYQDSYIVHYMD







DILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLKYLGTHIQG







DSVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTTGELKPLFEILN







PDSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSLCILKTEYT







PTACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIKGRHRSKELFSKDP







DYIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPLLTFTLQTA







IIFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKENTQNTAQQA







EIVAVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTKIYTELKHL







QRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILT





MMTVB_
MMTVB
P03365
derivative
3219
WVQEISDSRPMLHIYLNGRRFLGLLNTGADKTCIAGRDWPANWPIHQTES


P03365_




SLQGLGMACGVARSSQPLRWQHEDKSGIIHPFVIPTLPFTLWGRDIMKDI


2mutB




KVRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNOWPLKQEKLQ







ALQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLLQDLRAVNATMH







DMGALQPGLPSPVAPPKGWEIIIIDLQDCFFNIKLHPEDCKRFAFSVPSP







NFKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVRDKYQDSYIVHYMD







DILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLKYLGTHIQG







DSVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTTGELKPLFEILN







PDSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSLCILKTEYT







PTACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIK







GRHRSKELFSKDPDYIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHL







PKDPLLTFTLQTAIIFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREP







IIKENTQNTAQQAEIVAVITAFEEVSQPFNLYTDSKYVTGLFPEIETATL







SPRTKIYTELKHLQRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLT







RILT





MMTVB_
MMTVB
P03365
derivative
3220
VQEISDSRPMLHIYLNGRRFLGLLDTGADKTCIAGRDWPANWPIHQTESS


P03365_




LQGLGMACGVARSSQPLRWQHEDKSGIIHPFVIPTLPFTLWGRDIMKDIK


2mutB_




VRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQWPLKQEKLQA


WS




LQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLLQDLR







AVNATMHDMGALQPGLPSPPAVPKGWEIIIIDLQDCFFNIKLHPEDCKRF







AFSVPSPNFKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVRDKYQDS







YIVHYMDDILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLKY







LGTHIQGDSVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTTGELK







PLFEILNPDSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSLC







ILKTEYTPTACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIKGRHRSK







ELFSKDPDYIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPLL







TFTLQTAIIFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKENT







QNTAQQAEIVAVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTKI







YTELKHLQRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILTA





MMTVB_
MMTVB
P03365
derivative
3221
VQEISDSRPMLHIYLNGRRFLGLLDTGADKTCIAGRDWPANWPIHQTESS


P03365_




LQGLGMACGVARSSQPLRWQHEDKSGIIHPFVIPTLPFTLWGRDIMKDIK


2mut_




VRLMTDSPDDSQDLMIGAIESNLFADQISWKSDQPVWLNQWPLKQEKLQA


WS




LQQLVTEQLQLGHLEESNSPWNTPVFVIKKKSGKWRLLQDLRAVNATMHD







MGALQPGLPSPVAVPKGWEIIIIDLQDCFFNIKLHPEDCKRFAFSVPSPN







FKRPYQRFQWKVLPQGMKNSPTLCQKFVDKAILTVRDKYQDSYIVHYMDD







ILLAHPSRSIVDEILTSMIQALNKHGLVVSTEKIQKYDNLKYLGTHIQGD







SVSYQKLQIRTDKLRTLNDFQKLLGNINWIRPFLKLTTGELKPLFEILNP







DSNPISTRKLTPEACKALQLMNERLSTARVKRLDLSQPWSLCILKTEYTP







TACLWQDGVVEWIHLPHISPKVITPYDIFCTQLIIKGRHRSKELFSKDPD







YIVVPYTKVQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPLLTFTLQTAI







IFPHMTSTTPLEKGIVIFTDGSANGRSVTYIQGREPIIKENTQNTAQQAE







IVAVITAFEEVSQPFNLYTDSKYVTGLFPEIETATLSPRTKIYTELKHLQ







RLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYADSLTRILTA





MPMV_
MPMV
P07572
root
3222
LTAAIDILAPQQCAEPITWKSDEPVWVDQWPLTNDKLAAAQQLVQEQLEA


P07572




GHITESSSPWNTPIFVIKKKSGKWRLLQDLRAVNATMVLMGALQPGLPSP







VAIPQGYLKIIIDLKDCFFSIPLHPSDQKRFAFSLPSTNFKEPMQRFQWK







VLPQGMANSPTLCQKYVATAIHKVRHAWKQMYIIHYMDDILIAGKDGQQV







LQCFDQLKQELTAAGLHIAPEKVQLQDPYTYLGFELNGPKITNQKAVIRK







DKLQTLNDFQKLLGDINWLRPYLKLTTGDLKPLFDTLKGDSDPNSHRSLS







KEALASLEKVETAIAEQFVTHINYSLPLIFLIFNTALTPTGLFWQDNPIM







WIHLPASPKKVLLPYYDAIADLIILGRDHSKKYFGIEPSTIIQPYSKSQI







DWLMQNTEMWPIACASFVGILDNHYPPNKLIQFCKLHTFVFPQIISKTPL







NNALLVFTDGSSTGMAAYTLTDTTIKFQTNLNSAQLVELQALIAVLSAFP







NQPLNIYTDSAYLAHSIPLLETVAQIKHISETAKLFLQCQQLIYNRSIPF







YIGHVRAHSGLPGPIAQGNQRADLATKIVASNINT





MPMV_
MPMV
P07572
derivative
3223
LTAAIDILAPQQCAEPITWKSDEPVWVDQWPLTNDKLAAAQQLVQEQLEA


P07572_




GHITESSSPWNTPIFVIKKKSGKWRLLQDLRAVNATMVLMGALQPGLPSP


2mut




VAIPQGYLKIIIDLKDCFFSIPLHPSDQKRFAFSLPSTNFKEPMQRFQWK







VLPQGMANSPTLCQKYVATAIHKVRHAWKQMYIIHYMDDILIAGKDGQQV







LQCFDQLKQELTAAGLHIAPEKVQLQDPYTYLGFELNGPKITNQKAVIRK







DKLQTLNDFQKLLGDINWLRPYLKLTTGDLKPLFDTLKPDSDPNSHRSLS







KEALASLEKVETAIAEQFVTHINYSLPLIFLIFNTALTPTGLFWQDNPIM







WIHLPASPKKVLLPYYDAIADLIILGRDHSKKYFGIEPSTIIQPYSKSQI







DWLMQNTEMWPIACASFVGILDNHYPPNKLIQFCKLHTFVFPQIISKTPL







NNALLVFTDGSSTGMAAYTLTDTTIKFQTNLNSAQLVELQALIAVLSAFP







NQPLNIYTDSAYLAHSIPLLETVAQIKHISETAKLFLQCQQLIYNRSIPF







YIGHVRAHSGLPGPIAQGNQRADLATKIVASNINT





MPMV_
MPMV
P07572
derivative
3224
LTAAIDILAPQQCAEPITWKSDEPVWVDQWPLTNDKLAAAQQLVQEQLEA


P07572_




GHITESSSPWNTPIFVIKKKSGKWRLLQDLRAVNATMVLMGALQPGLPSP


2mutB




VAPPQGYLKIIIDLKDCFFSIPLHPSDQKRFAFSLPSTNFKEPMQRFQWK







VLPQGMANSPTLCQKYVATAIHKVRHAWKQMYIIHYMDDILIA







GKDGQQVLQCFDQLKQELTAAGLHIAPEKVQLQDPYTYLGFELNGPKITN







QKAVIRKDKLQTLNDFQKLLGDINWLRPYLKLTTGDLKPLFDTLKPDSDP







NSHRSLSKEALASLEKVETAIAEQFVTHINYSLPLIFLIFNTALTPTGLF







WQDNPIMWIHLPASPKKVLLPYYDAIADLIILGRDHSKKYFGIEPSTIIQ







PYSKSQIDWLMQNTEMWPIACASFVGILDNHYPPNKLIQFCKLHTFVFPQ







IISKTPLNNALLVFTDGSSTGMAAYTLTDTTIKFQTNLNSAQLVELQALI







AVLSAFPNQPLNIYTDSAYLAHSIPLLETVAQIKHISETAKLFLQCQQLI







YNRSIPFYIGHVRAHSGLPGPIAQGNQRADLATKIVASNINT





PERV_
PERV
Q4VFZ2
root
3225
LDDEYRLYSPLVKPDQNIQFWLEQFPQAWAETAGMGLAKQVPPQVIQLKA


Q4VFZ2_




SATPVSVRQYPLSKEAQEGIRPHVQRLIQQGILVPVQSPWNTPLLPVRKP


3mutA_




GTNDYRPVQDLREVNKRVQDIHPTVPNPYNLLCALPPQRSWYTVLDLKDA


WS




FFCLRLHPTSQPLFAFEWRDPGTGRTGQLTWTRLPQGFKNSPTIFNEALH







RDLANFRIQHPQVTLLQYVDDLLLAGATKQDCLEGTKALLLELSDLGYRA







SAKKAQICRREVTYLGYSLRDGQRWLTEARKKTVVQIPAPTTAKQVREFL







GKAGFCRLFIPGFATLAAPLYPLTKPKGEFSWAPEHQKAFDAIKKALLSA







PALALPDVTKPFTLYVDERKGVARGVLTQTLGPWRRPVAYLSKKLDPVAS







GWPVCLKAIAAVAILVKDADKLTLGQNITVIAPHALENIVRQPPDRWMTN







ARMTHYQSLLLTERVTFAPPAALNPATLLPEETDEPVTHDCHQLLIEETG







VRKDLTDIPLTGEVLTWFTDGSSYVVEGKRMAGAAVVDGTRTIWASSLPE







GTSAQKAELMALTQALRLAEGKSINIYTDSRYAFATAHVHGAIYKQRGWL







TSAGREIKNKEEILSLLEALHLPKRLAIIHCPGHQKAKDPISRGNQMADR







VAKQAAQGVNLLP





PERV_
PERV
Q4VFZ2
derivative
3226
TLQLDDEYRLYSPLVKPDQNIQFWLEQFPQAWAETAGMGLAKQVPPQVIQ


Q4VFZ2




LKASATPVSVRQYPLSKEAQEGIRPHVQRLIQQGILVPVQSPWNTPLLPV







RKPGTNDYRPVQDLREVNKRVQDIHPTVPNPYNLLCALPPQRSWYTVLDL







KDAFFCLRLHPTSQPLFAFEWRDPGTGRTGQLTWTRLPQGFKNSPTIFDE







ALHRDLANFRIQHPQVTLLQYVDDLLLAGATKQDCLEGTKALLLELSDLG







YRASAKKAQICRREVTYLGYSLRDGQRWLTEARKKTVVQIPAPTTAKQVR







EFLGTAGFCRLWIPGFATLAAPLYPLTKEKGEFSWAPEHQKAFDAIKKAL







LSAPALALPDVTKPFTLYVDERKGVARGVLTQTLGPWRRPVAYLSKKLDP







VASGWPVCLKAIAAVAILVKDADKLTLGQNITVIAPHALENIVRQPPDRW







MTNARMTHYQSLLLTERVTFAPPAALNPATL







LPEETDEPVTHDCHQLLIEETGVRKDLTDIPLTGEVLTWFTDGSSYVVEG







KRMAGAAVVDGTRTIWASSLPEGTSAQKAELMALTQALRLAEGKSINIYT







DSRYAFATAHVHGAIYKQRGLLTSAGREIKNKEEILSLLEALHLPKRLAI







IHCPGHQKAKDPISRGNQMADRVAKQAAQGVNLL





PERV_
PERV
Q4VFZ2
derivative
3227
TLQLDDEYRLYSPLVKPDQNIQFWLEQFPQAWAETAGMGLAKQVPPQVIQ


Q4VFZ2_




LKASATPVSVRQYPLSKEAQEGIRPHVQRLIQQGILVPVQSPWNTPLLPV


3mut




RKPGTNDYRPVQDLREVNKRVQDIHPTVPNPYNLLCALPPQRSWYTVLDL







KDAFFCLRLHPTSQPLFAFEWRDPGTGRTGQLTWTRLPQGFKNSPTIFNE







ALHRDLANFRIQHPQVTLLQYVDDLLLAGATKQDCLEGTKALLLELSDLG







YRASAKKAQICRREVTYLGYSLRDGQRWLTEARKKTVVQIPAPTTAKQVR







EFLGTAGFCRLWIPGFATLAAPLYPLTKPKGEFSWAPEHQKAFDAIKKAL







LSAPALALPDVTKPFTLYVDERKGVARGVLTQTLGPWRRPVAYLSKKLDP







VASGWPVCLKAIAAVAILVKDADKLTLGQNITVIAPHALENIVRQPPDRW







MTNARMTHYQSLLLTERVTFAPPAALNPATLLPEETDEPVTHDCHQLLIE







ETGVRKDLTDIPLTGEVLTWFTDGSSYVVEGKRMAGAAVVDGTRTIWASS







LPEGTSAQKAELMALTQALRLAEGKSINIYTDSRYAFATAHVHGAIYKQR







GWLTSAGREIKNKEEILSLLEALHLPKRLAIIHCPGHQKAKDPISRGNQM







ADRVAKQAAQGVNLL





PERV_
PERV
Q4VFZ2
root
3225
LDDEYRLYSPLVKPDQNIQFWLEQFPQAWAETAGMGLAKQVPPQVIQLKA


Q4VFZ2_




SATPVSVRQYPLSKEAQEGIRPHVQRLIQQGILVPVQSPWNTPLLPVRKP


3mutA_




GTNDYRPVQDLREVNKRVQDIHPTVPNPYNLLCALPPQRSWYTVLDLKDA


WS




FFCLRLHPTSQPLFAFEWRDPGTGRTGQLTWTRLPQGFKNSP







TIFNEALHRDLANFRIQHPQVTLLQYVDDLLLAGATKQDCLEGTKALLLE







LSDLGYRASAKKAQICRREVTYLGYSLRDGQRWLTEARKKTVVQIPAPTT







AKQVREFLGKAGFCRLFIPGFATLAAPLYPLTKPKGEFSWAPEHQKAFDA







IKKALLSAPALALPDVTKPFTLYVDERKGVARGVLTQTLGPWRRPVAYLS







KKLDPVASGWPVCLKAIAAVAILVKDADKLTLGQNITVIAPHALENIVRQ







PPDRWMTNARMTHYQSLLLTERVTFAPPAALNPATLLPEETDEPVTHDCH







QLLIEETGVRKDLTDIPLTGEVLTWFTDGSSYVVEGKRMAGAAVVDGTRT







IWASSLPEGTSAQKAELMALTQALRLAEGKSINIYTDSRYAFATAHVHGA







IYKQRGWLTSAGREIKNKEEILSLLEALHLPKRLAIIHCPGHQKAKDPIS







RGNQMADRVAKQAAQGVNLLP





PERV_
PERV
Q4VFZ2
derivative
3226
TLQLDDEYRLYSPLVKPDQNIQFWLEQFPQAWAETAGMGLAKQVPPQVIQ


Q4VFZ2




LKASATPVSVRQYPLSKEAQEGIRPHVQRLIQQGILVPVQSPWNTPLLPV







RKPGTNDYRPVQDLREVNKRVQDIHPTVPNPYNLLCALPPQRSWYTVLDL







KDAFFCLRLHPTSQPLFAFEWRDPGTGRTGQLTWTRLPQGFKNSPTIFDE







ALHRDLANFRIQHPQVTLLQYVDDLLLAGATKQDCLEGTKALLLELSDLG







YRASAKKAQICRREVTYLGYSLRDGQRWLTEARKKTVVQIPAPTTAKQVR







EFLGTAGFCRLWIPGFATLAAPLYPLTKEKGEFSWAPEHQKAFDAIKKAL







LSAPALALPDVTKPFTLYVDERKGVARGVLTQTLGPWRRPVAYLSKKLDP







VASGWPVCLKAIAAVAILVKDADKLTLGQNITVIAPHALENIVRQPPDRW







MTNARMTHYQSLLLTERVTFAPPAALNPATLLPEETDEPVTHDCHQLLIE







ETGVRKDLTDIPLTGEVLTWFTDGSSYVVEGKRMAGAAVVDGTRTIWASS







LPEGTSAQKAELMALTQALRLAEGKSINIYTDSRYAFATAHVHGAIYKQR







GLLTSAGREIKNKEEILSLLEALHLPKRLAIIHCPGHQKAKDPISRGNQM







ADRVAKQAAQGVNLL





PERV_
PERV
Q4VFZ2
derivative
3227
TLQLDDEYRLYSPLVKPDQNIQFWLEQFPQAWAETAGMGLAKQVPPQVIQ


Q4VFZ2_




LKASATPVSVRQYPLSKEAQEGIRPHVQRLIQQGILVPVQSPWNTPLLPV


3mut




RKPGTNDYRPVQDLREVNKRVQDIHPTVPNPYNLLCALPPQRSWYTVLDL







KDAFFCLRLHPTSQPLFAFEWRDPGTGRTGQLTWTRLPQGFKNSPTIFNE







ALHRDLANFRIQHPQVTLLQYVDDLLLAGATKQDCLEGTKALLLELSDLG







YRASAKKAQICRREVTYLGYSLRDGQRWLTEARKKTVVQIPAPTTAKQVR







EFLGTAGFCRLWIPGFATLAAPLYPLTKPKGEFSWAPEHQKAFDAIKKAL







LSAPALALPDVTKPFTLYVDERKGVARGVLTQTLGPWRRPVAYLSKKLDP







VASGWPVCLKAIAAVAILVKDADKLTLGQNITVIAPHALENIVRQPPDRW







MTNARMTHYQSLLLTERVTFAPPAALNPATLLPEETDEPVTHDCHQLLIE







ETGVRKDLTDIPLTGEVLTWFTDGSSYVVEGKRMAGAAVVDGTRTIWASS







LPEGTSAQKAELMALTQALRLAEGKSINIYTDSRYAFATAHVHGAIYKQR







GWLTSAGREIKNKEEILSLLEALHLPKRLAIIHCPGHQKAKDPISRGNQM







ADRVAKQAAQGVNLL





SFV1_
SFV1
P23074
root
3228
MDPLQLLQPLEAEIKGTKLKAHWNSGATITCVPEAFLEDERPIQTMLIKT


P23074




IHGEKQQDVYYLTFKVQGRKVEAEVLASPYDYILLNPSDVPWLMKKPLQL







TVLVPLHEYQERLLQQTALPKEQKELLQKLFLKYDALWQHWENQVGHRRI







KPHNIATGTLAPRPQKQYPINPKAKPSIQIVIDDLLKQGVLIQQNSTMNT







PVYPVPKPDGKWRMVLDYREVNKTIPLIAAQNQHSAGILSSIYRGKYKTT







LDLTNGFWAHPITPESYWLTAFTWQGKQYCWTRLPQGFLNSPALFTADVV







DLLKEIPNVQAYVDDIYISHDDPQEHLEQLEKIFSILLNAGYVVSLKKSE







IAQREVEFLGFNITKEGRGLTDTFKQKLLNITPPKDLKQLQSILGLLNFA







RNFIPNYSELVKPLYTIVANANGKFISWTEDNSNQLQHIISVLNQADNLE







ERNPETRLIIKVNSSPSAGYIRYYNEGSKRPIMYVNYIFSKAEAKFTQTE







KLLTTMHKGLIKAMDLAMGQEILVYSPIVSMTKIQRTPLPERKALPVRWI







TWMTYLEDPRIQFHYDKSLPELQQIPNVTEDVIAKTKHPSEFAMVFYTDG







SAIKHPDVNKSHSAGMGIAQVQFIPEYKIVHQWSIPLGDHTAQLAEIAAV







EFACKKALKISGPVLIVTDSFYVAESANKELPYWKSNGFLNNKKKPLRHV







SKWKSIAECLQLKPDIIIMHEKGHQQPMTTLHTEGNNLADKLATQGSYVV







H





SFV1_
SFV1
P23074
derivative
3229
VPWLMKKPLQLTVLVPLHEYQERLLQQTALPKEQKELLQKLFLKYDALWQ


P23074-




HWENQVGHRRIKPHNIATGTLAPRPQKQYPINPKAKPSIQIVIDDLLKQG


Pro




VLIQQNSTMNTPVYPVPKPDGKWRMVLDYREVNKTIPLIAAQNQHSAGIL







SSIYRGKYKTTLDLTNGFWAHPITPESYWLTAFTWQGKQYCWTRLPQGFL







NSPALFTADVVDLLKEIPNVQAYVDDIYISHDDPQEHLEQLEKIFSILLN







AGYVVSLKKSEIAQREVEFLGFNITKEGRGLTDTFKQKLLNITPPKDLKQ







LQSILGLLNFARNFIPNYSELVKPLYTIVANANGKFISWTEDNSNQLQHI







ISVLNQADNLEERNPETRLIIKVNSSPSAGYIRYYNEGSKRPIMYVNYIF







SKAEAKFTQTEKLLTTMHKGLIKAMDLAMGQEILVYSPIVSMTKIQRTPL







PERKALPVRWITWMTYLEDPRIQFHYDKSLPELQQIPNVTEDVIAKTKHP







SEFAMVFYTDGSAIKHPDVNKSHSAGMGIAQVQFIPEYKIVHQWSIPLGD







HTAQLAEIAAVEFACKKALKISGPVLIVTDSFYVAESANKELPYWKSNGF







LNNKKKPLRHVSKWKSIAECLQLKPDIIIMHEKGHQQPMTTLHTEGNNLA







DKLATQGSYVVH





SFV1_
SFV1
P23074
derivative
3230
VPWLMKKPLQLTVLVPLHEYQERLLQQTALPKEQKELLQKLFLKYDALWQ


P23074-




HWENQVGHRRIKPHNIATGTLAPRPQKQYPINPKAKPSIQIVIDDLLKQG


Pro_




VLIQQNSTMNTPVYPVPKPDGKWRMVLDYREVNKTIPLIAAQNQHSAGIL


2mut




SSIYRGKYKTTLDLTNGFWAHPITPESYWLTAFTWQGKQYCWTRLPQGFL







NSPALFNADVVDLLKEIPNVQAYVDDIYISHDDPQEHLEQLEKIFSILLN







AGYVVSLKKSEIAQREVEFLGFNITKEGRGLTDTFKQKLLNITPPKDLKQ







LQSILGLLNFARNFIPNYSELVKPLYTIVAPANGKFISWTEDNSNQLQHI







ISVLNQADNLEERNPETRLIIKVNSSPSAGYIRYYNEGSKRPIMYVNYIF







SKAEAKFTQTEKLLTTMHKGLIKAMDLAMGQEILVYSPIVSMTKIQRTPL







PERKALPVRWITWMTYLEDPRIQFHYDKSLPELQQIPN







VTEDVIAKTKHPSEFAMVFYTDGSAIKHPDVNKSHSAGMGIAQVQFIPEY







KIVHQWSIPLGDHTAQLAEIAAVEFACKKALKISGPVLIVTDSFYVAESA







NKELPYWKSNGFLNNKKKPLRHVSKWKSIAECLQLKPDIIIMHEKGHQQP







MTTLHTEGNNLADKLATQGSYVVH





SFV1_
SFV1
P23074
derivative
3231
VPWLMKKPLQLTVLVPLHEYQERLLQQTALPKEQKELLQKLFLKYDALWQ


P23074-




HWENQVGHRRIKPHNIATGTLAPRPQKQYPINPKAKPSIQIVIDDLLKQG


Pro_




VLIQQNSTMNTPVYPVPKPDGKWRMVLDYREVNKTIPLIAAQNQHSAGIL


2mutA




SSIYRGKYKTTLDLTNGFWAHPITPESYWLTAFTWQGKQYCWTRLPQGFL







NSPALFNADVVDLLKEIPNVQAYVDDIYISHDDPQEHLEQLEKIFSILLN







AGYVVSLKKSEIAQREVEFLGFNITKEGRGLTDTFKQKLLNITPPKDLKQ







LQSILGKLNFARNFIPNYSELVKPLYTIVAPANGKFISWTEDNSNQLQHI







ISVLNQADNLEERNPETRLIIKVNSSPSAGYIRYYNEGSKRPIMYVNYIF







SKAEAKFTQTEKLLTTMHKGLIKAMDLAMGQEILVYSPIVSMTKIQRTPL







PERKALPVRWITWMTYLEDPRIQFHYDKSLPELQQIPNVTEDVIAKTKHP







SEFAMVFYTDGSAIKHPDVNKSHSAGMGIAQVQFIPEYKIVHQWSIPLGD







HTAQLAEIAAVEFACKKALKISGPVLIVTDSFYVAESANKELPYWKSNGF







LNNKKKPLRHVSKWKSIAECLQLKPDIIIMHEKGHQQPMTTLHTEGNNLA







DKLATQGSYVVH





SFV1_
SFV1
P23074
derivative
3232
MDPLQLLQPLEAEIKGTKLKAHWNSGATITCVPEAFLEDERPIQTMLIKT


P23074_




IHGEKQQDVYYLTFKVQGRKVEAEVLASPYDYILLNPSDVPWLMKKPLQL


2mut




TVLVPLHEYQERLLQQTALPKEQKELLQKLFLKYDALWQHWENQVGHRRI







KPHNIATGTLAPRPQKQYPINPKAKPSIQIVIDDLLKQGVLIQQNSTMNT







PVYPVPKPDGKWRMVLDYREVNKTIPLIAAQNQHSAGILSSIYRGKYKTT







LDLTNGFWAHPITPESYWLTAFTWQGKQYCWTRLPQGFLNSPALFNADVV







DLLKEIPNVQAYVDDIYISHDDPQEHLEQLEKIFSILLNAGYVVSLKKSE







IAQREVEFLGFNITKEGRGLTDTFKQKLLNITPPKDLKQLQSILGLLNFA







RNFIPNYSELVKPLYTIVAPANGKFISWTEDNSNQLQHIISVLNQADNLE







ERNPETRLIIKVNSSPSAGYIRYYNEGSKRPIMYVNYIFSKAEAKFTQTE







KLLTTMHKGLIKAMDLAMGQEILVYSPIVSMTKIQRTPLPERKALPVRWI







TWMTYLEDPRIQFHYDKSLPELQQIPNVTEDVIAKT







KHPSEFAMVFYTDGSAIKHPDVNKSHSAGMGIAQVQFIPEYKIVHQWSIP







LGDHTAQLAEIAAVEFACKKALKISGPVLIVTDSFYVAESANKELPYWKS







NGFLNNKKKPLRHVSKWKSIAECLQLKPDIIIMHEKGHQQPMTTLHTEGN







NLADKLATQGSYVVH





SFV1_
SFV1
P23074
derivative
3233
MDPLQLLQPLEAEIKGTKLKAHWNSGATITCVPEAFLEDERPIQTMLIKT


P23074_




IHGEKQQDVYYLTFKVQGRKVEAEVLASPYDYILLNPSDVPWLMKKPLQL


2mutA




TVLVPLHEYQERLLQQTALPKEQKELLQKLFLKYDALWQHWENQVGHRRI







KPHNIATGTLAPRPQKQYPINPKAKPSIQIVIDDLLKQGVLIQQNSTMNT







PVYPVPKPDGKWRMVLDYREVNKTIPLIAAQNQHSAGILSSIYRGKYKTT







LDLTNGFWAHPITPESYWLTAFTWQGKQYCWTRLPQGFLNSPALFNADVV







DLLKEIPNVQAYVDDIYISHDDPQEHLEQLEKIFSILLNAGYVVSLKKSE







IAQREVEFLGFNITKEGRGLTDTFKQKLLNITPPKDLKQLQSILGKLNFA







RNFIPNYSELVKPLYTIVAPANGKFISWTEDNSNQLQHIISVLNQADNLE







ERNPETRLIIKVNSSPSAGYIRYYNEGSKRPIMYVNYIFSKAEAKFTQTE







KLLTTMHKGLIKAMDLAMGQEILVYSPIVSMTKIQRTPLPERKALPVRWI







TWMTYLEDPRIQFHYDKSLPELQQIPNVTEDVIAKTKHPSEFAMVFYTDG







SAIKHPDVNKSHSAGMGIAQVQFIPEYKIVHQWSIPLGDHTAQLAEIAAV







EFACKKALKISGPVLIVTDSFYVAESANKELPYWKSNGFLNNKKKPLRHV







SKWKSIAECLQLKPDIIIMHEKGHQQPMTTLHTEGNNLADKLATQGSYVV







H





SFV3L_
SFV3L
P27401
root
3234
MDPLQLLQPLEAEIKGTKLKAHWNSGATITCVPQAFLEEEVPIKNIWIKT


P27401




IHGEKEQPVYYLTFKIQGRKVEAEVISSPYDYILVSPSDIPWLMKKPLQL







TTLVPLQEYEERLLKQTMLTGSYKEKLQSLFLKYDALWQHWENQVGHRRI







KPHHIATGTVNPRPQKQYPINPKAKASIQTVINDLLKQGVLIQQNSIMNT







PVYPVPKPDGKWRMVLDYREVNKTIPLIAAQNQHSAGILSSIFRGKYKTT







LDLSNGFWAHSITPESYWLTAFTWLGQQYCWTRLPQGFLNSPALFTADVV







DLLKEVPNVQVYVDDIYISHDDPREHLEQLEKVFSLLLNAGYVVSLKKSE







IAQHEVEFLGFNITKEGRGLTETFKQKLLNITPPRDLKQLQSILGLLNFA







RNFIPNFSELVKPLYNIIATANGKYITWTTDNSQQLQNIISMLNSAENLE







ERNPEVRLIMKVNTSPSAGYIRFYNEFAKRPIMYLNYVYTKAEVKFTNTE







KLLTTIHKGLIKALDLGMGQEILVYSPIVSMTKIQKTPLPERKALPIRWI







TWMSYLEDPRIQFHYDKTLPELQQVPTVTDDIIAKIKHPSEFSMVFYTDG







SAIKHPNVNKSHNAGMGIAQVQFKPEFTVINTWSIPLGDHTAQLAEVAAV







EFACKKALKIDGPVLIVTDSFYVAESVNKELPYWQSNGFFNNKKKPLKHV







SKWKSIADCIQLKPDIIIIHEKGHQPTASTFHTEGNNLADKLATQGSYVV







N





SFV3L_
SFV3L
P27401
derivative
3235
IPWLMKKPLQLTTLVPLQEYEERLLKQTMLTGSYKEKLQSLFLKYDALWQ


P27401=




HWENQVGHRRIKPHHIATGTVNPRPQKQYPINPKAKASIQTVINDLLKQG


Pro




VLIQQNSIMNTPVYPVPKPDGKWRMVLDYREVNKTIPLIAAQNQHSAGIL







SSIFRGKYKTTLDLSNGFWAHSITPESYWLTAFTWLGQQYCWTRLPQGFL







NSPALFTADVVDLLKEVPNVQVYVDDIYISHDDPREHLEQLEKVFSLLLN







AGYVVSLKKSEIAQHEVEFLGFNITKEGRGLTETFKQKLLNITPPRDLKQ







LQSILGLLNFARNFIPNFSELVKPLYNIIATANGKYITWTTDNSQQLQNI







ISMLNSAENLEERNPEVRLIMKVNTSPSAGYIRFYNEFAKRPIMYLNYVY







TKAEVKFTNTEKLLTTIHKGLIKALDLGMGQEILVYSPIVSMTKIQKTPL







PERKALPIRWITWMSYLEDPRIQFHYDKTLPELQQVPTVTDDIIAKIKHP







SEFSMVFYTDGSAIKHPNVNKSHNAGMGIAQVQFKPEFTVINTWSIPLGD







HTAQLAEVAAVEFACKKALKIDGPVLIVTDSFYVAESVNKELPYWQSNGF







FNNKKKPLKHVSKWKSIADCIQLKPDIIIIHEKGHQPTASTFHTEGNNLA







DKLATQGSYVVN





SFV3L_
SFV3L
P27401
derivative
3236
IPWLMKKPLQLTTLVPLQEYEERLLKQTMLTGSYKEKLQSLFLKYDALWQ


P27401-




HWENQVGHRRIKPHHIATGTVNPRPQKQYPINPKAKASIQTVINDLLKQG


Pro_




VLIQQNSIMNTPVYPVPKPDGKWRMVLDYREVNKTIPLIAAQNQHSAGIL


2mut




SSIFRGKYKTTLDLSNGFWAHSITPESYWLTAFTWLGQQYCW







TRLPQGFLNSPALFNADVVDLLKEVPNVQVYVDDIYISHDDPREHLEQLE







KVFSLLLNAGYVVSLKKSEIAQHEVEFLGFNITKEGRGLTETFKQKLLNI







TPPRDLKQLQSILGLLNFARNFIPNFSELVKPLYNIIATAPGKYITWTTD







NSQQLQNIISMLNSAENLEERNPEVRLIMKVNTSPSAGYIRFYNEFAKRP







IMYLNYVYTKAEVKFTNTEKLLTTIHKGLIKALDLGMGQEILVYSPIVSM







TKIQKTPLPERKALPIRWITWMSYLEDPRIQFHYDKTLPELQQVPTVTDD







IIAKIKHPSEFSMVFYTDGSAIKHPNVNKSHNAGMGIAQVQFKPEFTVIN







TWSIPLGDHTAQLAEVAAVEFACKKALKIDGPVLIVTDSFYVAESVNKEL







PYWQSNGFFNNKKKPLKHVSKWKSIADCIQLKPDIIIIHEKGHQPTASTF







HTEGNNLADKLATQGSYVVN





SFV3L_
SFV3L
P27401
derivative
3237
IPWLMKKPLQLTTLVPLQEYEERLLKQTMLTGSYKEKLQSLFLKYDALWQ


P27401-




HWENQVGHRRIKPHHIATGTVNPRPQKQYPINPKAKASIQTVINDLLKQG


Pro_




VLIQQNSIMNTPVYPVPKPDGKWRMVLDYREVNKTIPLIAAQNQHSAGIL


2mutA




SSIFRGKYKTTLDLSNGFWAHSITPESYWLTAFTWLGQQYCWTRLPQGFL







NSPALFNADVVDLLKEVPNVQVYVDDIYISHDDPREHLEQLEKVFSLLLN







AGYVVSLKKSEIAQHEVEFLGFNITKEGRGLTETFKQKLL







NITPPRDLKQLQSILGKLNFARNFIPNFSELVKPLYNIIATAPGKYITWT







TDNSQQLQNIISMLNSAENLEERNPEVRLIMKVNTSPSAGYIRFYNEFAK







RPIMYLNYVYTKAEVKFTNTEKLLTTIHKGLIKALDLGMGQEILVYSPIV







SMTKIQKTPLPERKALPIRWITWMSYLEDPRIQFHYDKTLPELQQVPTVT







DDIIAKIKHPSEFSMVFYTDGSAIKHPNVNKSHNAGMGIAQVQFKPEFTV







INTWSIPLGDHTAQLAEVAAVEFACKKALKIDGPVLIVTDSFYVAESVNK







ELPYWQSNGFFNNKKKPLKHVSKWKSIADCIQLKPDIIIIHEKGHQPTAS







TFHTEGNNLADKLATQGSYVVN





SFV3L_
SFV3L
P27401
derivative
3238
MDPLQLLQPLEAEIKGTKLKAHWNSGATITCVPQAFLEEEVPIKNIWIKT


P27401_




IHGEKEQPVYYLTFKIQGRKVEAEVISSPYDYILVSPSDIPWLMKKPLQL


2mut




TTLVPLQEYEERLLKQTMLTGSYKEKLQSLFLKYDALWQHWENQVGHRRI







KPHHIATGTVNPRPQKQYPINPKAKASIQTVINDLLKQGVLIQQNSIMNT







PVYPVPKPDGKWRMVLDYREVNKTIPLIAAQNQHSAGILSSIFRGKYKTT







LDLSNGFWAHSITPESYWLTAFTWLGQQYCWTRLPQGFLNSPALFNADVV







DLLKEVPNVQVYVDDIYISHDDPREHLEQLEKVFSLLLNAGYVVSLKKSE







IAQHEVEFLGFNITKEGRGLTETFKQKLLNITPPRDLKQLQSILGLLNFA







RNFIPNFSELVKPLYNIIATAPGKYITWTTDNSQQLQNIISMLNSAENLE







ERNPEVRLIMKVNTSPSAGYIRFYNEFAKRPIMYLNYVYTKAEVKFTNTE







KLLTTIHKGLIKALDLGMGQEILVYSPIVSMTKIQKTPLPERKALPIRWI







TWMSYLEDPRIQFHYDKTLPELQQVPTVTDDIIAKIKHPSEFSMVFYTDG







SAIKHPNVNKSHNAGMGIAQVQFKPEFTVINTWSIPLGDHTAQLAEVAAV







EFACKKALKIDGPVLIVTDSFYVAESVNKELPYWQSNGFFNNKKKPLKHV







SKWKSIADCIQLKPDIIIIHEKGHQPTASTFHTEGNNLADKLATQGSYVV







N





SFV3L_
SFVL
P27401
derivative
3239
MDPLQLLQPLEAEIKGTKLKAHWNSGATITCVPQAFLEEEVPIKNIWIKT


P27401_




IHGEKEQPVYYLTFKIQGRKVEAEVISSPYDYILVSPSDIPWLMKKPLQL


2mutA




TTLVPLQEYEERLLKQTMLTGSYKEKLQSLFLKYDALWQHWENQVGHRRI







KPHHIATGTVNPRPQKQYPINPKAKASIQTVINDLLKQGVLIQQNSIMNT







PVYPVPKPDGKWRMVLDYREVNKTIPLIAAQNQHSAGILSSIFRGKYKTT







LDLSNGFWAHSITPESYWLTAFTWLGQQYCWTRLPQGFLNSPALFNADVV







DLLKEVPNVQVYVDDIYISHDDPREHLEQLEKVFSLLLNAGYVVSLKKSE







IAQHEVEFLGFNITKEGRGLTETFKQKLLNITPPRDLKQLQSILGKLNFA







RNFIPNFSELVKPLYNIIATAPGKYITWTTDNSQQLQNIISMLNSAENLE







ERNPEVRLIMKVNTSPSAGYIRFYNEFAKRPIMYLNYVYTKAEVKFTNTE







KLLTTIHKGLIKALDLGMGQEILVYSPIVSMTKIQKTPLPERKALPIRWI







TWMSYLEDPRIQFHYDKTLPELQQVPTVTDDIIAKIKHPSEFSMVFYTDG







SAIKHPNVNKSHNAGMGIAQVQFKPEFTVINTWSIPLGDHTAQLAEVAAV







EFACKKALKIDGPVLIVTDSFYVAESVNKELPY







WQSNGFFNNKKKPLKHVSKWKSIADCIQLKPDIIIIHEKGHQPTASTFHT







EGNNLADKLATQGSYVVN





SFVCP_
SFVCP
087040
root
3240
MNPLQLLQPLPAEVKGTKLLAHWNSGATITCIPESFLEDEQPIKQTLIKT


Q87040




IHGEKQQNVYYLTFKVKGRKVEAEVIASPYEYILLSPTDVPWLTQQPLQL







TILVPLQEYQDRILNKTALPEEQKQQLKALFTKYDNLWQHWENQVGHRKI







RPHNIATGDYPPRPQKQYPINPKAKPSIQIVIDDLLKQGVLTPQNSTMNT







PVYPVPKPDGRWRMVLDYREVNKTIPLTAAQNQHSAGILATIVRQKYKTT







LDLANGFWAHPITPDSYWLTAFTWQGKQYCWTRLPQGFLNSPALFTADAV







DLLKEVPNVQVYVDDIYLSHDNPHEHIQQLEKVFQILLQAGYVVSLKKSE







IGQRTVEFLGFNITKEGRGLTDTFKTKLLNVTPPKDLKQLQSILGLLNFA







RNFIPNFAELVQTLYNLIASSKGKYIEWTEDNTKQLNKVIEALNTASNLE







ERLPDQRLVIKVNTSPSAGYVRYYNESGKKPIMYLNYVFSKAELKFSMLE







KLLTTMHKALIKAMDLAMGQEILVYSPIVSMTKIQKTPLPERKALPIRWI







TWMTYLEDPRIQFHYDKTLPELKHIPDVYTSSIPPLKHPSQYEGVFCTDG







SAIKSPDPTKSNNAGMGIVHAIYNPEYKILNQWSIPLGHHTAQMAEIAAV







EFACKKALKVPGPVLVITDSFYVAESANKELPYWKSNGFVNNKKEPLKHI







SKWKSIAECLSIKPDITIQHEKGHQPINTSIHTEGNALADKLATQGSYVV







N





SFVCP_
SFVCP
Q87040
derivative
3241
VPWLTQQPLQLTILVPLQEYQDRILNKTALPEEQKQQLKALFTKYDNLWQ


Q87040-




HWENQVGHRKIRPHNIATGDYPPRPQKQYPINPKAKPSIQIVIDDLLKQG


Pro




VLTPQNSTMNTPVYPVPKPDGRWRMVLDYREVNKTIPLTAAQNQHSAGIL







ATIVRQKYKTTLDLANGFWAHPITPDSYWLTAFTWQGKQYCWTRLPQGFL







NSPALFTADAVDLLKEVPNVQVYVDDIYLSHDNPHEHIQQLEKVFQILLQ







AGYVVSLKKSEIGQRTVEFLGFNITKEGRGLTDTFKTKLLNVTPPKDLKQ







LQSILGLLNFARNFIPNFAELVQTLYNLIASSKGKYIEWTEDNTKQLNKV







IEALNTASNLEERLPDQRLVIKVNTSPSAGYVRYYNESGKKPIMYLNYVF







SKAELKFSMLEKLLTTMHKALIKAMDLAMGQEILVYSPIVSMTKIQKTPL







PERKALPIRWITWMTYLEDPRIQFHYDKTLPELKHIPDVYTSSIPPLKHP







SQYEGVFCTDGSAIKSPDPTKSNNAGMGIVHAIYNPEYKILNQWSIPLGH







HTAQMAEIAAVEFACKKALKVPGPVLVITDSFYVAESANKELPYWKSNGF







VNNKKEPLKHISKWKSIAECLSIKPDITIQHEKGHQPINTSIHTEGNALA







DKLATQGSYVVN





SFVCP_
SFVCP
087040
derivative
3242
VPWLTQQPLQLTILVPLQEYQDRILNKTALPEEQKQQLKALFTKYDNLWQ


Q87040-




HWENQVGHRKIRPHNIATGDYPPRPQKQYPINPKAKPSIQIVIDDLLKQG


Pro_




VLTPQNSTMNTPVYPVPKPDGRWRMVLDYREVNKTIPLTAAQNQHSAGIL


2mut




ATIVRQKYKTTLDLANGFWAHPITPDSYWLTAFTWQGKQYCWTRLPQGFL







NSPALFNADAVDLLKEVPNVQVYVDDIYLSHDNPHEHIQQLEKVFQILLQ







AGYVVSLKKSEIGQRTVEFLGFNITKEGRGLTDTFKTKLLNVTPPKDLKQ







LQSILGLLNFARNFIPNFAELVQTLYNLIASSPGKYIEWTEDNTKQLNKV







IEALNTASNLEERLPDQRLVIKVNTSPSAGYVRYYNESGKKPIMYLNYVF







SKAELKFSMLEKLLTTMHKALIKAMDLAMGQEILVYSPIVSMTKIQKTPL







PERKALPIRWITWMTYLEDPRIQFHYDKTLPELKHIPDVYTSSIPPLKHP







SQYEGVFCTDGSAIKSPDPTKSNNAGMGIVHAIYNPEYKILNQWSIPLGH







HTAQMAEIAAVEFACKKALKVPGPVLVITDSFYVAESANKELPYWKSNGF







VNNKKEPLKHISKWKSIAECLSIKPDITIQHEKGHQPINTSIHTEGNALA







DKLATQGSYVVN





SFVCP_
SFVCP
087040
derivative
3243
VPWLTQQPLQLTILVPLQEYQDRILNKTALPEEQKQQLKALFTKYDNLWQ


Q87040-




HWENQVGHRKIRPHNIATGDYPPRPQKQYPINPKAKPSIQIVIDDLLKQG


Pro_




VLTPQNSTMNTPVYPVPKPDGRWRMVLDYREVNKTIPLTAAQNQHSAGIL


2mutA




ATIVRQKYKTTLDLANGFWAHPITPDSYWLTAFTWQGKQYCWTRLPQGFL







NSPALFNADAVDLLKEVPNVQVYVDDIYLSHDNPHEHIQQLEKVFQILLQ







AGYVVSLKKSEIGQRTVEFLGFNITKEGRGLTDTFKTK







LLNVTPPKDLKQLQSILGKLNFARNFIPNFAELVQTLYNLIASSPGKYIE







WTEDNTKQLNKVIEALNTASNLEERLPDQRLVIKVNTSPSAGYVRYYNES







GKKPIMYLNYVFSKAELKFSMLEKLLTTMHKALIKAMDLAMGQEILVYSP







IVSMTKIQKTPLPERKALPIRWITWMTYLEDPRIQFHYDKTLPELKHIPD







VYTSSIPPLKHPSQYEGVFCTDGSAIKSPDPTKSNNAGMGIVHAIYNPEY







KILNQWSIPLGHHTAQMAEIAAVEFACKKALKVPGPVLVITDSFYVAESA







NKELPYWKSNGFVNNKKEPLKHISKWKSIAECLSIKPDITIQHEKGHQPI







NTSIHTEGNALADKLATQGSYVVN





SFVCP_
SFVCP
Q87040
derivative
3244
MNPLQLLQPLPAEVKGTKLLAHWNSGATITCIPESFLEDEQPIKQTLIKT


Q87040_




IHGEKQQNVYYLTFKVKGRKVEAEVIASPYEYILLSPTDVPWLTQQPLQL


2mut




TILVPLQEYQDRILNKTALPEEQKQQLKALFTKYDNLWQHWENQVGHRKI







RPHNIATGDYPPRPQKQYPINPKAKPSIQIVIDDLLKQGVLTPQNSTMNT







PVYPVPKPDGRWRMVLDYREVNKTIPLTAAQNQHSAGILATIVRQKYKTT







LDLANGFWAHPITPDSYWLTAFTWQGKQYCWTRLPQGFLNSPALFNADAV







DLLKEVPNVQVYVDDIYLSHDNPHEHIQQLEKVFQILLQAGYVVSLKKSE







IGQRTVEFLGFNITKEGRGLTDTFKTKLLNVTPPKDLKQLQSILGLLNFA







RNFIPNFAELVQTLYNLIASSPGKYIEWTEDNTKQLNKVIEALNTASNLE







ERLPDQRLVIKVNTSPSAGYVRYYNESGKKPIMYLNYVFSKAELKFSMLE







KLLTTMHKALIKAMDLAMGQEILVYSPIVSMTKIQKTPLPERKALPIRWI







TWMTYLEDPRIQFHYDKTLPELKHIPDVYTSSIPPLKHPSQYEGVFCTDG







SAIKSPDPTKSNNAGMGIVHAIYNPEYKILNQWSIPLGHHTAQMAEIAAV







EFACKKALKVPGPVLVITDSFYVAESANKELPYWKSNGFVNNKKEPLKHI







SKWKSIAECLSIKPDITIQHEKGHQPINTSIHTEGNALADKLATQGSYVV







N





SFVCP_
SFVCP
Q87040
derivative
3245
MNPLQLLQPLPAEVKGTKLLAHWNSGATITCIPESFLEDEQPIKQTLIKT


Q87040_




IHGEKQQNVYYLTFKVKGRKVEAEVIASPYEYILLSPTDVPWLTQQPLST


2mutA




MNTPVYPVPKPDGRWRMVLDYREVNKTIPLTAAQNQHSAGILATIVRQKY







KTTLDLANGFWAHPITPDSYWLTAFTWQGKQYCWTRLPQGFLNSPALFNA







DAVDLLKEVPNVQVYVDDIYLSHDNPHEHIQQLEKVFQILLQAGYVVSLK







KSEIGQRTVEFLGFNITKEGRGLTDTFKTKLLNVTPPKDLKQLQSILGKL







NFARNFIPNFAELVQTLYNLIASSPGKYIEWTEDNTKQLNKVIEALNTAS







NLEERLPDQRLVIKVNTSPSAGYVRYYNESGKKPIMYLNYVFSKAELKFS







MLEKLLTTMHKALIKAMDLAMGQEILVYSPIVSMTKIQKTPLPERKALPI







RWITWMTYLEDPRIQFHYDKTLPELKHIPDVYTSSIPPLKHPSQYEGVFC







TDGSAIKSPDPTKSNNAGMGIVHAIYNPEYKILNQWSIPLGHHTAQMAEI







AAVEFACKKALKVPGPVLVITDSFYVAESANKELPYWKSNGFVNNKKEPL







KHISKWKSIAECLSIKPDITIQHEKGHQPINTSIHTEGNALADKLATQGS







YVVN





SMRVH_
SMRVH
P03364
root
3246
PRSRAIDIPVPHADKISWKITDPVWVDQWPLTYEKTLAAIALVQEQLAAG


P03364




HIEPTNSPWNTPIFIIKKKSGSWRLLQDLRAVNKVMVPMGALQPGLPSPV







AIPLNYHKIVIDLKDCFFTIPLHPEDRPYFAFSVPQINFQSPMPRYQWKV







LPQGMANSPTLCQKFVAAAIAPVRSQWPEAYILHYMDDILLACDSAEAAK







ACYAHIISCLTSYGLKIAPDKVQVSEPFSYLGFELHHQQVFTPRVCLKTD







HLKTLNDFQKLLGDIQWLRPYLKLPTSALVPLNNILKGDPNPLSVRALTP







EAKQSLALINKAIQNQSVQQISYNLPLVLLLLPTPHTPTAVFWQPNGTDP







TKNGSPLLWLHLPASPSKVLLTYPSLLAMLIIKGRYTGRQLFGRDPHSII







IPYTQDQLTWLLQTSDEWAIALSSFTGDIDNHYPSDPVIQFAKLHQFIFP







KITKCAPIPQATLVFTDGSSNGIAAYVIDNQPISIKSPYLSAQLVELYAI







LQVFTVLAHQPFNLYTDSAYIAQSVPLLETVPFIKSSTNATPLFSKLQQL







ILNRQHPFFIGHLRAHLNLPGPLAEGNALADAATQIFPIISD





SMRVH_
SMRVH
P03364
derivative
3247
PRSRAIDIPVPHADKISWKITDPVWVDQWPLTYEKTLAAIALVQEQLAAG


P03364_




HIEPTNSPWNTPIFIIKKKSGSWRLLQDLRAVNKVMVPMGALQPGLPSPV


2mut




AIPLNYHKIVIDLKDCFFTIPLHPEDRPYFAFSVPQINFQSPMPRYQWKV







LPQGMANSPTLCQKFVAAAIAPVRSQWPEAYILHYMDDILLACDS







AEAAKACYAHIISCLTSYGLKIAPDKVQVSEPFSYLGFELHHQQVFTPRV







CLKTDHLKTLNDFQKLLGDIQWLRPYLKLPTSALVPLNNILKPDPNPLSV







RALTPEAKQSLALINKAIQNQSVQQISYNLPLVLLLLPTPHTPTAVFWQP







NGTDPTKNGSPLLWLHLPASPSKVLLTYPSLLAMLIIKGRYTGRQLFGRD







PHSIIIPYTQDQLTWLLQTSDEWAIALSSFTGDIDNHYPSDPVIQFAKLH







QFIFPKITKCAPIPQATLVFTDGSSNGIAAYVIDNQPISIKSPYLSAQLV







ELYAILQVFTVLAHQPFNLYTDSAYIAQSVPLLETVPFIKSSTNATPLFS







KLQQLILNRQHPFFIGHLRAHLNLPGPLAEGNALADAATQIFPIISD





SMRVH_
SMRVH
P03364
derivative
3248
PRSRAIDIPVPHADKISWKITDPVWVDQWPLTYEKTLAAIALVQEQLAAG


P03364_2




HIEPTNSPWNTPIFIIKKKSGSWRLLQDLRAVNKVMVPMGALQPGLPSPV


mutB




APPLNYHKIVIDLKDCFFTIPLHPEDRPYFAFSVPQINFQSPMPRYQWKV







LPQGMANSPTLCQKFVAAAIAPVRSQWPEAYILHYMDDILLACDSAEAAK







ACYAHIISCLTSYGLKIAPDKVQVSEPFSYLGFELHHQQVFTPRVCLKTD







HLKTLNDFQKLLGDIQWLRPYLKLPTSALVPLNNILKPDPNPLSVRALTP







EAKQSLALINKAIQNQSVQQISYNLPLVLLLLPTPHTPTAVFWQPNGTDP







TKNGSPLLWLHLPASPSKVLLTYPSLLAMLIIKGRYTGRQLFGRDPHSII







IPYTQDQLTWLLQTSDEWAIALSSFTGDIDNHYPSDPVIQFAKLHQFIFP







KITKCAPIPQATLVFTDGSSNGIAAYVIDNQPISIKSPYLSAQLVELYAI







LQVFTVLAHQPFNLYTDSAYIAQSVPLLETVPFIKSSTNATPLFSKLQQL







ILNRQHPFFIGHLRAHLNLPGPLAEGNALADAATQIFPIISD





SRV1_
SRV1
P04025
root
3249
LTAAIDMLAPQQCAEPITWKSDEPVWVDQWPLTSEKLAAAQQLVQEQLEA


P04025




GHITESNSPWNTPIFVIKKKSGKWRLLQDLRAVNATMVLMGALQPGLPSP







VAIPQGYLKIIIDLKDCFFSIPLHPSDQKRFAFSLPSTNFKEPMQRFQWK







VLPQRMANSPTLCQKYVATAIHKVRHAWKQMYIIHYMDDILIAGKDGQQV







LQCFDQLKQELTIAGLHIAPEKIQLQDPYTYLGFELNGPKITNQKAVIRK







DKLQTLNDFQKLLGDINWLRPYLKLTTADLKPLFDTLKGDSNPNSHRSLS







KEALALLDKVETAIAEQFVTHINYSLPLMFLIFNTALTPTGLFWQNNPIM







WVHLPASPKKVLLPYYDAIADLIILGRDHSKKYFGIEPSVIIQPYSKSQI







DWLMQNTEMWPIACASYVGILDNHYPPNKLIQFCKLHAFIFPQIISKTPL







NNALLVFTDGSSTGMAAYTLADTTIKFQTNLNSAQLVELQALIAVLSAFP







NQPLNIYTDSAYLAHSIPLLETVAQIKHISETAKLFLQCQQLIYNRSIPF







YIGHVRAHSGLPGPIAHGNQKADLATKTVASNINT





SRV1_
SRV1
P04025
derivative
3250
LTAAIDMLAPQQCAEPITWKSDEPVWVDQWPLTSEKLAAAQQLVQEQLEA


P04025_




GHITESNSPWNTPIFVIKKKSGKWRLLQDLRAVNATMVLMGALQPGLPSP


2mutB




VAPPQGYLKIIIDLKDCFFSIPLHPSDQKRFAFSLPSTNFKEPMQRFQWK







VLPQRMANSPTLCQKYVATAIHKVRHAWKQMYIIHYMDDILIAGKDGQQV







LQCFDQLKQELTIAGLHIAPEKIQLQDPYTYLGFELNGPKITNQKAVIRK







DKLQTLNDFQKLLGDINWLRPYLKLTTADLKPLFDTLKGDSNPNSHRSLS







KEALALLDKVETAIAEQFVTHINYSLPLMFLIFNTALTPTGLFWQNNPIM







WVHLPASPKKVLLPYYDAIADLIILGRDHSKKYFGIEPSVIIQPYSKSQI







DWLMQNTEMWPIACASYVGILDNHYPPNKLIQFCKLHAFIFPQIISKTPL







NNALLVFTDGSSTGMAAYTLADTTIKFQTNLNSAQLVELQALIAVLSAFP







NQPLNIYTDSAYLAHSIPLLETVAQIKHISETAKLFLQCQQLIYNRSIPF







YIGHVRAHSGLPGPIAHGNQKADLATKTVASNINT





SRV2_
SRV2
P51517
root
3251
LATAVDILAPQRYADPITWKSDEPVWVDQWPLTQEKLAAAQQLVQEQLQA


P51517




GHIIESNSPWNTPIFVIKKKSGKWRLLQDLRAVNATMVLMGALQPGLPSP







VAIPQGYFKIVIDLKDCFFTIPLQPVDQKRFAFSLPSTNFKQPMKRYQWK







VLPQGMANSPTLCQKYVAAAIEPVRKSWAQMYIIHYMDDILIAGKLGEQV







LQCFAQLKQALTTTGLQIAPEKVQLQDPYTYLGFQINGPKITNQKAVIRR







DKLQTLNDFQKLLGDINWLRPYLHLTTGDLKPLFDILKGDSNPNSPRSLS







EAALASLQKVETAIAEQFVTQIDYTQPLTFLIFNTTLTPTGLFWQNNPVM







WVHLPASPKKVLLPYYDAIADLIILGRDNSKKYFGLEPS







PWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILA







PHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPVVALN







LQALIAVLSAFPHRALNVYTDSAYLAHSIPLLETVSHIKHISDTAKFFLQ







CQQLIYNRSIPFYLGHIRAHSGLPGPLSQGNHITDLATKVVATTLTT





SRV2_
SRV2
P51517
derivative
3252
LATAVDILAPQRYADPITWKSDEPVWVDQWPLTQEKLAAAQQLVQEQLQA


P51517_




GHIIESNSPWNTPIFVIKKKSGKWRLLQDLRAVNATMVLMGALQPGLPSP


2mutB




VAPPQGYFKIVIDLKDCFFTIPLQPVDQKRFAFSLPSTNFKQPMKRYQWK







VLPQGMANSPTLCQKYVAAAIEPVRKSWAQMYIIHYMDDILIAGKLGEQV







LQCFAQLKQALTTTGLQIAPEKVQLQDPYTYLGFQINGPKITNQKAVIRR







DKLQTLNDFQKLLGDINWLRPYLHLTTGDLKPLFDILKGDSNPNSPRSLS







EAALASLQKVETAIAEQFVTQIDYTQPLTFLIFNTTLTPTGLFWQNNPVM







WVHLPASPKKVLLPYYDAIADLIILGRDNSKKYFGLEPSTIIQPYSKSQI







HWLMQNTETWPIACASYAGNIDNHYPPNKLIQFCKLHAVVFPRIISKTPL







DNALLVFTDGSSTGIAAYTFEKTTVRFKTSHTSAQLVELQALIAVLSAFP







HRALNVYTDSAYLAHSIPLLETVSHIKHISDTAKFFLQCQQLIYNRSIPF







YLGHIRAHSGLPGPLSQGNHITDLATKVVATTLTT





WDSV_
WDSV
O92815
root
3253
SCQTKNTLNIDEYLLQFPDQLWASLPTDIGRMLVPPITIKIKDNASLPSI


O92815




RQYPLPKDKTEGLRPLISSLENQGILIKCHSPCNTPIFPIKKAGRDEYRM







IHDLRAINNIVAPLTAVVASPTTVLSNLAPSLHWFTVIDLSNAFFSVPIH







KDSQYLFAFTFEGHQYTWTVLPQGFIHSPTLFSQALYQSLHKIKFKISSE







ICIYMDDVLIASKDRDTNLKDTAVMLQHLASEGHKVSKKKLQLCQQEVVY







LGQLLTPEGRKILPDRKVTVSQFQQPTTIRQIRAFLGLVGYCRHWIPEFS







IHSKFLEKQLKKDTAEPFQLDDQQVEAFNKLKHAITTAPVLVVPDPAKPF







QLYTSHSEHASIAVLTQKHAGRTRPIAFLSSKFDAIESGLPPCLKACASI







HRSLTQADSFILGAPLIIYTTHAICTLLQRDRSQLVTASRFSKWEADLLR







PELTFVACSAVSPAHLYMQSCENNIPPHDCVLLTHTISRPRPDLSDLPIP







DPDMTLFSDGSYTTGRGGAAVVMHRPVTDDFIIIHQQPGGASAQTAELLA







LAAACHLATDKTVNIYTDSRYAYGVVHDFGHLWMHRGFVTSAGTPIKNHK







EIEYLLKQIMKPKQVSVIKIEAHTKGVSMEVRGNAAADEAAKNAVFLVQR


WDSV_
WDSV
O92815
derivative
3254
SCQTKNTLNIDEYLLQFPDQLWASLPTDIGRMLVPPITIKIKDNASLPSI


O92815_




RQYPLPKDKTEGLRPLISSLENQGILIKCHSPCNTPIFPIKKAGRDEYRM


2mut




IHDLRAINNIVAPLTAVVASPTTVLSNLAPSLHWFTVIDLSNAFFSVPIH







KDSQYLFAFTFEGHQYTWTVLPQGFIHSPTLFNQALYQSLHKIKFKISSE







ICIYMDDVLIASKDRDTNLKDTAVMLQHLASEGHKVSKKKLQLCQQEVVY







LGQLLTPEGRKILPDRKVTVSQFQQPTTIRQIRAFLGLVGYCRHWIPEFS







IHSKFLEKQLKPDTAEPFQLDDQQVEAFNKLKHAITTAPVLVVPDPAKPF







QLYTSHSEHASIAVLTQKHAGRTRPIAFLSSKFDAIESGLPPCLKACASI







HRSLTQADSFILGAPLIIYTTHAICTLLQRDRSQLVTASRFSKWEADLLR







PELTFVACSAVSPAHLYMQSCENNIPPHDCVLLTHTISRPRPDLSDLPIP







DPDMTLFSDGSYTTGRGGAAVVMHRPVTDDFIIIHQQPGGASAQTAELLA







LAAACHLATDKTVNIYTDSRYAYGVVHDFGHLWMHRGFVTSAGTPIKNHK







EIEYLLKQIMKPKQVSVIKIEAHTKGVSMEVRGNAAADEAAKNAVFLVQR


WDSV_
WDSV
O92815
derivative
3255
SCQTKNTLNIDEYLLQFPDQLWASLPTDIGRMLVPPITIKIKDNASLPSI


O92815_




RQYPLPKDKTEGLRPLISSLENQGILIKCHSPCNTPIFPIKKAGRDEYRM


2mutA




IHDLRAINNIVAPLTAVVASPTTVLSNLAPSLHWFTVIDLSNAFFSVPIH







KDSQYLFAFTFEGHQYTWTVLPQGFIHSPTLFNQALYQSLHKIKFKISSE







ICIYMDDVLIASKDRDTNLKDTAVMLQHLASEGHKVSKKKLQLCQQEVVY







LGQLLTPEGRKILPDRKVTVSQFQQPTTIRQIRAFLGKVGYCRHFIPEFS







IHSKFLEKQLKPDTAEPFQLDDQQVEAFNKLKHAITTAPVLVVPDPAKPF







QLYTSHSEHASIAVLTQKHAGRTRPIAFLSSKFDAIESGLPPCLKACASI







HRSLTQADSFILGAPLIIYTTHAICTLLQRDRSQLVTASRFSKWEADLLR







PELTFVACSAVSPAHLYMQSCENNIPPHDCVLLTHTISRPRPDLSDLPIP







DPDMTLFSDGSYTTGRGGAAVVMHRPVTDDFIIIHQQPGGASAQTAELLA







LAAACHLATDKTVNIYTDSRYAYGVVHDFGHLWMHRGFVTSAGTPIKNHK







EIEYLLKQIMKPKQVSVIKIEAHTKGVSMEVRGNAAADEAAKNAVFLVQR





WMSV_
WMSV
P03359
root
3256
VLNLEEEYRLHEKPVPSSIDPSWLQLFPTVWAERAGMGLANQVPPVVVEL


P03359




RSGASPVAVRQYPMSKEAREGIRPHIQRFLDLGVLVPCQSPWNTPLLPVK







KPGTNDYRPVQDLREINKRVQDIHPTVPNPYNLLSSLPPSHTWYSVLDLK







DAFFCLKLHPNSQPLFAFEWRDPEKGNTGQLTWTRLPQGFKNSPTLFDEA







LHRDLAPFRALNPQVVLLQYVDDLLVAAPTYRDCKEGTQKLLQELSKLGY







RVSAKKAQLCQKEVTYLGYLLKEGKRWLTPARKATVMKIPPPTTPRQVRE







FLGTAGFCRLWIPGFASLAAPLYPLTKESIPFIWTEEHQKAFDRIKEALL







SAPALALPDLTKPFTLYVDERAGVARGVLTQTLGPWRRPVAYLSKKLDPV







ASGWPTCLKAVAAVALLLKDADKLTLGQNVTVIASHSLESIVRQPPDRWM







TNARMTHYQSLLLNERVSFAPPAVLNPATLLPVESEATPVHRCSEILAEE







TGTRRDLKDQPLPGVPAWYTDGSSFIAEGKRRAGAAIVDGKRTVWASSLP







EGTSAQKAELVALTQALRLAEGKDINIYTDSRYAFATAHIHGAIYKQRGL







LTSAGKDIKNKEEILALLEAIHLPKRVAIIHCPGHQKGNDPVATGNRRAD







EAAKQAALSTRVLAETTKP





WMSV_
WMSV
P03359
derivative
3257
VLNLEEEYRLHEKPVPSSIDPSWLQLFPTVWAERAGMGLANQVPPVVVEL


P03359_




RSGASPVAVRQYPMSKEAREGIRPHIQRFLDLGVLVPCQSPWNTPLLPVK


3mut




KPGTNDYRPVQDLREINKRVQDIHPTVPNPYNLLSSLPPSHTWYSVLDLK







DAFFCLKLHPNSQPLFAFEWRDPEKGNTGQLTWTRLPQGFKNSPTLFNEA







LHRDLAPFRALNPQVVLLQYVDDLLVAAPTYRDCKEGTQKLLQELSKLGY







RVSAKKAQLCQKEVTYLGYLLKEGKRWLTPARKATVMKIPPPTTPRQVRE







FLGTAGFCRLWIPGFASLAAPLYPLTKPSIPFIWTEEHQKAFDRIKEALL







SAPALALPDLTKPFTLYVDERAGVARGVLTQTLGPWRRPVAYLSKKLDPV







ASGWPTCLKAVAAVALLLKDADKLTLGQNVTVIASHSLESIVRQPPDRWM







TNARMTHYQSLLLNERVSFAPPAVLNPATLLPVESEATPVHRCSEILAEE







TGTRRDLKDQPLPGVPAWYTDGSSFIAEGKRRAGAAIVDGKRTVWASSLP







EGTSAQKAELVALTQALRLAEGKDINIYTDSRYAFATAHIHGAIYKQRGW







LTSAGKDIKNKEEILALLEAIHLPKRVAIIHCPGHQKGNDPVATGNRRAD







EAAKQAALSTRVLAETTKP





WMSV_
WMSV
P03359
derivative
3258
VLNLEEEYRLHEKPVPSSIDPSWLQLFPTVWAERAGMGLANQVPPVVVEL


P03359_




RSGASPVAVRQYPMSKEAREGIRPHIQRFLDLGVLVPCQSPWNTPLLPVK


3mutA




KPGTNDYRPVQDLREINKRVQDIHPTVPNPYNLLSSLPPSHTWYSVLDLK







DAFFCLKLHPNSQPLFAFEWRDPEKGNTGQLTWTRLPQGFKNSPTLFNEA







LHRDLAPFRALNPQVVLLQYVDDLLVAAPTYRDCKEGTQKLLQELSKLGY







RVSAKKAQLCQKEVTYLGYLLKEGKRWLTPARKATVMKIPPPTTPRQVRE







FLGKAGFCRLFIPGFASLAAPLYPLTKPSIPFIWTEEHQKAFDRIKEALL







SAPALALPDLTKPFTLYVDERAGVARGVLTQTLGPWRRPVAYLSKKLDPV







ASGWPTCLKAVAAVALLLKDADKLTLGQNVTVIASHSLESIVRQPPDRWM







TNARMTHYQSLLLNERVSFAPPAVLNPATLLPVESEATPVHRCSEILAEE







TGTRRDLKDQPLPGVPAWYTDGSSFIAEGKRRAGAAIVDGKRTVWASSLP







EGTSAQKAELVALTQALRLAEGKDINIYTDSRYAFATAHIHGAIYKQRGW







LTSAGKDIKNKEEILALLEAIHLPKRVAIIHCPGHQKGNDPVATGNRRAD







EAAKQAALSTRVLAETTKP





XMRV6_
XMRV6
A1Z651
root
3259
TLNIEDEYRLHETSKEPDVPLGSTWLSDFPQAWAETGGMGLAVRQAPLII


A1Z651




PLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP







VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLD







LKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFD







EALHRDLADFRIQHPDLILLQYVDDLLLAATSEQDCQRGTRALLQTLGNL







GYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQL







REFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA







LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD







PVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDR







WLSNARMTHYQAMLLDTDRVQFGPVVAL







NPATLLPLPEKEAPHDCLEILAETHGTRPDLTDQPIPDADYTWYTDGSSF







LQEGQRRAGAAVTTETEVIWARALPAGTSAQRAELIALTQALKMAEGKKL







NVYTDSRYAFATAHVHGEIYRRRGLLTSEGREIKNKNEILALLKALFLPK







RLSIIHCPGHQKGNSAEARGNRMADQAAREAAMKAVLETSTLL





XMRV6_
XMRV6
A1Z651
derivative
3260
TLNIEDEYRLHETSKEPDVPLGSTWLSDFPQAWAETGGMGLAVRQAPLII


A1Z651_




PLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP


3mut




VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLD







LKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFN







EALHRDLADFRIQHPDLILLQYVDDLLLAATSEQDCQRGTRALLQTLGNL







GYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQL







REFLGTAGFCRLWIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQA







LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD







PVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDR







WLSNARMTHYQAMLLDTDRVQFGPVVALNPATLLPLPEKEAPHDCLEILA







ETHGTRPDLTDQPIPDADYTWYTDGSSFLQEGQRRAGAAVTTETEVIWAR







ALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHVHGEIYRR







RGWLTSEGREIKNKNEILALLKALFLPKRLSIIHCPGHQKGNSAEARGNR







MADQAAREAAMKAVLETSTLL





XMRV6_
XMRV6
A1Z651
derivative
3261
TLNIEDEYRLHETSKEPDVPLGSTWLSDFPQAWAETGGMGLAVRQAPLIIP


AA1Z651_




LKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLPV


3mut




KKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDL







KDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNE







ALHRDLADFRIQHPDLILLQYVDDLLLAATSEQDCQRGTRALLQTLGNLG







YRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLR







EFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQAL







LTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDP







VAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDRW







LSNARMTHYQAMLLDTDRVQFGPVVALNPATLLPLPEKEAPHDCLEILAE







THGTRPDLTDQPIPDADYTWYTDGSSFLQEGQRRAGAAVTTETEVIWARA







LPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHVHGEIYRRR







GWLTSEGREIKNKNEILALLKALFLPKRLSIIHCPGHQKGNSAEARGNRM







ADQAAREAAMKAVLETSTLL









In some embodiments, reverse transcriptase domains are modified, for example by site-specific mutation. In some embodiments, reverse transcriptase domains are engineered to have improved properties, e.g. SuperScript IV (SSIV) reverse transcriptase derived from the MMLV RT. In some embodiments, the reverse transcriptase domain may be engineered to have lower error rates, e.g., as described in WO2001068895, incorporated herein by reference. In some embodiments, the reverse transcriptase domain may be engineered to be more thermostable. In some embodiments, the reverse transcriptase domain may be engineered to be more processive. In some embodiments, the reverse transcriptase domain may be engineered to have tolerance to inhibitors. In some embodiments, the reverse transcriptase domain may be engineered to be faster. In some embodiments, the reverse transcriptase domain may be engineered to better tolerate modified nucleotides in the RNA template. In some embodiments, the reverse transcriptase domain may be engineered to insert modified DNA nucleotides. In some embodiments, the reverse transcriptase domain is engineered to bind a template RNA. In some embodiments, one or more mutations are chosen from D200N, L603W, T330P, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, W313F, L435G, N454K, H594Q, L671P, E69K, or D653N in the RT domain of murine leukemia virus reverse transcriptase or a corresponding mutation at a corresponding position of another RT domain. In some embodiments, one or more mutations are chosen as described in WO2018089860A1, incorporated herein by reference (e.g., a C952S, and/or C956S, and/or C952S, C956S (double mutant), and/or C969S, and/or H970Y, and/or R979Q, and/or R976Q, and/or R1071S, and/or R328A, and/or R329A, and/or Q336A, and/or R328A, R329A, Q336A (triple mutant), and/or G426A, and/or D428A, and/or G426A, D428A (double mutant) mutation, and/or any combination thereof, positions relative to WO2018089860A1 SEQ ID NO: 52), in the RT domain of R2Bm retrotransposase or a corresponding mutation at a corresponding position of another RT domain.


In some embodiments, the RT domain possesses proofreading activity. In some embodiments, the RT domain is evolved from a DNA-dependent DNA polymerase and has gained RNA-dependent DNA polymerase activity. The synthetic evolved proofreading RT known as reverse transcription xenopolymerase (RTX, Genbank: QFN49000.1) was previously generated by taking a DNA-dependent DNA polymerase (KOD, Genbank: ABN15964.1) and selecting for RNA-dependent DNA polymerase activity (Ellefson et al 2016). In some embodiments, the engineered RT may comprise DNA-dependent DNA polymerase signatures as the result of the wild-type enzyme, e.g., IPR006134, PF00136, cd05536.


In some embodiments, the reverse transcription domain only recognizes and reverse transcribes a specific template. In some embodiments, the template comprises of specific sequences. In some embodiments, the template comprises inclusion of a UTR that associates the nucleic acid with the reverse transcriptase domain (e.g. an untranslated region (UTR) from a retrotransposon, e.g. the 3′ UTR of an R2 retrotransposon).


The writing domain may also comprise DNA-dependent DNA polymerase activity, e.g., comprise enzymatic activity capable of writing DNA into the genome from a template DNA sequence. In some embodiments, the DNA-dependent DNA polymerase activity is provided by a DNA polymerase domain in the polypeptide. In some embodiments, the DNA-dependent DNA polymerase activity is provided by a reverse transcriptase domain that is also capable of DNA-dependent DNA polymerization, e.g., second strand synthesis.


In some embodiments, a writing domain (e.g., RT domain) comprises an RNA-binding domain, e.g., that specifically binds to an RNA sequence. In some embodiments, a template RNA comprises an RNA sequence that is specifically bound by the RNA-binding domain of the writing domain.


In contrast to other types of reverse transcription machines, e.g., retroviral RTs and LTR retrotransposons, reverse transcription in non-LTR retrotransposons like R2 is performed only on RNA templates containing specific recognition sequences. The R2 retrotransposase requires its template to contain a minimal 3′ UTR region in order to initiate TPRT (Luan and Eickbush Mol Cell Biol 15, 3882-91 (1995)). In some embodiments, the Gene Writer polypeptide is derived from a retrotransposase with a required binding motif and the template RNA is designed to contain said binding motif, such that there is specific retrotransposition of only the desired template. In some embodiments, the Gene Writer polypeptide is derived from a retrotransposon selected from Table 3 and the 3′ UTR on the RNA template comprises the 3′ UTR from the same retrotransposon in Table 3.


Template Nucleic Acid Binding Domain:


The Gene Writer™ polypeptide typically contains regions capable of associating with the Gene Writer™ template nucleic acid (e.g., template RNA). In some embodiments, the template nucleic acid binding domain is an RNA binding domain. In some embodiments, the RNA binding domain is a modular domain that can associate with RNA molecules containing specific signatures, e.g., structural motifs, e.g., secondary structures present in the 3′ UTR in non-LTR retrotransposons. In other embodiments, the template nucleic acid binding domain (e.g., RNA binding domain) is contained within the reverse transcription domain, e.g., the reverse transcriptase-derived component has a known signature for RNA preference, e.g., secondary structures present in the 3′ UTR in non-LTR retrotransposons. In other embodiments, the template nucleic acid binding domain (e.g., RNA binding domain) is contained within the DNA binding domain. For example, in some embodiments, the DNA binding domain is a CRISPR-associated protein that recognizes the structure of a template nucleic acid (e.g., template RNA) comprising a gRNA. In some embodiments, the gRNA is a short synthetic RNA composed of a scaffold sequence that participates in CRISPR-associated protein binding and a user-defined ˜20 nucleotide targeting sequence for a genomic target. The structure of a complete gRNA was described by Nishimasu et al. Cell 156, P935-949 (2014). The gRNA (also referred to as sgRNA for single-guide RNA) consists of crRNA- and tracrRNA-derived sequences connected by an artificial tetraloop. The crRNA sequence can be divided into guide (20 nt) and repeat (12 nt) regions, whereas the tracrRNA sequence can be divided into anti-repeat (14 nt) and three tracrRNA stem loops (Nishimasu et al. Cell 156, P935-949 (2014)). In practice, guide RNA sequences are generally designed to have a length of between 17-24 nucleotides (e.g., 19, 20, or 21 nucleotides) and be complementary to a targeted nucleic acid sequence. Custom gRNA generators and algorithms are available commercially for use in the design of effective guide RNAs. In some embodiments, the gRNA comprises two RNA components from the native CRISPR system, e.g. crRNA and tracrRNA. As is well known in the art, the gRNA may also comprise a chimeric, single guide RNA (sgRNA) containing sequence from both a tracrRNA (for binding the nuclease) and at least one crRNA (to guide the nuclease to the sequence targeted for editing/binding). Chemically modified sgRNAs have also been demonstrated to be effective for use with CRISPR-associated proteins; see, for example, Hendel et al. (2015) Nature Biotechnol., 985-991. In some embodiments, a gRNA comprises a nucleic acid sequence that is complementary to a DNA sequence associated with a target gene. In some embodiments, a polypeptide comprises a DNA-binding domain comprising a CRISPR-associated protein that associates with a gRNA that allows the DNA-binding domain to bind a target genomic DNA sequence. In some embodiments, the gRNA is comprised within the template nucleic acid (e.g., template RNA), thus the DNA-binding domain is also the template nucleic acid binding domain. In some embodiments, the polypeptide possesses RNA binding function in multiple domains, e.g., can bind a gRNA structure in a CRISPR-associated DNA binding domain and a 3′ UTR structure in a non-LTR retrotransposon derived reverse transcription domain.


Endonuclease Domain:


In some embodiments, a Gene Writer™ polypeptide possesses the function of DNA target site cleavage via an endonuclease domain. In some embodiments, the endonuclease domain is also a DNA-binding domain. In some embodiments, the endonuclease domain is also a template nucleic acid (e.g., template RNA) binding domain. For example, in some embodiments a polypeptide comprises a CRISPR-associated endonuclease domain that binds a template RNA comprising a gRNA, binds a target DNA sequence (e.g., with complementarity to a portion of the gRNA), and cuts the target DNA sequence. In certain embodiments, the endonuclease/DNA binding domain of an APE-type retrotransposon or the endonuclease domain of an RLE-type retrotransposon can be used or can be modified (e.g., by insertion, deletion, or substitution of one or more residues) in a Gene Writer™ system described herein. In some embodiments the endonuclease domain or endonuclease/DNA binding domain is altered from its natural sequence to have altered codon usage, e.g. improved for human cells. In some embodiments the endonuclease element is a heterologous endonuclease element, such as Fok1 nuclease, a type-II restriction 1-like endonuclease (RLE-type nuclease), or another RLE-type endonuclease (also known as REL). In some embodiments the heterologous endonuclease activity has nickase activity and does not form double stranded breaks. The amino acid sequence of an endonuclease domain of a Gene Writer™ system described herein may be at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to the amino acid sequence of an endonuclease domain of a retrotransposon whose DNA sequence is referenced in Table 1 or 3. A person having ordinary skill in the art is capable of identifying endonuclease domains based upon homology to other known endonuclease domains using tools such as Basic Local Alignment Search Tool (BLAST). In certain embodiments, the heterologous endonuclease is Fok1 or a functional fragment thereof. In certain embodiments, the heterologous endonuclease is a Holliday junction resolvase or homolog thereof, such as the Holliday junction resolving enzyme from Sulfolobus solfataricus-Ssol Hje (Govindaraju et al., Nucleic Acids Research 44:7, 2016). In certain embodiments, the heterologous endonuclease is the endonuclease of the large fragment of a spliceosomal protein, such as Prp8 (Mahbub et al., Mobile DNA 8:16, 2017). In certain embodiments, the heterologous endonuclease is derived from a CRISPR-associated protein, e.g., Cas9. In certain embodiments, the heterologous endonuclease is engineered to have only ssDNA cleavage activity, e.g., only nickase activity, e.g., be a Cas9 nickase. For example, a Gene Writer™ polypeptide described herein may comprise a reverse transcriptase domain from an APE- or RLE-type retrotransposon and an endonuclease domain that comprises Fok1 or a functional fragment thereof. In still other embodiments, homologous endonuclease domains are modified, for example by site-specific mutation, to alter DNA endonuclease activity. In still other embodiments, endonuclease domains are modified to remove any latent DNA-sequence specificity.


In some embodiments the endonuclease domain has nickase activity and does not form double stranded breaks. In some embodiments, the endonuclease domain forms single stranded breaks at a higher frequency than double stranded breaks, e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% of the breaks are single stranded breaks, or less than 10%, 5%, 4%, 3%, 2%, or 1% of the breaks are double stranded breaks. In some embodiments, the endonuclease forms substantially no double stranded breaks. In some embodiments, the enonuclease does not form detectable levels of double stranded breaks.


In some embodiments, the endonuclease domain has nickase activity that nicks the target site DNA of the first strand; e.g., in some embodiments, the endonuclease domain cuts the genomic DNA of the target site near to the site of alteration on the strand that will be extended by the writing domain. In some embodiments, the endonuclease domain has nickase activity that nicks the target site DNA of the first strand and does not nick the target site DNA of the second strand. For example, when a polypeptide comprises a CRISPR-associated endonuclease domain having nickase activity and that does not form double stranded breaks, in some embodiments said CRISPR-associated endonuclease domain nicks the target site DNA strand containing the PAM site (e.g., and does not nick the target site DNA strand that does not contain the PAM site). As a further example, when a polypeptide comprises a CRISPR-associated endonuclease domain having nickase activity and that does not form double stranded breaks, in some embodiments said CRISPR-associated endonuclease domain nicks the target site DNA strand not containing the PAM site (e.g., and does not nick the target site DNA strand that contains the PAM site).


In some other embodiments, the endonuclease domain has nickase activity that nicks the target site DNA of the first strand and the second strand. Without wishing to be bound by theory, after a writing domain (e.g., RT domain) of a polypeptide described herein polymerizes (e.g., reverse transcribes) from the heterologous object sequence of a template nucleic acid (e.g., template RNA), the cellular DNA repair machinery must repair the nick on the first DNA strand. The target site DNA now contains two different sequences for the first DNA strand: one corresponding to the original genomic DNA and a second corresponding to that polymerized from the heterologous object sequence. It is thought that the two different sequences equilibrate with one another, first one hybridizing the second strand, then the other, and which the cellular DNA repair apparatus incorporates into its repaired target site is thought to be random. Without wishing to be bound by theory, it is thought that introducing an additional nick to the second strand may bias the cellular DNA repair machinery to adopt the heterologous object sequence-based sequence more frequently than the original genomic sequence. In some embodiments, the additional nick is positioned at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 nucleotides 5′ or 3′ of the target site modification (e.g., the insertion, deletion, or substitution) or to the nick on the first strand.


Alternatively or additionally, without wishing to be bound by theory, it is thought that an additional nick to the second strand may promote second strand synthesis. In some embodiments, where the Gene Writer™ has inserted or substituted a portion of the first strand, synthesis of a new sequence corresponding to the insertion/substitution in the second strand is necessary.


In some embodiments, the polypeptide comprises a single domain having endonuclease activity (e.g., a single endonuclease domain) and said domain nicks both the first strand and the second strand. For example, in such an embodiment the endonuclease domain may be a CRISPR-associated endonuclease domain, and the template nucleic acid (e.g., template RNA) comprises a gRNA that directs nicking of the first strand and an additional gRNA that directs nicking of the second strand. In some embodiments, the polypeptide comprises a plurality of domains having endonuclease activity, and a first endonuclease domain nicks the first strand and a second endonuclease domain nicks the second strand (optionally, the first endonuclease domain does not (e.g., cannot) nick the second strand and the second endonuclease domain does not (e.g., cannot) nick the first strand).


In some embodiments, the endonuclease domain is capable of nicking a first strand and a second strand. In some embodiments, the first and second strand nicks occur at the same position in the target site but on opposite strands. In some embodiments, the second strand nick occurs in a staggered location, e.g., upstream or downstream, from the first nick. In some embodiments, the endonuclease domain generates a target site deletion if the second strand nick is upstream of the first strand nick. In some embodiments, the endonuclease domain generates a target site duplication if the second strand nick is downstream of the first strand nick. In some embodiments, the endonuclease domain generates no duplication and/or deletion if the first and second strand nicks occur in the same position of the target site (e.g., as described in Gladyshev and Arkhipova Gene 2009, incorporated by reference herein in its entirety). In some embodiments, the endonuclease domain has altered activity depending on protein conformation or RNA-binding status, e.g., which promotes the nicking of the first or second strand (e.g., as described in Christensen et al. PNAS 2006; incorporated by reference herein in its entirety).


In some embodiments, a Gene Writer polypeptide comprises a modification to an endonuclease domain, e.g., relative to the wild-type polypeptide. In some embodiments, the endonuclease domain comprises an addition, deletion, replacement, or modification to the amino acid sequence of the original endonuclease domain. In some embodiments, the endonuclease domain is modified to include a heterologous functional domain that binds specifically to and/or induces endonuclease cleavage of a target nucleic acid (e.g., DNA) sequence of interest. In some embodiments, the endonuclease domain comprises a zinc finger. In some embodiments, the endonuclease domain comprises a Cas domain (e.g., a Cas9 or a mutant or variant thereof). In embodiments, the endonuclease domain comprising the Cas domain is associated with a guide RNA (gRNA), e.g., as described herein. In some embodiments, the endonuclease domain is modified to include a functional domain that does not target a specific target nucleic acid (e.g., DNA) sequence. In embodiments, the endonuclease domain comprises a Fok1 domain.


In some embodiments, the endonuclease domain comprises a meganuclease, or a functional fragment thereof. In some embodiments, the endonuclease domain comprises a homing endonuclease, or a functional fragment thereof. In some embodiments, the endonuclease domain comprises a meganuclease from the LAGLIDADG (SEQ ID NO: 1577), GIY-YIG, HNH, His-Cys Box, or PD-(D/E) XK families, or a functional fragment or variant thereof, e.g., which possess conserved amino acid motifs, e.g., as indicated in the family names. In some embodiments, the endonuclease domain comprises a meganuclease, or fragment thereof, chosen from, e.g., I-SmaMI (Uniprot F7WD42), I-SceI (Uniprot P03882), I-Anil (Uniprot P03880), I-DmoI (Uniprot P21505), I-CreI (Uniprot P05725), I-TevI (Uniprot P13299), I-OnuI (Uniprot Q4VWW5), or I-BmoI (Uniprot Q9ANR6). In some embodiments, the meganuclease is naturally monomeric, e.g., I-SceI, I-TevI, or dimeric, e.g., I-CreI, in its functional form. For example, the LAGLIDADG (SEQ ID NO: 1577) meganucleases with a single copy of the LAGLIDADG motif (SEQ ID NO: 1577) generally form homodimers, whereas members with two copies of the LAGLIDADG motif (SEQ ID NO: 1577) are generally found as monomers. In some embodiments, a meganuclease that normally forms as a dimer is expressed as a fusion, e.g., the two subunits are expressed as a single ORF and, optionally, connected by a linker, e.g., an I-CreI dimer fusion (Rodriguez-Fornes et al. Gene Therapy 2020; incorporated by reference herein in its entirety). In some embodiments, a meganuclease, or a functional fragment thereof, is altered to favor nickase activity for one strand of a double-stranded DNA molecule, e.g., I-SceI (K122I and/or K223I) (Niu et al. J Mol Biol 2008), I-Anil (K227M) (McConnell Smith et al. PNAS 2009), I-DmoI (Q42A and/or K120M) (Molina et al. J Biol Chem 2015). In some embodiments, a meganuclease or functional fragment thereof possessing this preference for single-strand cleavage is used as an endonuclease domain, e.g., with nickase activity. In some embodiments, an endonuclease domain comprises a meganuclease, or a functional fragment thereof, which naturally targets or is engineered to target a safe harbor site, e.g., an I-CreI targeting SH6 site (Rodriguez-Fornes et al., supra). In some embodiments, an endonuclease domain comprises a meganuclease, or a functional fragment thereof, with a sequence tolerant catalytic domain, e.g., I-TevI recognizing the minimal motif CNNNG (Kleinstiver et al. PNAS 2012). In some embodiments, a target sequence tolerant catalytic domain is fused to a DNA binding domain, e.g., to direct activity, e.g., by fusing I-TevI to: (i) zinc fingers to create Tev-ZFEs (Kleinstiver et al. PNAS 2012), (ii) other meganucleases to create MegaTevs (Wolfs et al. Nucleic Acids Res 2014), and/or (iii) Cas9 to create TevCas9 (Wolfs et al. PNAS 2016).


In some embodiments, the endonuclease domain comprises a restriction enzyme, e.g., a Type IIS or Type IIP restriction enzyme. In some embodiments, the endonuclease domain comprises a Type IIS restriction enzyme, e.g., FokI, or a fragment or variant thereof. In some embodiments, the endonuclease domain comprises a Type IIP restriction enzyme, e.g., PvuII, or a fragment or variant thereof. In some embodiments, a dimeric restriction enzyme is expressed as a fusion such that it functions as a single chain, e.g., a FokI dimer fusion (Minczuk et al. Nucleic Acids Res 36(12):3926-3938 (2008)).


The use of additional endonuclease domains is described, for example, in Guha and Edgell Int J Mol Sci 18(22):2565 (2017), which is incorporated herein by reference in its entirety.


In some embodiments, an endonuclease domain comprises a CRISPR/Cas domain (also referred to herein as a CRISPR-associated protein). In some embodiments, a DNA-binding domain comprises a CRISPR/Cas domain. In some embodiments, a CRISPR/Cas domain comprises a protein involved in the clustered regulatory interspaced short palindromic repeat (CRISPR) system, e.g., a Cas protein, and optionally binds a guide RNA, e.g., single guide RNA (sgRNA).


CRISPR systems are adaptive defense systems originally discovered in bacteria and archaea. CRISPR systems use RNA-guided nucleases termed CRISPR-associated or “Cas” endonucleases (e. g., Cas9 or Cpf1) to cleave foreign DNA. For example, in a typical CRISPR/Cas system, an endonuclease is directed to a target nucleotide sequence (e. g., a site in the genome that is to be sequence-edited) by sequence-specific, non-coding “guide RNAs” that target single- or double-stranded DNA sequences. Three classes (I-III) of CRISPR systems have been identified. The class II CRISPR systems use a single Cas endonuclease (rather than multiple Cas proteins). One class II CRISPR system includes a type II Cas endonuclease such as Cas9, a CRISPR RNA (“crRNA”), and a trans-activating crRNA (“tracrRNA”). The crRNA contains a “guide RNA”, typically about 20-nucleotide RNA sequence that corresponds to a target DNA sequence. In the wild-type system, and in some engineered systems, crRNA also contains a region that binds to the tracrRNA to form a partially double-stranded structure which is cleaved by RNase III, resulting in a crRNA/tracrRNA hybrid. A crRNA/tracrRNA hybrid then directs Cas9 endonuclease to recognize and cleave a target DNA sequence. A target DNA sequence is generally adjacent to a “protospacer adjacent motif” (“PAM”) that is specific for a given Cas endonuclease; however, PAM sequences appear throughout a given genome. CRISPR endonucleases identified from various prokaryotic species have unique PAM sequence requirements; examples of PAM sequences include 5′-NGG (Streptococcus pyogenes), 5′-NNAGAA (Streptococcus thermophilus CRISPR1), 5′-NGGNG (Streptococcus thermophilus CRISPR3), and 5′-NNNGATT (Neisseria meningiditis). Some endonucleases, e.g., Cas9 endonucleases, are associated with G-rich PAM sites, e. g., 5′-NGG, and perform blunt-end cleaving of the target DNA at a location 3 nucleotides upstream from (5′ from) the PAM site. Another class II CRISPR system includes the type V endonuclease Cpf1, which is smaller than Cas9; examples include AsCpf1 (from Acidaminococcus sp.) and LbCpf1 (from Lachnospiraceae sp.). Cpf1-associated CRISPR arrays are processed into mature crRNAs without the requirement of a tracrRNA; in other words, a Cpf1 system, in some embodiments, comprises only Cpf1 nuclease and a crRNA to cleave a target DNA sequence. Cpf1 endonucleases, are typically associated with T-rich PAM sites, e. g., 5′-TTN. Cpf1 can also recognize a 5′-CTA PAM motif. Cpf1 typically cleaves a target DNA by introducing an offset or staggered double-strand break with a 4- or 5-nucleotide 5′ overhang, for example, cleaving a target DNA with a 5-nucleotide offset or staggered cut located 18 nucleotides downstream from (3′ from) from a PAM site on the coding strand and 23 nucleotides downstream from the PAM site on the complimentary strand; the 5-nucleotide overhang that results from such offset cleavage allows more precise genome editing by DNA insertion by homologous recombination than by insertion at blunt-end cleaved DNA. See, e.g., Zetsche et al. (2015) Cell, 163:759-771.


A variety of CRISPR associated (Cas) genes or proteins can be used in the technologies provided by the present disclosure and the choice of Cas protein will depend upon the particular conditions of the method. Specific examples of Cas proteins include class II systems including Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cpf1, C2C1, or C2C3. In some embodiments, a Cas protein, e.g., a Cas9 protein, may be from any of a variety of prokaryotic species. In some embodiments a particular Cas protein, e.g., a particular Cas9 protein, is selected to recognize a particular protospacer-adjacent motif (PAM) sequence. In some embodiments, a DNA-binding domain or endonuclease domain includes a sequence targeting polypeptide, such as a Cas protein, e.g., Cas9. In certain embodiments a Cas protein, e.g., a Cas9 protein, may be obtained from a bacteria or archaea or synthesized using known methods. In certain embodiments, a Cas protein may be from a gram positive bacteria or a gram negative bacteria. In certain embodiments, a Cas protein may be from a Streptococcus (e.g., a S. pyogenes, or a S. thermophilus), a Francisella (e.g., an F. novicida), a Staphylococcus (e.g., an S. aureus), an Acidaminococcus (e.g., an Acidaminococcus sp. BV3L6), a Neisseria (e.g., an N. meningitidis), a Cryptococcus, a Corynebacterium, a Haemophilus, a Eubacterium, a Pasteurella, a Prevotella, a Veillonella, or a Marinobacter.


In some embodiments, a Cas protein requires a protospacer adjacent motif (PAM) to be present in or adjacent to a target DNA sequence for the Cas protein to bind and/or function. In some embodiments, the PAM is or comprises, from 5′ to 3′, NGG, YG, NNGRRT, NNNRRT, NGA, TYCV, TATV, NTTN, or NNNGATT, where N stands for any nucleotide, Y stands for C or T, R stands for A or G, and V stands for A or C or G. In some embodiments, a Cas protein is a protein listed in Table 4. In some embodiments, a Cas protein comprises one or more mutations altering its PAM. In some embodiments, a Cas protein comprises E1369R, E1449H, and R1556A mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises E782K, N968K, and R1015H mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises D1135V, R1335Q, and T1337R mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises S542R and K607R mutations or analogous substitutions to the amino acids corresponding to said positions. In some embodiments, a Cas protein comprises S542R, K548V, and N552R mutations or analogous substitutions to the amino acids corresponding to said positions.









TABLE 4







CRISPR/Cas Proteins, Species, and Mutations
















# of

Mutations to alter
Mutations to make


Name
Enzyme
Species
AAs
PAM
PAM recognition
catalytically dead
















FnCas9
Cas9

Francisella

1629
5′-NGG-3′
Wt
D11A/H969A/N995A





novicida







FnCas9
Cas9

Francisella

1629
5′-YG-3′
E1369R/E1449H/
D11A/H969A/N995A


RHA


novicida



R1556A



SaCas9
Cas9

Staphylococcus

1053
5′-NNGRRT-3′
Wt
D10A/H557A





aureus







SaCas9
Cas9

Staphylococcus

1053
5′-NNNRRT-3′
E782K/N968K/
D10A/H557A


KKH


aureus



R1015H



SpCas9
Cas9

Streptococcus

1368
5′-NGG-3′
Wt
D10A/D839A/H840A/





pyogenes




N863A


SpCas9
Cas9

Streptococcus

1368
5′-NGA-3′
D1135V/R1335Q/
D10A/D839A/H840A/


VQR


pyogenes



T1337R
N863A


AsCpf1
Cpf1

Acidaminococcus

1307
5′-TYCV-3′
S542R/K607R
E993A


RR

sp. BV3L6






AsCpf1
Cpf1

Acidaminococcus

1307
5′-TATV-3′
S542R/K548V/
E993A


RVR

sp. BV3L6


N552R



FnCpf1
Cpf1

Francisella

1300
5′-NTTN-3′
Wt
D917A/E1006A/





novicida




D1255A


NmCas9
Cas9

Neisseria

1082
5′-NNNGATT-3′
Wt
D16A/D587A/H588A/





meningitidis




N611A









In some embodiments, the Cas protein is catalytically active and cuts one or both strands of the target DNA site. In some embodiments, cutting the target DNA site is followed by formation of an alteration, e.g., an insertion or deletion, e.g., by the cellular repair machinery.


In some embodiments, the Cas protein is modified to deactivate or partially deactivate the nuclease, e.g., nuclease-deficient Cas9. Whereas wild-type Cas9 generates double-strand breaks (DSBs) at specific DNA sequences targeted by a gRNA, a number of CRISPR endonucleases having modified functionalities are available, for example: a “nickase” version of Cas9 that has been partially deactivated generates only a single-strand break; a catalytically inactive Cas9 (“dCas9”) does not cut target DNA. In some embodiments, dCas9 binding to a DNA sequence may interfere with transcription at that site by steric hindrance. In some embodiments, dCas9 binding to an anchor sequence may interfere with (e.g., decrease or prevent) genomic complex (e.g., ASMC) formation and/or maintenance. In some embodiments, a DNA-binding domain comprises a catalytically inactive Cas9, e.g., dCas9. Many catalytically inactive Cas9 proteins are known in the art. In some embodiments, dCas9 comprises mutations in each endonuclease domain of the Cas protein, e.g., D10A and H840A or N863A mutations. In some embodiments, a catalytically inactive or partially inactive CRISPR/Cas domain comprises a Cas protein comprising one or more mutations, e.g., one or more of the mutations listed in Table 4. In some embodiments, a Cas protein described on a given row of Table 4 comprises one, two, three, or all of the mutations listed in the same row of Table 4. In some embodiments, a Cas protein, e.g., not described in Table 4, comprises one, two, three, or all of the mutations listed in a row of Table 4 or a corresponding mutation at a corresponding site in that Cas protein.


In some embodiments, a catalytically inactive, e.g., dCas9, or partially deactivated Cas9 protein comprises a D11 mutation (e.g., D11A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a H969 mutation (e.g., H969A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a N995 mutation (e.g., N995A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises mutations at one, two, or three of positions D11, H969, and N995 (e.g., D11A, H969A, and N995A mutations) or analogous substitutions to the amino acids corresponding to said positions.


In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D10 mutation (e.g., a D10A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a H557 mutation (e.g., a H557A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D10 mutation (e.g., a D10A mutation) and a H557 mutation (e.g., a H557A mutation) or analogous substitutions to the amino acids corresponding to said positions.


In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D839 mutation (e.g., a D839A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a H840 mutation (e.g., a H840A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a N863 mutation (e.g., a N863A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D10 mutation (e.g., D10A), a D839 mutation (e.g., D839A), a H840 mutation (e.g., H840A), and a N863 mutation (e.g., N863A) or analogous substitutions to the amino acids corresponding to said positions.


In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a E993 mutation (e.g., a E993A mutation) or an analogous substitution to the amino acid corresponding to said position.


In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a a D917 mutation (e.g., a D917A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a a E1006 mutation (e.g., a E1006A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D1255 mutation (e.g., a D1255A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D917 mutation (e.g., D917A), a E1006 mutation (e.g., E1006A), and a D1255 mutation (e.g., D1255A) or analogous substitutions to the amino acids corresponding to said positions.


In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a a D16 mutation (e.g., a D16A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a D587 mutation (e.g., a D587A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a H588 mutation (e.g., a H588A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, or partially deactivated Cas9 protein comprises a N611 mutation (e.g., a N611A mutation) or an analogous substitution to the amino acid corresponding to said position. In some embodiments, a catalytically inactive Cas9 protein, e.g., dCas9, comprises a D16 mutation (e.g., D16A), a D587 mutation (e.g., D587A), a H588 mutation (e.g., H588A), and a N611 mutation (e.g., N611A) or analogous substitutions to the amino acids corresponding to said positions.


In some embodiments, a DNA-binding domain or endonuclease domain may comprise a Cas molecule comprising or linked (e.g., covalently) to a gRNA (e.g., a template nucleic acid, e.g., template RNA, comprising a gRNA).


In some embodiments, an endonuclease domain or DNA binding domain comprises a Streptococcus pyogenes Cas9 (SpCas9) or a functional fragment or variant thereof. In some embodiments, the endonuclease domain or DNA binding domain comprises a modified SpCas9. In embodiments, the modified SpCas9 comprises a modification that alters protospacer-adjacent motif (PAM) specificity. In embodiments, the PAM has specificity for the nucleic acid sequence 5′-NGT-3′. In embodiments, the modified SpCas9 comprises one or more amino acid substitutions, e.g., at one or more of positions L1111, D1135, G1218, E1219, A1322, of R1335, e.g., selected from L1111R, D1135V, G1218R, E1219F, A1322R, R1335V. In embodiments, the modified SpCas9 comprises the amino acid substitution T1337R and one or more additional amino acid substitutions, e.g., selected from L1111, D1135L, S1136R, G1218S, E1219V, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, R1335Q, T1337, T1337L, T1337Q, T1337I, T1337V, T1337F, T1337S, T1337N, T1337K, T1337H, T1337Q, and T1337M, or corresponding amino acid substitutions thereto. In embodiments, the modified SpCas9 comprises: (i) one or more amino acid substitutions selected from D1135L, S1136R, G1218S, E1219V, A1322R, R1335Q, and T1337; and (ii) one or more amino acid substitutions selected from L1111R, G1218R, E1219F, D1332A, D1332S, D1332T, D1332V, D1332L, D1332K, D1332R, T1337L, T1337I, T1337V, T1337F, T1337S, T1337N, T1337K, T1337R, T1337H, T1337Q, and T1337M, or corresponding amino acid substitutions thereto.


In some embodiments, a Gene Writer may comprise a Cas protein as listed in Table 40A. The predicted or validated nickase mutations for installing Nickase activity in the Cas protein as shown in Table 40A, are based on the signature of the SpCas9(N863A) mutation. In some embodiments, system described herein comprises a GeneWriter protein of Table 3 and a Cas protein of Table 40A. In some embodiments, a protein or domain of Table 3, 41, or 44 is fused to a Cas protein of Table 40A.









TABLE 40A







CRISPR/Cas Proteins, Species, and Mutations













SEQ






ID

Nickase


Variant
Parental Host
NO:
Protein Sequence
Mutation





Nme2Cas9

Neisseria

3262
MAAFKPNPINYILGLDIGIASVGWAMVEIDEEENPIRLIDLGVRVFERAEVPKTGDSLAMARRLAR
N611A




meningitidis


SVRRLTRRRAHRLLRARRLLKREGVLQAADFDENGLIKSLPNTPWQLRAAALDRKLTPLEWSAVL






LHLIKHRGYLSQRKNEGETADKELGALLKGVANNAHALQTGDFRTPAELALNKFEKESGHIRNQR






GDYSHTFSRKDLQAELILLFEKQKEFGNPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFE






PAEPKAAKNTYTAERFIWLTKLNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGL






EDTAFFKGLRYGKDNAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSSELQDEIGTAFSLFKT






DEDITGRLKDRVQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKN






TEEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRKEIEKRQEE






NRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLVRLNEKGYVEIDHAL






PFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFKARVETSRFPRSKKQRILLQ






KFDEDGFKECNLNDTRYVNRFLCQFVADHILLTGKGKRRVFASNGQITNLLRGFWGLRKVRAEN






DRHHALDAVVVACSTVAMQQKITRFVRYKEMNAFDGKTIDKETGKVLHQKTHFPQPWEFFAQE






VMIRVFGKPDGKPEFEEADTPEKLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSGAHKDTLRS






AKRFVKHNEKISVKRVWLTEIKLADLENMVNYKNGREIELYEALKARLEAYGGNAKQAFDPKDN






PFYKKGGQLVKAVRVEKTQESGVLLNKKNAYTIADNGDMVRVDVFCKVDKKGKNQYFIVPIYA






WQVAENILPDIDCKGYRIDDSYTFCFSLHKYDLIAFQKDEKSKVEFAYYINCDSSNGRFYLAWHD






KGSKEQQFRISTQNLVLIQKYQVNELGKEIRPCRLKKRPPVR






PpnCas9

Pasteurella

3263
MQNNPLNYILGLDLGIASIGWAVVEIDEESSPIRLIDVGVRTFERAEVAKTGESLALSRRLARSSRR
N605A




pneumotropica


LIKRRAERLKKAKRLLKAEKILHSIDEKLPINVWQLRVKGLKEKLERQEWAAVLLHLSKHRGYLS






QRKNEGKSDNKELGALLSGIASNHQMLQSSEYRTPAEIAVKKFQVEEGHIRNQRGSYTHTFSRLDL






LAEMELLFORQAELGNSYTSTTLLENLTALLMWQKPALAGDAILKMLGKCTFEPSEYKAAKNSY






SAERFVWLTKLNNLRILENGTERALNDNERFALLEQPYEKSKLTYAQVRAMLALSDNAIFKGVRY






LGEDKKTVESKTTLIEMKFYHQIRKTLGSAELKKEWNELKGNSDLLDEIGTAFSLYKTDDDICRYL






EGKLPERVLNALLENLNFDKFIQLSLKALHQILPLMLQGQRYDEAVSAIYGDHYGKKSTETTRLLP






TIPADEIRNPVVLRTLTQARKVINAVVRLYGSPARIHIETAREVGKSYQDRKKLEKQQEDNRKQRE






SAVKKFKEMFPHFVGEPKGKDILKMRLYELQQAKCLYSGKSLELHRLLEKGYVEVDHALPFSRT






WDDSFNNKVLVLANENQNKGNLTPYEWLDGKNNSERWQHFVVRVQTSGFSYAKKQRILNHKLD






EKGFIERNLNDTRYVARFLCNFIADNMLLVGKGKRNVFASNGQITALLRHRWGLQKVREQNDRH






HALDAVVVACSTVAMQQKITRFVRYNEGNVFSGERIDRETGEIIPLHFPSPWAFFKENVEIRIFSEN






PKLELENRLPDYPQYNHEWVQPLFVSRMPTRKMTGQGHMETVKSAKRLNEGLSVLKVPLTQLKL






SDLERMVNRDREIALYESLKARLEQFGNDPAKAFAEPFYKKGGALVKAVRLEQTQKSGVLVRDG






NGVADNASMVRVDVFTKGGKYFLVPIYTWQVAKGILPNRAATQGKDENDWDIMDEMATFQFSL






CQNDLIKLVTKKKTIFGYFNGLNRATSNINIKEHDLDKSKGKLGIYLEVGVKLAISLEKYQVDELG






KNIRPCRPTKRQHVR






SauCas9

Staphylococcus

3264
MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQR
N580A




aureus


VKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNE






LSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQS






FIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALN






VYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSL






KAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIK






KYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGK






CLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISY






ETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVN






NLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQ






MFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGN






TLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETG






NYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFV






TVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRI






EVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG






SauCas9-

Staphylococcus

3265
MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQR
N580A


KKH

aureus


VKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNE






LSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQS






FIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALN






VYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSL






KAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIK






KYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGK






CLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISY






ETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVN






NLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQ






MFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKG






NTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEET






GNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKF






VTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYKNDLIKINGELYRVIGVNNDLLNR






IEVNMIDITYREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG






SauriCas9

Staphylococcus

3266
MQENQQKQNYILGLDIGITSVGYGLIDSKTREVIDAGVRLFPEADSENNSNRRSKRGARRLKRRRI
N588A




auricularis


HRLNRVKDLLADYQMIDLNNVPKSTDPYTIRVKGLREPLTKEEFAIALLHIAKRRGLHNISVSMGD






EEQDNELSTKQQLQKNAQQLQDKYVCELQLERLTNINKVRGEKNRFKTEDFVKEVKQLCETQRQ






YHNIDDQFIQQYIDLVSTRREYFEGPGNGSPYGWDGDLLKWYEKLMGRCTYFPEELRSVKYAYS






ADLFNALNDLNNLVVTRDDNPKLEYYEKYHIIENVFKQKKNPTLKQIAKEIGVQDYDIRGYRITKS






GKPQFTSFKLYHDLKNIFEQAKYLEDVEMLDEIAKILTIYQDEISIKKALDQLPELLTESEKSQIAQL






TGYTGTHRLSLKCIHIVIDELWESPENQMEIFTRLNLKPKKVEMSEIDSIPTTLVDEFILSPVVKRAFI






KIKLHDMQEGKCLYSLEAIPLEDLLSNPTHYEVDHIIPRSVSFDNSLNNKVLVKQSENSKKGNRTP






YQYLSSNESKISYNQFKQHILNLSKAKDRISKKKRDMLLEERDINKFEVQKEFINRNLVDTRYATR






ELSNLLKTYFSTHDYAVKVKTINGGFTNHLRKVWDFKKHRNHGYKHHAEDALVIANADFLFKTH






KALRRTDKILEQPGLEVNDTTVKVDTEEKYQELFETPKQVKNIKQFRDFKYSHRVDKKPNRQLIN






DTLYSTREIDGETYVVQTLKDLYAKDNEKVKKLFTERPQKILMYQHDPKTFEKLMTILNQYAEAK






NPLAAYYEDKGEYVTKYAKKGNGPAIHKIKYIDKKLGSYLDVSNKYPETQNKLVKLSLKSFRFDI






YKCEQGYKMVSIGYLDVLKKDNYYYIPKDKYEAEKQKKKIKESDLFVGSFYYNDLIMYEDELFR






VIGVNSDINNLVELNMVDITYKDFCEVNNVTGEKRIKKTIGKRVVLIEKYTTDILGNLYKTPLPKKP






QLIFKRGEL






SauriCas9-

Staphylococcus

3267
MQENQQKQNYILGLDIGITSVGYGLIDSKTREVIDAGVRLFPEADSENNSNRRSKRGARRLKRRRI
N588A


KKH

auricularis


HRLNRVKDLLADYQMIDLNNVPKSTDPYTIRVKGLREPLTKEEFAIALLHIAKRRGLHNISVSMGD






EEQDNELSTKQQLQKNAQQLQDKYVCELQLERLTNINKVRGEKNRFKTEDFVKEVKQLCETQRQ






YHNIDDQFIQQYIDLVSTRREYFEGPGNGSPYGWDGDLLKWYEKLMGRCTYFPEELRSVKYAYS






ADLFNALNDLNNLVVTRDDNPKLEYYEKYHIIENVFKQKKNPTLKQIAKEIGVQDYDIRGYRITKS






GKPQFTSFKLYHDLKNIFEQAKYLEDVEMLDEIAKILTIYQDEISIKKALDQLPELLTESEKSQIAQL






TGYTGTHRLSLKCIHIVIDELWESPENQMEIFTRLNLKPKKVEMSEIDSIPTTLVDEFILSPVVKRAFI






QSIKVINAVINRFGLPEDITIELAREKNSKDRRKFINKLQKQNEATRKKIEQLLAKYGNTNAKYMIE






KIKLHDMQEGKCLYSLEAIPLEDLLSNPTHYEVDHIIPRSVSFDNSLNNKVLVKQSENSKKGNRTP






YQYLSSNESKISYNQFKQHILNLSKAKDRISKKKRDMLLEERDINKFEVQKEFINRNLVDTRYATR






ELSNLLKTYFSTHDYAVKVKTINGGFTNHLRKVWDFKKHRNHGYKHHAEDALVIANADFLFKTH






KALRRTDKILEQPGLEVNDTTVKVDTEEKYQELFETPKQVKNIKQFRDFKYSHRVDKKPNRKLIN






DTLYSTREIDGETYVVQTLKDLYAKDNEKVKKLFTERPQKILMYQHDPKTFEKLMTILNQYAEAK






NPLAAYYEDKGEYVTKYAKKGNGPAIHKIKYIDKKLGSYLDVSNKYPETQNKLVKLSLKSFRFDI






YKCEQGYKMVSIGYLDVLKKDNYYYIPKDKYEAEKQKKKIKESDLFVGSFYKNDLIMYEDELFR






VIGVNSDINNLVELNMVDITYKDFCEVNNVTGEKHIKKTIGKRVVLIEKYTTDILGNLYKTPLPKK






PQLIFKRGEL






ScaCas9-

Streptococcus

3268
MEKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTNRKSIKKNLMGALLFDSGETAEATRLK
N872A


Sc++

canis


RTARRRYTRRKNRIRYLQEIFANEMAKLDDSFFQRLEESFLVEEDKKNERHPIFGNLADEVAYHRN






YPTIYHLRKKLADSPEKADLRLIYLALAHIIKFRGHFLIEGKLNAENSDVAKLFYQLIQTYNQLFEE






SPLDEIEVDAKGILSARLSKSKRLEKLIAVFPNEKKNGLFGNIIALALGLTPNFKSNFDLTEDAKLQL






SKDTYDDDLDELLGQIGDQYADLFSAAKNLSDAILLSDILRSNSEVTKAPLSASMVKRYDEHHQD






LALLKTLVRQQFPEKYAEIFKDDTKNGYAGYVGADKKLRKRSGKLATEEEFYKFIKPILEKMDGA






EELLAKLNRDDLLRKQRTFDNGSIPHQIHLKELHAILRRQEEFYPFLKENREKIEKILTFRIPYYVGP






LARGNSRFAWLTRKSEEAITPWNFEEVVDKGASAQSFIERMTNFDEQLPNKKVLPKHSLLYEYFT






VYNELTKVKYVTERMRKPEFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEIIGVED






RFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL






KRRHYTGWGRLSRKMINGIRDKQSGKTILDFLKSDGFSNRNFMQLIHDDSLTFKEEIEKAQVSGQG






DSLHEQIADLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIEMARENQTTTKGLQQSRERKKRI






EEGIKELESQILKENPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDD






SIDNKVLTRSVENRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSEADKA






GFIKRQLVETRQITKHVARILDSRMNTKRDKNDKPIREVKVITLKSKLVSDFRKDFQLYKVRDINN






YHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKRFFYSNIMN






FFKTEVKLANGEIRKRPLIETNGETGEVVWNKEKDFATVRKVLAMPQVNIVKKTEVQTGGFSKES






ILSKRESAKLIPRKKGWDTRKYGGFGSPTVAYSILVVAKVEKGKAKKLKSVKVLVGITIMEKGSY






EKDPIGFLEAKGYKDIKKELIFKLPKYSLFELENGRRRMLASAKELQKANELVLPQHLVRLLYYTQ






NISATTGSNNLGYIEQHREEFKEIFEKIIDFSEKYILKNKVNSNLKSSFDEQFAVSDSILLSNSFVSLL






KYTSFGASGGFTFLDLDVKQGRLRYQTVTEVLDATLIYQSITGLYETRTDLSQLGGD






SpyCas9

Streptococcus

3269
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKR
N863A




pyogenes


TARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY






PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE






NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ






LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQD






LTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED






LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW






MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKY






VTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTY






HDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWG






RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANL






AGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG






SQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVL






TRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQL






VETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHD






AYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL






ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDK






LIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEA






KGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSP






EDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLT






NLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD






SpyCas9-

Streptococcus

3270
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKR
N863A


NG

pyogenes


TARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY






PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE






NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ






LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQD






LTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED






LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW






MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKY






VTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTY






HDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWG






RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANL






AGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG






SQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVL






TRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQL






VETROITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHD






AYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL






ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIRPKRNSDK






LIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEA






KGYKEVKKDLIIKLPKYSLFELENGRKRMLASARFLQKGNELALPSKYVNFLYLASHYEKLKGSP






EDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLT






NLGAPRAFKYFDTTIDRKVYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD






SpyCas9-

Streptococcus

3271
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAERTRLKR
N863A


SpRY

pyogenes


TARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY






PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE






NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ






LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQD






LTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED






LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW






MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKY






VTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTY






HDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWG






RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANL






AGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG






SQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVL






TRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQL






VETROITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHD






AYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL






ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIRPKRNSDK






LIARKKDWDPKKYGGFLWPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLE






AKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAKQLQKGNELALPSKYVNFLYLASHYEKLKGS






PEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLT






RLGAPRAFKYFDTTIDPKQYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD






St1Cas9

Streptococcus

3272
MSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQAENNLVRRTNRQGRRLARRKKHRRVRL
N622A




thermophilus


NRLFEESGLITDFTKISINLNPYQLRVKGLTDELSNEELFIALKNMVKHRGISYLDDASDDGNSSVG






DYAQIVKENSKQLETKTPGQIQLERYQTYGQLRGDFTVEKDGKKHRLINVFPTSAYRSEALRILQT






QQEFNPQITDEFINRYLEILTGKRKYYHGPGNEKSRTDYGRYRTSGETLDNIFGILIGKCTFYPDEFR






AAKASYTAQEFNLLNDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLFKYIAKLLSCDVA






DIKGYRIDKSGKAEIHTFEAYRKMKTLETLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFAD






GSFSQKQVDELVQFRKANSSIFGKGWHNFSVKLMMELIPELYETSEEQMTILTRLGKQKTTSSSNK






TKYIDEKLLTEEIYNPVVAKSVRQAIKIVNAAIKEYGDFDNIVIEMARETNEDDEKKAIQKIQKANK






DEKDAAMLKAANQYNGKAELPHSVFHGHKQLATKIRLWHQQGERCLYTGKTISIHDLINNSNQF






EVDHILPLSITFDDSLANKVLVYATANQEKGQRTPYQALDSMDDAWSFRELKAFVRESKTLSNKK






KEYLLTEEDISKFDVRKKFIERNLVDTRYASRVVLNALQEHFRAHKIDTKVSVVRGQFTSQLRRH






WGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKNTLVSYSEDQLLDIETGELISDDEYKESVFKAP






YQHFVDTLKSKEFEDSILFSYQVDSKFNRKISDATIYATROAKVGKDKADETYVLGKIKDIYTQDG






YDAFMKIYKKDKSKFLMYRHDPQTFEKVIEPILENYPNKQINEKGKEVPCNPFLKYKEEHGYIRKY






SKKGNGPEIKSLKYYDSKLGNHIDITPKDSNNKVVLQSVSPWRADVYFNKTTGKYEILGLKYADL






QFEKGTGTYKISQEKYNDIKKKEGVDSDSEFKFTLYKNDLLLVKDTETKEQQLFRFLSRTMPKQK






HYVELKPYDKQKFEGGEALIKVLGNVANSGQCKKGLGKSNISIYKVRTDVLGNQHIIKNEGDKPK






LDF






BlatCas9

Brevibacillus

3273
MAYTMGIDVGIASCGWAIVDLERQRIIDIGVRTFEKAENPKNGEALAVPRREARSSRRRLRRKKHR
N607A




laterosporus


IERLKHMFVRNGLAVDIQHLEQTLRSQNEIDVWQLRVDGLDRMLTQKEWLRVLIHLAQRRGFQS






NRKTDGSSEDGQVLVNVTENDRLMEEKDYRTVAEMMVKDEKFSDHKRNKNGNYHGVVSRSSL






LVEIHTLFETQRQHHNSLASKDFELEYVNIWSAQRPVATKDQIEKMIGTCTFLPKEKRAPKASWHF






QYFMLLQTINHIRITNVQGTRSLNKEEIEQVVNMALTKSKVSYHDTRKILDLSEEYQFVGLDYGKE






DEKKKVESKETIIKLDDYHKLNKIFNEVELAKGETWEADDYDTVAYALTFFKDDEDIRDYLQNKY






KDSKNRLVKNLANKEYTNELIGKVSTLSFRKVGHLSLKALRKIIPFLEQGMTYDKACQAAGFDFQ






GISKKKRSVVLPVIDQISNPVVNRALTQTRKVINALIKKYGSPETIHIETARELSKTFDERKNITKDY






KENRDKNEHAKKHLSELGIINPTGLDIVKYKLWCEQQGRCMYSNQPISFERLKESGYTEVDHIIPY






SRSMNDSYNNRVLVMTRENREKGNQTPFEYMGNDTQRWYEFEQRVTTNPQIKKEKRONLLLKG






FTNRRELEMLERNLNDTRYITKYLSHFISTNLEFSPSDKKKKVVNTSGRITSHLRSRWGLEKNRGQ






NDLHHAMDAIVIAVTSDSFIQQVTNYYKRKERRELNGDDKFPLPWKFFREEVIARLSPNPKEQIEA






LPNHFYSEDELADLQPIFVSRMPKRSITGEAHQAQFRRVVGKTKEGKNITAKKTALVDISYDKNGD






FNMYGRETDPATYEAIKERYLEFGGNVKKAFSTDLHKPKKDGTKGPLIKSVRIMENKTLVHPVNK






GKGVVYNSSIVRTDVFQRKEKYYLLPVYVTDVTKGKLPNKVIVAKKGYHDWIEVDDSFTFLFSLY






PNDLIFIRQNPKKKISLKKRIESHSISDSKEVQEIHAYYKGVDSSTAAIEFIIHDGSYYAKGVGVQNL






DCFEKYQVDILGNYFKVKGEKRLELETSDSNHKGKDVNSIKSTSR






cCas9-

Staphylococcus

3274
MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQR
N580A


v16

aureus


VKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNE






LSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQS






FIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALN






DLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLK






VYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSL






KAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIK






KYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGK






CLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISY






ETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVN






NLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQ






MFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKG






NTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEET






GNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKF






VTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYKNDLIKINGELYRVIGVNSDKNNL






IEVNMIDITYREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG






cCas9-

Staphylococcus

3275
MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQR
N580A


v17

aureus


VKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNE






LSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQS






FIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALN






DLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLK






VYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSL






KAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIK






KYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGK






CLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISY






ETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVN






NLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQ






MFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKG






NTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEET






GNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKF






VTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYKNDLIKINGELYRVIGVNNSTRNI






VELNMIDITYREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG






cCas9-

Staphylococcus

3276
MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQR
N580A


v21

aureus


VKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNE






LSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQS






FIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALN






DLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLK






VYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSL






KAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIK






KYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGK






CLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISY






ETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVN






NLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQ






MFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKG






NTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEET






GNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKF






VTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYKNDLIKINGELYRVIGVNSDDRNII






ELNMIDITYREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG






cCas9-

Staphylococcus

3277
MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQR
N580A


v42

aureus


VKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDTGNE






LSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQS






FIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALN






VYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSL






KAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIK






KYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGK






CLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISY






ETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVN






NLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQ






MFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKG






NTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEET






GNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKF






VTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYKNDLIKINGELYRVIGVNNNRLNK






IELNMIDITYREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG






CdiCas9

Corynebacterium

3278
MKYHVGIDVGTFSVGLAAIEVDDAGMPIKTLSLVSHIHDSGLDPDEIKSAVTRLASSGIARRTRRL
H573A




diphtheriae


YRRKRRRLQQLDKFIQRQGWPVIELEDYSDPLYPWKVRAELAASYIADEKERGEKLSVALRHIAR
(Alternate)





HRGWRNPYAKVSSLYLPDGPSDAFKAIREEIKRASGQPVPETATVGQMVTLCELGTLKLRGEGGV






LSARLQQSDYAREIQEICRMQEIGQELYRKIIDVVFAAESPKGSASSRVGKDPLQPGKNRALKASD






AFQRYRIAALIGNLRVRVDGEKRILSVEEKNLVFDHLVNLTPKKEPEWVTIAEILGIDRGQLIGTAT






MTDDGERAGARPPTHDTNRSIVNSRIAPLVDWWKTASALEQHAMVKALSNAEVDDFDSPEGAK






VQAFFADLDDDVHAKLDSLHLPVGRAAYSEDTLVRLTRRMLSDGVDLYTARLQEFGIEPSWTPPT






PRIGEPVGNPAVDRVLKTVSRWLESATKTWGAPERVIIEHVREGFVTEKRAREMDGDMRRRAAR






NAKLFQEMQEKLNVQGKPSRADLWRYQSVQRQNCQCAYCGSPITFSNSEMDHIVPRAGQGSTNT






RENLVAVCHRCNQSKGNTPFAIWAKNTSIEGVSVKEAVERTRHWVTDTGMRSTDFKKFTKAVVE






RFQRATMDEEIDARSMESVAWMANELRSRVAQHFASHGTTVRVYRGSLTAEARRASGISGKLKF






FDGVGKSRLDRRHHAIDAAVIAFTSDYVAETLAVRSNLKQSQAHRQEAPQWREFTGKDAEHRAA






WRVWCQKMEKLSALLTEDLRDDRVVVMSNVRLRLGNGSAHKETIGKLSKVKLSSQLSVSDIDKA






SSEALWCALTREPGFDPKEGLPANPERHIRVNGTHVYAGDNIGLFPVSAGSIALRGGYAELGSSFH






HARVYKITSGKKPAFAMLRVYTIDLLPYRNQDLFSVELKPQTMSMRQAEKKLRDALATGNAEYL






GWLVVDDELVVDTSKIATDQVKAVEAELGTIRRWRVDGFFSPSKLRLRPLQMSKEGIKKESAPEL






SKIIDRPGWLPAVNKLFSDGNVTVVRRDSLGRVRLESTAHLPVTWKVQ






CjeCas9

Campylobacter

3279
MARILAFDIGISSIGWAFSENDELKDCGVRIFTKVENPKTGESLALPRRLARSARKRLARRKARLN
N582A




jejuni


HLKHLIANEFKLNYEDYQSFDESLAKAYKGSLISPYELRFRALNELLSKQDFARVILHIAKRRGYD






DIKNSDDKEKGAILKAIKQNEEKLANYQSVGEYLYKEYFQKFKENSKEFTNVRNKKESYERCIAQ






SFLKDELKLIFKKQREFGFSFSKKFEEEVLSVAFYKRALKDFSHLVGNCSFFTDEKRAPKNSPLAFM






FVALTRIINLLNNLKNTEGILYTKDDLNALLNEVLKNGTLTYKQTKKLLGLSDDYEFKGEKGTYFI






EFKKYKEFIKALGEHNLSQDDLNEIAKDITLIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNIS






FKALKLVTPLMLEGKKYDEACNELNLKVAINEDKKDFLPAFNETYYKDEVTNPVVLRAIKEYRK






VLNALLKKYGKVHKINIELAREVGKNHSQRAKIEKEQNENYKAKKDAELECEKLGLKINSKNILK






LRLFKEQKEFCAYSGEKIKISDLQDEKMLEIDHIYPYSRSFDDSYMNKVLVFTKQNQEKLNQTPFE






AFGNDSAKWQKIEVLAKNLPTKKQKRILDKNYKDKEQKNFKDRNLNDTRYIARLVLNYTKDYLD






FLPLSDDENTKLNDTQKGSKVHVEAKSGMLTSALRHTWGFSAKDRNNHLHHAIDAVIIAYANNSI






VKAFSDFKKEQESNSAELYAKKISELDYKNKRKFFEPFSGFRQKVLDKIDEIFVSKPERKKPSGALH






EETFRKEEEFYQSYGGKEGVLKALELGKIRKVNGKIVKNGDMFRVDIFKHKKTNKFYAVPIYTMD






FALKVLPNKAVARSKKGEIKDWILMDENYEFCFSLYKDSLILIQTKDMQEPEFVYYNAFTSSTVSLI






VSKHDNKFETLSKNQKILFKNANEKEVIAKSIGIQNLKVFEKYIVSALGEVTKAEFRQREDFKK






GeoCas9

Geobacillus

3280
MRYKIGLDIGITSVGWAVMNLDIPRIEDLGVRIFDRAENPQTGESLALPRRLARSARRRLRRRKHR
N605A




stearother


LERIRRLVIREGILTKEELDKLFEEKHEIDVWQLRVEALDRKLNNDELARVLLHLAKRRGFKSNRK





mophilus


SERSNKENSTMLKHIEENRAILSSYRTVGEMIVKDPKFALHKRNKGENYTNTIARDDLEREIRLIFS






KQREFGNMSCTEEFENEYITIWASQRPVASKDDIEKKVGFCTFEPKEKRAPKATYTFQSFIAWEHIN






KLRLISPSGARGLTDEERRLLYEQAFQKNKITYHDIRTLLHLPDDTYFKGIVYDRGESRKQNENIRF






LELDAYHQIRKAVDKVYGKGKSSSFLPIDFDTFGYALTLFKDDADIHSYLRNEYEQNGKRMPNLA






NKVYDNELIEELLNLSFTKFGHLSLKALRSILPYMEQGEVYSSACERAGYTFTGPKKKQKTMLLPN






IPPIANPVVMRALTQARKVVNAIIKKYGSPVSIHIELARDLSQTFDERRKTKKEQDENRKKNETAIR






QLMEYGLTLNPTGHDIVKFKLWSEQNGRCAYSLQPIEIERLLEPGYVEVDHVIPYSRSLDDSYTNK






VLVLTRENREKGNRIPAEYLGVGTERWQQFETFVLTNKQFSKKKRDRLLRLHYDENEETEFKNRN






LNDTRYISRFFANFIREHLKFAESDDKQKVYTVNGRVTAHLRSRWEFNKNREESDLHHAVDAVIV






ACTTPSDIAKVTAFYQRREQNKELAKKTEPHFPQPWPHFADELRARLSKHPKESIKALNLGNYDD






QKLESLQPVFVSRMPKRSVTGAAHQETLRRYVGIDERSGKIQTVVKTKLSEIKLDASGHFPMYGK






ESDPRTYEAIRQRLLEHNNDPKKAFQEPLYKPKKNGEPGPVIRTVKIIDTKNQVIPLNDGKTVAYN






SNIVRVDVFEKDGKYYCVPVYTMDIMKGILPNKAIEPNKPYSEWKEMTEDYTFRFSLYPNDLIRIE






LPREKTVKTAAGEEINVKDVFVYYKTIDSANGGLELISHDHRFSLRGVGSRTLKRFEKYQVDVLG






NIYKVRGEKRVGLASSAHSKPGKTIRPLQSTRD






iSpyMac

Streptococcus

3281
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKR
N863A


Cas9

spp.


TARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY






PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE






NPINASGVDAKAILSARLSKSRKLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ






LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQD






LTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLKRED






LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW






MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKY






VTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTY






HDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWG






RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANL






AGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG






SQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVL






TRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQL






VETROITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHD






AYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL






ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEIQTVGQNGGLFDDNPKSPL






EVTPSKLVPLKKELNPKKYGGYQKPTTAYPVLLITDTKQLIPISVMNKKQFEQNPVKFLRDRGYQQ






VGKNDFIKLPKYTLVDIGDGIKRLWASSKEIHKGNQLVVSKKSQILLYHAHHLDSDLSNDYLQNH






NQQFDVLFNEIISFSKKCKLGKEHIQKIENVYSNKKNSASIEELAESFIKLLGFTQLGATSPFNFLGV






KLNQKQYKGKKDYILPCTEGTLIRQSITGLYETRVDLSKIGEDSGGSGGSKRTADGSEFES






NmeCas9

Neisseria

3282
MAAFKPNSINYILGLDIGIASVGWAMVEIDEEENPIRLIDLGVRVFERAEVPKTGDSLAMARRLAR
N611A




meningitidis


SVRRLTRRRAHRLLRTRRLLKREGVLQAANFDENGLIKSLPNTPWQLRAAALDRKLTPLEWSAVL






LHLIKHRGYLSQRKNEGETADKELGALLKGVAGNAHALQTGDFRTPAELALNKFEKESGHIRNQR






SDYSHTFSRKDLQAELILLFEKQKEFGNPHVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEP






AEPKAAKNTYTAERFIWLTKLNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLE






DTAFFKGLRYGKDNAEASTLMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTD






EDITGRLKDRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNTE






EKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRKEIEKRQEENR






KDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLGRLNEKGYVEIDHALPFS






RTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFKARVETSRFPRSKKQRILLQKF






DEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKRVFASNGQITNLLRGFWGLRKVRAEND






RHHALDAVVVACSTVAMQQKITRFVRYKEMNAFDGKTIDKETGEVLHQKTHFPQPWEFFAQEV






MIRVFGKPDGKPEFEEADTLEKLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVK






SAKRLDEGVSVLRVPLTQLKLKDLEKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDK






AGNRTQQVKAVRVEQVQKTGVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGIL






PDRAVVQGKDEEDWQLIDDSFNFKFSLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDH






KIGKNGILEGIGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR






ScaCas9

Streptococcus

3283
MEKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTNRKSIKKNLMGALLFDSGETAEATRLK
N872A




canis


RTARRRYTRRKNRIRYLQEIFANEMAKLDDSFFQRLEESFLVEEDKKNERHPIFGNLADEVAYHRN






YPTIYHLRKKLADSPEKADLRLIYLALAHIIKFRGHFLIEGKLNAENSDVAKLFYQLIQTYNQLFEE






SPLDEIEVDAKGILSARLSKSKRLEKLIAVFPNEKKNGLFGNIIALALGLTPNFKSNFDLTEDAKLQL






SKDTYDDDLDELLGQIGDQYADLFSAAKNLSDAILLSDILRSNSEVTKAPLSASMVKRYDEHHQD






LALLKTLVRQQFPEKYAEIFKDDTKNGYAGYVGIGIKHRKRTTKLATQEEFYKFIKPILEKMDGAE






ELLAKLNRDDLLRKQRTFDNGSIPHQIHLKELHAILRRQEEFYPFLKENREKIEKILTFRIPYYVGPL






ARGNSRFAWLTRKSEEAITPWNFEEVVDKGASAQSFIERMTNFDEQLPNKKVLPKHSLLYEYFTV






YNELTKVKYVTERMRKPEFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEIIGVEDR






FNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLK






RRHYTGWGRLSRKMINGIRDKQSGKTILDFLKSDGFSNRNFMQLIHDDSLTFKEEIEKAQVSGQGD






SLHEQIADLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIEMARENQTTTKGLQQSRERKKRIEE






GIKELESQILKENPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSI






DNKVLTRSVENRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSEADKAG






FIKRQLVETRQITKHVARILDSRMNTKRDKNDKPIREVKVITLKSKLVSDFRKDFQLYKVRDINNY






HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKRFFYSNIMNF






FKTEVKLANGEIRKRPLIETNGETGEVVWNKEKDFATVRKVLAMPQVNIVKKTEVQTGGFSKESI






LSKRESAKLIPRKKGWDTRKYGGFGSPTVAYSILVVAKVEKGKAKKLKSVKVLVGITIMEKGSYE






KDPIGFLEAKGYKDIKKELIFKLPKYSLFELENGRRRMLASATELQKANELVLPQHLVRLLYYTQN






ISATTGSNNLGYIEQHREEFKEIFEKIIDFSEKYILKNKVNSNLKSSFDEQFAVSDSILLSNSFVSLLK






YTSFGASGGFTFLDLDVKQGRLRYQTVTEVLDATLIYQSITGLYETRTDLSQLGGD






ScaCas9-

Streptococcus

3284
MEKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTNRKSIKKNLMGALLFDSGETAEATRLK
N872A


HiFi-

canis


RTARRRYTRRKNRIRYLQEIFANEMAKLDDSFFQRLEESFLVEEDKKNERHPIFGNLADEVAYHRN



Sc++


YPTIYHLRKKLADSPEKADLRLIYLALAHIIKFRGHFLIEGKLNAENSDVAKLFYQLIQTYNQLFEE






SPLDEIEVDAKGILSARLSKSKRLEKLIAVFPNEKKNGLFGNIIALALGLTPNFKSNFDLTEDAKLQL






SKDTYDDDLDELLGQIGDQYADLFSAAKNLSDAILLSDILRSNSEVTKAPLSASMVKRYDEHHQD






LALLKTLVRQQFPEKYAEIFKDDTKNGYAGYVGADKKLRKRSGKLATEEEFYKFIKPILEKMDGA






EELLAKLNRDDLLRKQRTFDNGSIPHQIHLKELHAILRRQEEFYPFLKENREKIEKILTFRIPYYVGP






LARGNSRFAWLTRKSEEAITPWNFEEVVDKGASAQSFIERMTNFDEQLPNKKVLPKHSLLYEYFT






VYNELTKVKYVTERMRKPEFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEIIGVED






RFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL






KRRHYTGWGRLSRKMINGIRDKQSGKTILDFLKSDGFSNANFMQLIHDDSLTFKEEIEKAQVSGQ






GDSLHEQIADLAGSPAIKKGILQTVKIVDELVKVMGHKPENIVIEMARENQTTTKGLQQSRERKKR






IEEGIKELESQILKENPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFIKD






DSIDNKVLTRSVENRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSEADK






AGFIKRQLVETRQITKHVARILDSRMNTKRDKNDKPIREVKVITLKSKLVSDFRKDFQLYKVRDIN






NYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKRFFYSNIM






NFFKTEVKLANGEIRKRPLIETNGETGEVVWNKEKDFATVRKVLAMPQVNIVKKTEVQTGGFSKE






SILSKRESAKLIPRKKGWDTRKYGGFGSPTVAYSILVVAKVEKGKAKKLKSVKVLVGITIMEKGSY






EKDPIGFLEAKGYKDIKKELIFKLPKYSLFELENGRRRMLASAKELQKANELVLPQHLVRLLYYTQ






NISATTGSNNLGYIEQHREEFKEIFEKIIDFSEKYILKNKVNSNLKSSFDEQFAVSDSILLSNSFVSLL






KYTSFGASGGFTFLDLDVKQGRLRYQTVTEVLDATLIYQSITGLYETRTDLSQLGGD






SpyCas9-

Streptococcus

3285
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKR
N863A


3var-

pyogenes


TARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY



NRRH


PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE






NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ






LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMVKRYDEHHQ






DLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE






DLLRKQRTFDNGIIPHQIHLGELHAILRRQGDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW






MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKY






VTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTY






HDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRLRYTGWG






RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANL






AGSPAIKKGILQTVKVVDELVKVMGGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG






SQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVL






TRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQL






VETROITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHD






AYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL






ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKGNSDK






LIARKKDWDPKKYGGFNSPTAAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIGFLEA






KGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLHKGNELALPSKYVNFLYLASHYEKLKGSP






EDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLT






NLGVPAAFKYFDTTIDKKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD






SpyCas9-

Streptococcus

3286
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKR
N863A


3var-

pyogenes


TARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY



NRTH


PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE






NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ






LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMVKRYDEHHQ






DLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE






DLLRKQRTFDNGIIPHQIHLGELHAILRRQGDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW






MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKY






VTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTY






HDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRLRYTGWG






RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANL






AGSPAIKKGILQTVKVVDELVKVMGGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG






SQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVL






TRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQL






VETROITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHD






AYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL






ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKGNSDK






LIARKKDWDPKKYGGFNSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIGFLEA






KGYKEVKKDLIIKLPKYSLFELENGRKRMLASASVLHKGNELALPSKYVNFLYLASHYEKLKGSS






EDNKQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLT






NLGASAAFKYFDTTIGRKLYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD






SpyCas9-

Streptococcus

3287
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKR
N863A


3var-

pyogenes


TARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY



NRCH


PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE






NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ






LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMVKRYDEHHQ






DLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRE






DLLRKQRTFDNGIIPHQIHLGELHAILRRQGDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW






MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKY






VTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTY






HDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRLRYTGWG






RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANL






AGSPAIKKGILQTVKVVDELVKVMGGHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG






SQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVL






TRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQL






VETROITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHD






AYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL






ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKGNSDK






LIARKKDWDPKKYGGFNSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEA






KGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNELALPSKYVNFLYLASHYEKLKGSP






EDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLT






NLGAPAAFKYFDTTINRKQYNTTKEVLDATLIRQSITGLYETRIDLSQLGGD






SpyCas9-

Streptococcus

3269
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKR
N863A


HF1

pyogenes


TARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY






PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE






NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ






LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQD






LTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED






LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW






MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKY






VTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTY






HDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWG






RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANL






AGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG






SQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVL






TRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQL






VETROITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHD






AYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL






ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDK






LIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEA






KGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSP






EDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLT






NLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD






SpyCas9-

Streptococcus

3288
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKR
N863A


QQR1

pyogenes


TARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY






PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE






NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ






LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQD






LTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED






LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW






MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKY






VTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTY






HDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWG






RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANL






AGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG






SQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVL






TRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQL






VETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHD






AYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL






ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDK






LIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEA






KGYKEVKKDLIIKLPKYSLFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSP






EDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADAQLDKVLSAYNKHRDKPIREQAENIIHLFTLT






NLGAPAAFKYFDTTFKQKQYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD






SpyCas9-

Streptococcus

3289
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKR
N863A


SpG

pyogenes


TARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY






PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE






NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ






LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQD






LTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED






LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW






MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKY






VTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTY






HDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWG






RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANL






AGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG






SQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVL






TRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQL






VETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHD






AYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL






ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDK






LIARKKDWDPKKYGGFLWPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLE






AKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAKQLQKGNELALPSKYVNFLYLASHYEKLKGS






PEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLT






NLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD






SpyCas9-

Streptococcus

3290
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKR
N863A


VQR

pyogenes


TARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY






PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE






NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ






LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQD






LTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED






LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW






MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKY






VTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTY






HDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWG






RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANL






AGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG






SQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVL






TRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQL






VETROITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHD






AYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL






ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDK






LIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEA






KGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSP






EDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLT






NLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD






SpyCas9-

Streptococcus

3291
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKR
N863A


VRER

pyogenes


TARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY






PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE






NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ






LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQD






LTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED






LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW






MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKY






VTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTY






HDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWG






RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANL






AGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG






SQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVL






TRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQL






VETROITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHD






AYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL






ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDK






LIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEA






KGYKEVKKDLIIKLPKYSLFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSP






EDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLT






NLGAPAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD






SpyCas9-

Streptococcus

3292
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKR
N863A


xCas

pyogenes


TARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY






PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE






NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDTKLQ






LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKLYDEHHQD






LTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED






LLRKQRTFDNGIIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW






MTRKSEETITPWNFEKVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKY






VTEGMRKPAFLSGDQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTY






HDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWG






RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFIQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANL






AGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELG






SQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVL






TRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQL






VETROITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHD






AYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL






ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDK






LIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEA






KGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGVLQKGNELALPSKYVNFLYLASHYEKLKGSP






EDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLT






NLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD






SpyCas9-

Streptococcus

3293
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKR
N863A


xCas-NG

pyogenes


TARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY






PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE






NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDTKLQ






LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKLYDEHHQD






LTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED






LLRKQRTFDNGIIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW






MTRKSEETITPWNFEKVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKY






VTEGMRKPAFLSGDQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTY






HDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWG






RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFIQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANL






SQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVL






TRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQL






VETROITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHD






AYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITL






ANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIRPKRNSDK






LIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEA






KGYKEVKKDLIIKLPKYSLFELENGRKRMLASARFLQKGNELALPSKYVNFLYLASHYEKLKGSP






EDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLT






NLGAPRAFKYFDTTIDRKVYRSTKEVLDATLIHQSITGLYETRIDLSQLGGD






St1Cas9-

Streptococcus

3294
MSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQAENNLVRRTNRQGRRLARRKKHRRVRL
N622A


CNRZ1066

thermophilus


NRLFEESGLITDFTKISINLNPYQLRVKGLTDELSNEELFIALKNMVKHRGISYLDDASDDGNSSVG






DYAQIVKENSKQLETKTPGQIQLERYQTYGQLRGDFTVEKDGKKHRLINVFPTSAYRSEALRILQT






QQEFNPQITDEFINRYLEILTGKRKYYHGPGNEKSRTDYGRYRTSGETLDNIFGILIGKCTFYPDEFR






AAKASYTAQEFNLLNDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLFKYIAKLLSCDVA






DIKGYRIDKSGKAEIHTFEAYRKMKTLETLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFAD






GSFSQKQVDELVQFRKANSSIFGKGWHNFSVKLMMELIPELYETSEEQMTILTRLGKQKTTSSSNK






TKYIDEKLLTEEIYNPVVAKSVRQAIKIVNAAIKEYGDFDNIVIEMARETNEDDEKKAIQKIQKANK






DEKDAAMLKAANQYNGKAELPHSVFHGHKQLATKIRLWHQQGERCLYTGKTISIHDLINNSNQF






EVDHILPLSITFDDSLANKVLVYATANQEKGQRTPYQALDSMDDAWSFRELKAFVRESKTLSNKK






KEYLLTEEDISKFDVRKKFIERNLVDTRYASRVVLNALQEHFRAHKIDTKVSVVRGQFTSQLRRH






WGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKNTLVSYSEEQLLDIETGELISDDEYKESVFKAP






YQHFVDTLKSKEFEDSILFSYQVDSKFNRKISDATIYATROAKVGKDKKDETYVLGKIKDIYTQDG






YDAFMKIYKKDKSKFLMYRHDPQTFEKVIEPILENYPNKQMNEKGKEVPCNPFLKYKEEHGYIRK






YSKKGNGPEIKSLKYYDSKLLGNPIDITPENSKNKVVLQSLKPWRTDVYFNKATGKYEILGLKYA






DLQFEKGTGTYKISQEKYNDIKKKEGVDSDSEFKFTLYKNDLLLVKDTETKEQQLFRFLSRTLPKQ






KHYVELKPYDKQKFEGGEALIKVLGNVANGGQCIKGLAKSNISIYKVRTDVLGNQHIIKNEGDKP






KLDF






St1Cas9-

Streptococcus

3295
MSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQAENNLVRRTNRQGRRLARRKKHRRVRL
N622A


LMG1831

thermophilus


NRLFEESGLITDFTKISINLNPYQLRVKGLTDELSNEELFIALKNMVKHRGISYLDDASDDGNSSVG






DYAQIVKENSKQLETKTPGQIQLERYQTYGQLRGDFTVEKDGKKHRLINVFPTSAYRSEALRILQT






QQEFNPQITDEFINRYLEILTGKRKYYHGPGNEKSRTDYGRYRTSGETLDNIFGILIGKCTFYPDEFR






AAKASYTAQEFNLLNDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLFKYIAKLLSCDVA






DIKGYRIDKSGKAEIHTFEAYRKMKTLETLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFAD






GSFSQKQVDELVQFRKANSSIFGKGWHNFSVKLMMELIPELYETSEEQMTILTRLGKQKTTSSSNK






TKYIDEKLLTEEIYNPVVAKSVRQAIKIVNAAIKEYGDFDNIVIEMARETNEDDEKKAIQKIQKANK






DEKDAAMLKAANQYNGKAELPHSVFHGHKQLATKIRLWHQQGERCLYTGKTISIHDLINNSNQF






EVDHILPLSITFDDSLANKVLVYATANQEKGQRTPYQALDSMDDAWSFRELKAFVRESKTLSNKK






KEYLLTEEDISKFDVRKKFIERNLVDTRYASRVVLNALQEHFRAHKIDTKVSVVRGQFTSQLRRH






WGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKNTLVSYSEEQLLDIETGELISDDEYKESVFKAP






YQHFVDTLKSKEFEDSILFSYQVDSKFNRKISDATIYATROAKVGKDKKDETYVLGKIKDIYTQDG






YDAFMKIYKKDKSKFLMYRHDPQTFEKVIEPILENYPNKQMNEKGKEVPCNPFLKYKEEHGYIRK






YSKKGNGPEIKSLKYYDSKLLGNPIDITPENSKNKVVLQSLKPWRTDVYFNKNTGKYEILGLKYA






DLQFEKKTGTYKISQEKYNGIMKEEGVDSDSEFKFTLYKNDLLLVKDTETKEQQLFRFLSRTMPN






VKYYVELKPYSKDKFEKNESLIEILGSADKSGRCIKGLGKSNISIYKVRTDVLGNQHIIKNEGDKPK






LDF






St1Cas9-

Streptococcus

3296
MSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQAENNLVRRTNRQGRRLARRKKHRRVRL
N622A


MTH17C

thermophilus


NRLFEESGLITDFTKISINLNPYQLRVKGLTDELSNEELFIALKNMVKHRGISYLDDASDDGNSSVG



L396


DYAQIVKENSKQLETKTPGQIQLERYQTYGQLRGDFTVEKDGKKHRLINVFPTSAYRSEALRILQT






QQEFNPQITDEFINRYLEILTGKRKYYHGPGNEKSRTDYGRYRTSGETLDNIFGILIGKCTFYPDEFR






AAKASYTAQEFNLLNDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLFKYIAKLLSCDVA






DIKGYRIDKSGKAEIHTFEAYRKMKTLETLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFAD






GSFSQKQVDELVQFRKANSSIFGKGWHNFSVKLMMELIPELYETSEEQMTILTRLGKQKTTSSSNK






TKYIDEKLLTEEIYNPVVAKSVRQAIKIVNAAIKEYGDFDNIVIEMARETNEDDEKKAIQKIQKANK






DEKDAAMLKAANQYNGKAELPHSVFHGHKQLATKIRLWHQQGERCLYTGKTISIHDLINNSNQF






EVDHILPLSITFDDSLANKVLVYATANQEKGQRTPYQALDSMDDAWSFRELKAFVRESKTLSNKK






KEYLLTEEDISKFDVRKKFIERNLVDTRYASRVVLNALQEHFRAHKIDTKVSVVRGQFTSQLRRH






WGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKNTLVSYSEDQLLDIETGELISDDEYKESVFKAP






YQHFVDTLKSKEFEDSILFSYQVDSKFNRKISDATIYATRQAKVGKDKADETYVLGKIKDIYTQDG






YDAFMKIYKKDKSKFLMYRHDPQTFEKVIEPILENYPNKQINEKGKEVPCNPFLKYKEEHGYIRKY






SKKGNGPEIKSLKYYDSKLGNHIDITPKDSNNKVVLQSLKPWRTDVYFNKNTGKYEILGLKYSDM






QFEKGTGKYSISKEQYENIKVREGVDENSEFKFTLYKNDLLLLKDSENGEQILLRFTSRNDTSKHY






VELKPYNRQKFEGSEYLIKSLGTVAKGGQCIKGLGKSNISIYKVRTDVLGNQHIIKNEGDKPKLDF






St1Cas9-

Streptococcus

3297
MSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQAENNLVRRTNRQGRRLARRKKHRRVRL
N622A


TH1477

thermophilus


NRLFEESGLITDFTKISINLNPYQLRVKGLTDELSNEELFIALKNMVKHRGISYLDDASDDGNSSVG






DYAQIVKENSKQLETKTPGQIQLERYQTYGQLRGDFTVEKDGKKHRLINVFPTSAYRSEALRILQT






QQEFNPQITDEFINRYLEILTGKRKYYHGPGNEKSRTDYGRYRTSGETLDNIFGILIGKCTFYPDEFR






AAKASYTAQEFNLLNDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLFKYIAKLLSCDVA






DIKGYRIDKSGKAEIHTFEAYRKMKTLETLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFAD






GSFSQKQVDELVQFRKANSSIFGKGWHNFSVKLMMELIPELYETSEEQMTILTRLGKQKTTSSSNK






TKYIDEKLLTEEIYNPVVAKSVRQAIKIVNAAIKEYGDFDNIVIEMARETNEDDEKKAIQKIQKANK






DEKDAAMLKAANQYNGKAELPHSVFHGHKQLATKIRLWHQQGERCLYTGKTISIHDLINNSNQF






EVDHILPLSITFDDSLANKVLVYATANQEKGQRTPYQALDSMDDAWSFRELKAFVRESKTLSNKK






KEYLLTEEDISKFDVRKKFIERNLVDTRYASRVVLNALQEHFRAHKIDTKVSVVRGQFTSQLRRH






WGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKNTLVSYSEDQLLDIETGELISDDEYKESVFKAP






YQHFVDTLKSKEFEDSILFSYQVDSKFNRKISDATIYATROAKVGKDKADETYVLGKIKDIYTQDG






YDAFMKIYKKDKSKFLMYRHDPQTFEKVIEPILENYPNKQINEKGKEVPCNPFLKYKEEHGYIRKY






SKKGNGPEIKSLKYYDSKLGNHIDITPKDSNNKVVLQSLKPWRTDVYFNKNTGKYEILGLKYSDM






QFEKGTGKYSISKEQYENIKVREGVDENSEFKFTLYKNDLLLLKDSENGEQILLRFTSRNDTSKHY






VELKPYNRQKFEGSEYLIKSLGTVVKGGRCIKGLGKSNISIYKVRTDVLGNQHIIKNEGDKPKLDF









In some embodiments, the endonuclease domain or DNA binding domain comprises a Cas domain, e.g., a Cas9 domain. In embodiments, the endonuclease domain or DNA binding domain comprises a nuclease-active Cas domain, a Cas nickase (nCas) domain, or a nuclease-inactive Cas (dCas) domain. In embodiments, the endonuclease domain or DNA binding domain comprises a nuclease-active Cas9 domain, a Cas9 nickase (nCas9) domain, or a nuclease-inactive Cas9 (dCas9) domain. In some embodiments, the endonuclease domain or DNA binding domain comprises a Cas9 domain of Cas9 (e.g., dCas9 and nCas9), Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, or Cas12i. In some embodiments, the endonuclease domain or DNA binding domain comprises a Cas9 (e.g., dCas9 and nCas9), Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, or Cas12i. In some embodiments, the endonuclease domain or DNA binding domain comprises an S. pyogenes or an S. thermophilus Cas9, or a functional fragment thereof. In some embodiments, the endonuclease domain or DNA binding domain comprises a Cas9 sequence, e.g., as described in Chylinski, Rhun, and Charpentier (2013) RNA Biology 10:5, 726-737; incorporated herein by reference. In some embodiments, the endonuclease domain or DNA binding domain comprises the HNH nuclease subdomain and/or the RuvC1 subdomain of a Cas, e.g., Cas9, e.g., as described herein, or a variant thereof. In some embodiments, the endonuclease domain or DNA binding domain comprises Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, or Cas12i. In some embodiments, the endonuclease domain or DNA binding domain comprises a Cas polypeptide (e.g., enzyme), or a functional fragment thereof. In embodiments, the Cas polypeptide (e.g., enzyme) is selected from Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (e.g., Csn1 or Csx12), Cas10, Cas10d, Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Type II Cas effector proteins, Type V Cas effector proteins, Type VI Cas effector proteins, CARF, DinG, Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12b/C2c1, Cas12c/C2c3, SpCas9(K855A), eSpCas9(1.1), SpCas9-HF1, hyper accurate Cas9 variant (HypaCas9), homologues thereof, modified or engineered versions thereof, and/or functional fragments thereof. In embodiments, the Cas9 comprises one or more substiutions, e.g., selected from H840A, D10A, P475A, W476A, N477A, D1125A, Wi 126A, and D1127A. In embodiments, the Cas9 comprises one or more mutations at positions selected from: D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and/or A987, e.g., one or more substitutions selected from D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and/or D986A. In some embodiments, the endonuclease domain or DNA binding domain comprises a Cas (e.g., Cas9) sequence from Corynebacterium ulcerans, Corynebacterium diphtheria, Spiroplasma syrphidicola, Prevotella intermedia, Spiroplasma taiwanense, Streptococcus iniae, Belliella baltica, Psychroflexus torquis, Streptococcus thermophilus, Listeria innocua, Campylobacter jejuni, Neisseria meningitidis, Streptococcus pyogenes, or Staphylococcus aureus, or a fragment or variant thereof.


In some embodiments, the endonuclease domain or DNA binding domain comprises a Cpf1 domain, e.g., comprising one or more substitutions, e.g., at position D917, E1006A, D1255 or any combination thereof, e.g., selected from D917A, E1006A, D1255A, D917A/E1006A, D917A/D1255A, E1006A/D1255A, and D917A/E1006A/D1255A.


In some embodiments, the endonuclease domain or DNA binding domain comprises spCas9, spCas9-VRQR, spCas9-VRER, xCas9 (sp), saCas9, saCas9-KKH, spCas9-MQKSER, spCas9-LRKIQK, or spCas9-LRVSQL.


In some embodiments, the endonuclease domain or DNA-binding domain comprises an amino acid sequence as listed in Table 37 below, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the endonuclease domain or DNA-binding domain comprises an amino acid sequence that has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 differences (e.g., mutations) relative to any of the amino acid sequences described herein.









TABLE 37







Each of the Reference Sequences are incorporated by reference in their entirety.








Name
Amino Acid Sequence or Reference Sequence






Streptococcus pyogenes




Cas9






Exemplary Linker
SGSETPGTSESATPES (SEQ ID NO: 1023)





Exemplary Linker Motif
(SGGS)n (SEQ ID NO: 1583)





Exemplary Linker Motif
(GGGS)n (SEQ ID NO: 1584)





Exemplary Linker Motif
(GGGGS)n (SEQ ID NO: 1535)





Exemplary Linker Motif
(G)n





Exemplary Linker Motif
(EAAAK)n (SEQ ID NO: 1534)





Exemplary Linker Motif
(GGS)n





Exemplary Linker Motif
(XP)n





Cas9 from Streptococcus
NCBI Reference Sequence: NC_002737.2 and Uniprot



pyogenes

Reference Sequence: Q99ZW2





Cas9 from Corynebacterium
NCBI Refs: NC_015683.1, NC_017317.1



ulcerans







Cas9 from Corynebacterium
NCBI Refs: NC_016782.1, NC_016786.1



diphtheria







Cas9 from Spiroplasma
NCBI Ref: NC_021284.1



syrphidicola







Cas9 from Prevotella
NCBI Ref: NC_017861.1



intermedia







Cas9 from Spiroplasma
NCBI Ref: NC_021846.1



taiwanense







Cas9 from Streptococcus
NCBI Ref: NC_021314.1



iniae







Cas9 from Belliella baltica
NCBI Ref: NC_018010.1





Cas9 from Psychroflexus
NCBI Ref: NC_018721.1



torquisI







Cas9 from Streptococcus
NCBI Ref: YP_820832.1



thermophilus







Cas9 from Listeria innocua
NCBI Ref: NP_472073.1





Cas9 from Campylobacter
NCBI Ref: YP_002344900.1



jejuni







Cas9 from Neisseria
NCBI Ref: YP_002342100.1



meningitidis







dCas9 (D10A and H840A)






Catalytically inactive Cas9



(dCas9)






Cas9 nickase (nCas9)






Catalytically active Cas9






CasY
((ncbi.nlm.nih.gov/protein/APG80656.1)



>APG80656.1 CRISPR-associated protein CasY [uncultured



Parcubacteria group bacterium])





CasX
uniprot.org/uniprot/F0NN87; uniprot.org/uniprot/F0NH53





CasX
>tr|F0NH53|F0NH53_SULIR CRISPR associated protein, Casx



OS = Sulfolobus islandicus (strain REY15A) GN = SiRe_0771



PE = 4 SV = 1





Deltaproteobacteria CasX






Cas12b/C2c1
((uniprot.org/uniprot/T0D7A2#2) sp|T0D7A2|C2C1_ALIAG



CRISPR- associated endonuclease C2c1 OS = Alicyclobacillus




acido-terrestris (strain ATCC 49025/DSM 3922/CIP 106132/



NCIMB 13137/GD3B) GN = c2c1 PE = 1 SV = 1)


BhCas12b ((Bacillus
NCBI Reference Sequence: WP_095142515



hisashii)






BvCas12b (Bacillus sp. V3-
NCBI Reference Sequence: WP_101661451.1


13)






Wild-type Francisella




novicida Cpf1







Francisella novicida Cpf1



D917A







Francisella novicida Cpf1



E1006A







Francisella novicida Cpf1



D1255A







Francisella novicida Cpf1



D917A/E1006A







Francisella novicida Cpf1



D917A/D1255A







Francisella novicida Cpf1



E1006A/D1255A







Francisella novicida Cpf1



D917A/E1006A






SaCas9






SaCas9n






PAM-binding SpCas9






PAM-binding SpCas9n






PAM-binding SpEQR Cas9






PAM-binding SpVQR Cas9






PAM-binding SpVRER



Cas9






PAM-binding SpVRQR



Cas9






SpyMacCas9









In some embodiments, a Gene Writing polypeptide has an endonuclease domain comprising a Cas9 nickase, e.g., Cas9 H840A. In embodiments, the Cas9 H840A has the following amino acid sequence:









Cas9 nickase (H840A):


(SEQ ID NO: 1585)


DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA





LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFH





RLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDK





ADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFE





ENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSL





GLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKN





LSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLP





EKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKL





NREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK





ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSF





IERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFL





SGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNA





SLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKT





YAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDG





FANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKG





ILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIE





EGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLS





DYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYW





RQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVA





QILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNY





HHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIG





KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRD





FATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDP





KKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKN





PIDFLEAKGYKEVKKFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDAT





LIHQSITGLYETRIDLSQLGGD






In some embodiments, a Gene Writing polypeptide comprises the RT domain from a retroviral reverse transcriptase, e.g., a wild-type M-MLV RT, e.g., comprising the following sequence:









M-MLV (WT):


(SEQ ID NO: 1586)


TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLII





PLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP





VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLD





LKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFD





EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNL





GYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQL





REFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA





LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD





PVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDR





WLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILA





EAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAK





ALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRR





RGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNR





MADQAARKAAITETPDTSTLLI 






In some embodiments, a Gene Writing polypeptide comprises the RT domain from a retroviral reverse transcriptase, e.g., an M-MLV RT, e.g., comprising the following sequence:









(SEQ ID NO: 1548)


TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLII





PLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP





VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLD





LKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFD





EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNL





GYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQL





REFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA





LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD





PVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDR





WLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILA





EAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAK





ALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRR





RGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNR





MADQAARKAAITETPDTSTLL






In some embodiments, a Gene Writing polypeptide comprises the RT domain from a retroviral reverse transcriptase comprising the sequence of amino acids 659-1329 of NP_057933. In embodiments, the Gene Writing polypeptide further comprises one additional amino acid at the N-terminus of the sequence of amino acids 659-1329 of NP_057933, e.g., as shown below:









(SEQ ID NO: 1587)


TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLII





PLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP






VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLD







LKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFD







EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNL







GYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQL





REFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA





LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD





PVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDR





WLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILA





EAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAK






ALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRR







RGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNR







MADQAARKAA







Core RT (bold), annotated per above


RNAseH (underlined), annotated per above


In embodiments, the Gene Writing polypeptide further comprises one additional amino acid at the C-terminus of the sequence of amino acids 659-1329 of NP_057933. In embodiments, the Gene Writing polypeptide comprises an RNaseH1 domain (e.g., amino acids 1178-1318 of NP_057933).


In some embodiments, a retroviral reverse transcriptase domain, e.g., M-MLV RT, may comprise one or more mutations from a wild-type sequence that may improve features of the RT, e.g., thermostability, processivity, and/or template binding. In some embodiments, an M-MLV RT domain comprises, relative to the M-MLV (WT) sequence above, one or more mutations, e.g., selected from D200N, L603W, T330P, T306K, W313F, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, L435G, N454K, H594Q, D653N, R110S, K103L, e.g., a combination of mutations, such as D200N, L603W, and T330P, optionally further including T306K and W313F. In some embodiments, an M-HLV RT used herein comprises the mutations D200N, L603W, T330P, T306K and W313F. In embodiments, the mutant M-MLV RT comprises the following amino acid sequence:









M-MLV (PE2):


(SEQ ID NO: 1588)


TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLII





PLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP





VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLD





LKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFN





EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNL





GYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQL





REFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQA





LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD





PVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDR





WLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILA





EAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAK





ALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRR





RGWLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNR





MADQAARKAAITETPDTSTLLI 






In some embodiments, a Gene Writer polypeptide may comprise a linker, e.g., a peptide linker, e.g., a linker as described in Table 38. In some embodiments, a Gene Writer polypeptide comprises a flexible linker between the endonuclease and the RT domain, e.g., a linker comprising the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 1589). In some embodiments, an RT domain of a Gene Writer polypeptide may be located C-terminal to the endonuclease domain. In some embodiments, an RT domain of a Gene Writer polypeptide may be located N-terminal to the endonuclease domain.









TABLE 38







Exemplary linker sequences









SEQ



ID


Amino Acid Sequence
NO:





GGS






GGSGGS
3298





GGSGGSGGS
3299





GGSGGSGGSGGS
3300





GGSGGSGGSGGSGGS
3301





GGSGGSGGSGGSGGSGGS
3302





GGGGS
1535





GGGGSGGGGS
3303





GGGGSGGGGSGGGGS
3304





GGGGSGGGGSGGGGSGGGGS
3305





GGGGSGGGGSGGGGSGGGGSGGGGS
3306





GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS
3307





GGG






GGGG
3308





GGGGG
3309





GGGGGG
3310





GGGGGGG
3311





GGGGGGGG
3312





GSS






GSSGSS
1736





GSSGSSGSS
3313





GSSGSSGSSGSS
3314





GSSGSSGSSGSSGSS
3315





GSSGSSGSSGSSGSSGSS
3316





EAAAK
1534





EAAAKEAAAK
3317





EAAAKEAAAKEAAAK
3318





EAAAKEAAAKEAAAKEAAAK
3319





EAAAKEAAAKEAAAKEAAAKEAAAK
3320





EAAAKEAAAKEAAAKEAAAKEAAAKEAAAK
3321





PAP






PAPAP
3322





PAPAPAP
3323





PAPAPAPAP
3324





PAPAPAPAPAP
3325





PAPAPAPAPAPAP
3326





GGSGGG
3327





GGGGGS
3328





GGSGSS
3329





GSSGGS
3330





GGSEAAAK
3331





EAAAKGGS
3332





GGSPAP
3333





PAPGGS
3334





GGGGSS
3335





GSSGGG
3336





GGGEAAAK
3337





EAAAKGGG
3338





GGGPAP
3339





PAPGGG
3340





GSSEAAAK
3341





EAAAKGSS
3342





GSSPAP
3343





PAPGSS
3344





EAAAKPAP
3345





PAPEAAAK
3346





GGSGGGGSS
3347





GGSGSSGGG
3348





GGGGGSGSS
3349





GGGGSSGGS
3350





GSSGGSGGG
3351





GSSGGGGGS
3352





GGSGGGEAAAK
3353





GGSEAAAKGGG
3354





GGGGGSEAAAK
3355





GGGEAAAKGGS
3356





EAAAKGGSGGG
3357





EAAAKGGGGGS
3358





GGSGGGPAP
3359





GGSPAPGGG
3360





GGGGGSPAP
3361





GGGPAPGGS
3362





PAPGGSGGG
3363





PAPGGGGGS
3364





GGSGSSEAAAK
3365





GGSEAAAKGSS
3366





GSSGGSEAAAK
3367





GSSEAAAKGGS
3368





EAAAKGGSGSS
3369





EAAAKGSSGGS
3370





GGSGSSPAP
3371





GGSPAPGSS
3372





GSSGGSPAP
3373





GSSPAPGGS
3374





PAPGGSGSS
3375





PAPGSSGGS
3376





GGSEAAAKPAP
3377





GGSPAPEAAAK
3378





EAAAKGGSPAP
3379





EAAAKPAPGGS
3380





PAPGGSEAAAK
3381





PAPEAAAKGGS
3382





GGGGSSEAAAK
3383





GGGEAAAKGSS
3384





GSSGGGEAAAK
3385





GSSEAAAKGGG
3386





EAAAKGGGGSS
3387





EAAAKGSSGGG
3388





GGGGSSPAP
3389





GGGPAPGSS
3390





GSSGGGPAP
3391





GSSPAPGGG
3392





PAPGGGGSS
3393





PAPGSSGGG
3394





GGGEAAAKPAP
3395





GGGPAPEAAAK
3396





EAAAKGGGPAP
3397





EAAAKPAPGGG
3398





PAPGGGEAAAK
3399





PAPEAAAKGGG
3400





GSSEAAAKPAP
3401





GSSPAPEAAAK
3402





EAAAKGSSPAP
3403





EAAAKPAPGSS
3404





PAPGSSEAAAK
3405





PAPEAAAKGSS
3406





AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKA
3407





GGGGSEAAAKGGGGS
3408





EAAAKGGGGSEAAAK
3409





SGSETPGTSESATPES
1023





GSAGSAAGSGEF
3410





SGGSSGGSSGSETPGTSESATPESSGGSSGGSS
1589









In some embodiments, a Gene Writer polypeptide comprises a dCas9 sequence comprising a D10A and/or H840A mutation, e.g., the following sequence:









(SEQ ID NO: 1590)


SMDKKYSIGLAIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKKNLIG





ALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFH





RLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKA





DLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEEN





PINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLT





PNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDA





ILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKE





IFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL





RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIP





YYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFD





KNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIV





DLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLK





IIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMK





QLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHD





DSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVK





VMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEH





PVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKD





DSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDN





LTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKL





IREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIK





KYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE





ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTE





VQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKV





EKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLP





KYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSP





EDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRD





KPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIH





QSITGLYETRIDLSQLGGD






In some embodiments, a template RNA molecule for use in the system comprises, from 5′ to 3′ (1) a gRNA spacer; (2) a gRNA scaffold; (3) heterologous object sequence (4) 3′ homology domain. In some embodiments:

    • (1) Is a Cas9 spacer of ˜18-22 nt, e.g., is 20 nt
    • (2) Is a gRNA scaffold comprising one or more hairpin loops, e.g., 1, 2, of 3 loopd for associating the template with a nickase Cas9 domain. In some embodiments, the gRNA scaffold carries the sequence, from 5′ to 3′,









(SEQ ID NO: 1591)


GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAAC





TTGAAAAAGTGGGACCGAGTCGGTCC.








    • (3) In some embodiments, the heterologous object sequence is, e.g., 7-74, e.g., 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, or 70-80 nt or, 80-90 nt in length. In some embodiments, the first (most 5′) base of the sequence is not C.

    • (4) In some embodiments, the 3′ homology domain that binds the target priming sequence after nicking occurs is e.g., 3-20 nt, e.g., 7-15 nt, e.g., 12-14 nt. In some embodiments, the 3′ homology domain has 40-60% GC content.





A second gRNA associated with the system may help drive complete integration. In some embodiments, the second gRNA may target a location that is 0-200 nt away from the first-strand nick, e.g., 0-50, 50-100, 100-200 nt away from the first-strand nick. In some embodiments, the second gRNA can only bind its target sequence after the edit is made, e.g., the gRNA binds a sequence present in the heterologous object sequence, but not in the initial target sequence.


In some embodiments, a Gene Writing system described herein is used to make an edit in HEK293, K562, U2OS, or HeLa cells. In some embodiment, a Gene Writing system is used to make an edit in primary cells, e.g., primary cortical neurons from E18.5 mice.


In some embodiments, a reverse transcriptase or RT domain (e.g., as described herein) comprises a MoMLV RT sequence or variant thereof. In embodiments, the MoMLV RT sequence comprises one or more mutations selected from D200N, L603W, T330P, T306K, W313F, D524G, E562Q, D583N, P51L, S67R, E67K, T197A, H204R, E302K, F309N, L435G, N454K, H594Q, D653N, R110S, and K103L. In embodiments, the MoMLV RT sequence comprises a combination of mutations, such as D200N, L603W, and T330P, optionally further including T306K and/or W313F.


In some embodiments, an endonuclease domain (e.g., as described herein) comprises nCAS9, e.g., comprising the H840A mutation.


In some embodiments, the heterologous object sequence (e.g., of a system as described herein) is about 1-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000, or more, nucleotides in length.


In some embodiments, the RT and endonuclease domains are joined by a flexible linker, e.g., comprising the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 1589).


In some embodiments, the endonuclease domain is N-terminal relative to the RT domain.


In some embodiments, the endonuclease domain is C-terminal relative to the RT domain.


In some embodiments, the system incorporates a heterologous object sequence into a target site by TPRT, e.g., as described herein.


In some embodiments, a system or method described herein involves a CRISPR DNA targeting enzyme or system described in US Pat. App. Pub. No. 20200063126, 20190002889, or 20190002875 (each of which is incorporated by reference herein in its entirety) or a functional fragment or variant thereof. For instance, in some embodiments, a GeneWriter polypeptide or Cas endonuclease described herein comprises a polypeptide sequence of any of the applications mentioned in this paragraph, and in some embodiments a template RNA or guide RNA comprises a nucleic acid sequence of any of the applications mentioned in this paragraph.


In some embodiments, an endonuclease domain or DNA-binding domain comprises a TAL effector molecule. A TAL effector molecule, e.g., a TAL effector molecule that specifically binds a DNA sequence, typically comprises a plurality of TAL effector domains or fragments thereof, and optionally one or more additional portions of naturally occurring TAL effectors (e.g., N- and/or C-terminal of the plurality of TAL effector domains). Many TAL effectors are known to those of skill in the art and are commercially available, e.g., from Thermo Fisher Scientific.


Naturally occurring TALEs are natural effector proteins secreted by numerous species of bacterial pathogens including the plant pathogen Xanthomonas which modulates gene expression in host plants and facilitates bacterial colonization and survival. The specific binding of TAL effectors is based on a central repeat domain of tandemly arranged nearly identical repeats of typically 33 or 34 amino acids (the repeat-variable di-residues, RVD domain).


Members of the TAL effectors family differ mainly in the number and order of their repeats. The number of repeats typically ranges from 1.5 to 33.5 repeats and the C-terminal repeat is usually shorter in length (e.g., about 20 amino acids) and is generally referred to as a “half-repeat”. Each repeat of the TAL effector generally features a one-repeat-to-one-base-pair correlation with different repeat types exhibiting different base-pair specificity (one repeat recognizes one base-pair on the target gene sequence). Generally, the smaller the number of repeats, the weaker the protein-DNA interactions. A number of 6.5 repeats has been shown to be sufficient to activate transcription of a reporter gene (Scholze et al., 2010).


Repeat to repeat variations occur predominantly at amino acid positions 12 and 13, which have therefore been termed “hypervariable” and which are responsible for the specificity of the interaction with the target DNA promoter sequence, as shown in Table 5 listing exemplary repeat variable diresidues (RVD) and their correspondence to nucleic acid base targets.









TABLE 5







RVDs and Nucleic Acid Base Specificity








Target
Possible RVD Amino Acid Combinations























A
NI
NN
CI
HI
KI










G
NN
GN
SN
VN
LN
DN
QN
EN
HN
RH
NK
AN
FN


C
HD
RD
KD
ND
AD










T
NG
HG
VG
IG
EG
MG
YG
AA
EP
VA
QG
KG
RG









Accordingly, it is possible to modify the repeats of a TAL effector to target specific DNA sequences. Further studies have shown that the RVD NK can target G. Target sites of TAL effectors also tend to include a T flanking the 5′ base targeted by the first repeat, but the exact mechanism of this recognition is not known. More than 113 TAL effector sequences are known to date. Non-limiting examples of TAL effectors from Xanthomonas include, Hax2, Hax3, Hax4, AvrXa7, AvrXa10 and AvrBs3.


Accordingly, the TAL effector domain of a TAL effector molecule described herein may be derived from a TAL effector from any bacterial species (e.g., Xanthomonas species such as the African strain of Xanthomonas oryzae pv. Oryzae (Yu et al. 2011), Xanthomonas campestris pv. raphani strain 756C and Xanthomonas oryzae pv. oryzicolas train BLS256 (Bogdanove et al. 2011). In some embodiments, the TAL effector domain comprises an RVD domain as well as flanking sequence(s) (sequences on the N-terminal and/or C-terminal side of the RVD domain) also from the naturally occurring TAL effector. It may comprise more or fewer repeats than the RVD of the naturally occurring TAL effector. The TAL effector molecule can be designed to target a given DNA sequence based on the above code and others known in the art. The number of TAL effector domains (e.g., repeats (monomers or modules)) and their specific sequence can be selected based on the desired DNA target sequence. For example, TAL effector domains, e.g., repeats, may be removed or added in order to suit a specific target sequence. In an embodiment, the TAL effector molecule of the present invention comprises between 6.5 and 33.5 TAL effector domains, e.g., repeats. In an embodiment, TAL effector molecule of the present invention comprises between 8 and 33.5 TAL effector domains, e.g., repeats, e.g., between 10 and 25 TAL effector domains, e.g., repeats, e.g., between 10 and 14 TAL effector domains, e.g., repeats.


In some embodiments, the TAL effector molecule comprises TAL effector domains that correspond to a perfect match to the DNA target sequence. In some embodiments, a mismatch between a repeat and a target base-pair on the DNA target sequence is permitted as along as it allows for the function of the polypeptide comprising the TAL effector molecule. In general, TALE binding is inversely correlated with the number of mismatches. In some embodiments, the TAL effector molecule of a polypeptide of the present invention comprises no more than 7 mismatches, 6 mismatches, 5 mismatches, 4 mismatches, 3 mismatches, 2 mismatches, or 1 mismatch, and optionally no mismatch, with the target DNA sequence. Without wishing to be bound by theory, in general the smaller the number of TAL effector domains in the TAL effector molecule, the smaller the number of mismatches will be tolerated and still allow for the function of the polypeptide comprising the TAL effector molecule. The binding affinity is thought to depend on the sum of matching repeat-DNA combinations. For example, TAL effector molecules having 25 TAL effector domains or more may be able to tolerate up to 7 mismatches.


In addition to the TAL effector domains, the TAL effector molecule of the present invention may comprise additional sequences derived from a naturally occurring TAL effector. The length of the C-terminal and/or N-terminal sequence(s) included on each side of the TAL effector domain portion of the TAL effector molecule can vary and be selected by one skilled in the art, for example based on the studies of Zhang et al. (2011). Zhang et al., have characterized a number of C-terminal and N-terminal truncation mutants in Hax3 derived TAL-effector based proteins and have identified key elements, which contribute to optimal binding to the target sequence and thus activation of transcription. Generally, it was found that transcriptional activity is inversely correlated with the length of N-terminus. Regarding the C-terminus, an important element for DNA binding residues within the first 68 amino acids of the Hax 3 sequence was identified. Accordingly, in some embodiments, the first 68 amino acids on the C-terminal side of the TAL effector domains of the naturally occurring TAL effector is included in the TAL effector molecule. Accordingly, in an embodiment, a TAL effector molecule comprises 1) one or more TAL effector domains derived from a naturally occurring TAL effector; 2) at least 70, 80, 90, 100, 110, 120, 130, 140, 150, 170, 180, 190, 200, 220, 230, 240, 250, 260, 270, 280 or more amino acids from the naturally occurring TAL effector on the N-terminal side of the TAL effector domains; and/or 3) at least 68, 80, 90, 100, 110, 120, 130, 140, 150, 170, 180, 190, 200, 220, 230, 240, 250, 260 or more amino acids from the naturally occurring TAL effector on the C-terminal side of the TAL effector domains.


In some embodiments, an endonuclease domain or DNA-binding domain is or comprises a Zn finger molecule. A Zn finger molecule comprises a Zn finger protein, e.g., a naturally occurring Zn finger protein or engineered Zn finger protein, or fragment thereof. Many Zn finger proteins are known to those of skill in the art and are commercially available, e.g., from Sigma-Aldrich.


In some embodiments, a Zn finger molecule comprises a non-naturally occurring Zn finger protein that is engineered to bind to a target DNA sequence of choice. See, for example, Beerli, et al. (2002) Nature Biotechnol. 20:135-141; Pabo, et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan, et al. (2001) Nature Biotechnol. 19:656-660; Segal, et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo, et al. (2000) Curr. Opin. Struct. Biol. 10:411-416; U.S. Pat. Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent Publication Nos. 2005/0064474; 2007/0218528; 2005/0267061, all incorporated herein by reference in their entireties.


An engineered Zn finger protein may have a novel binding specificity, compared to a naturally-occurring Zn finger protein. Engineering methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, using databases comprising triplet (or quadruplet) nucleotide sequences and individual Zn finger amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence. See, for example, U.S. Pat. Nos. 6,453,242 and 6,534,261, incorporated by reference herein in their entireties.


Exemplary selection methods, including phage display and two-hybrid systems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; as well as International Patent Publication Nos. WO 98/37186; WO 98/53057; WO 00/27878; and WO 01/88197 and GB 2,338,237. In addition, enhancement of binding specificity for zinc finger proteins has been described, for example, in International Patent Publication No. WO 02/077227.


In addition, as disclosed in these and other references, zinc finger domains and/or multi-fingered zinc finger proteins may be linked together using any suitable linker sequences, including for example, linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 or more amino acids in length. The proteins described herein may include any combination of suitable linkers between the individual zinc fingers of the protein. In addition, enhancement of binding specificity for zinc finger binding domains has been described, for example, in co-owned International Patent Publication No. WO 02/077227.


Zn finger proteins and methods for design and construction of fusion proteins (and polynucleotides encoding same) are known to those of skill in the art and described in detail in U.S. Pat. Nos. 6,140,0815; 789,538; 6,453,242; 6,534,261; 5,925,523; 6,007,988; 6,013,453; and 6,200,759; International Patent Publication Nos. WO 95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970; WO 01/88197; WO 02/099084; WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536; and WO 03/016496.


In addition, as disclosed in these and other references, Zn finger proteins and/or multi-fingered Zn finger proteins may be linked together, e.g., as a fusion protein, using any suitable linker sequences, including for example, linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 or more amino acids in length. The Zn finger molecules described herein may include any combination of suitable linkers between the individual zinc finger proteins and/or multi-fingered Zn finger proteins of the Zn finger molecule.


In certain embodiments, the DNA-binding domain or endonuclease domain comprises a Zn finger molecule comprising an engineered zinc finger protein that binds (in a sequence-specific manner) to a target DNA sequence. In some embodiments, the Zn finger molecule comprises one Zn finger protein or fragment thereof. In other embodiments, the Zn finger molecule comprises a plurality of Zn finger proteins (or fragments thereof), e.g., 2, 3, 4, 5, 6 or more Zn finger proteins (and optionally no more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 Zn finger proteins). In some embodiments, the Zn finger molecule comprises at least three Zn finger proteins. In some embodiments, the Zn finger molecule comprises four, five or six fingers. In some embodiments, the Zn finger molecule comprises 8, 9, 10, 11 or 12 fingers. In some embodiments, a Zn finger molecule comprising three Zn finger proteins recognizes a target DNA sequence comprising 9 or 10 nucleotides. In some embodiments, a Zn finger molecule comprising four Zn finger proteins recognizes a target DNA sequence comprising 12 to 14 nucleotides. In some embodiments, a Zn finger molecule comprising six Zn finger proteins recognizes a target DNA sequence comprising 18 to 21 nucleotides.


In some embodiments, a Zn finger molecule comprises a two-handed Zn finger protein. Two handed zinc finger proteins are those proteins in which two clusters of zinc finger proteins are separated by intervening amino acids so that the two zinc finger domains bind to two discontinuous target DNA sequences. An example of a two handed type of zinc finger binding protein is SIP1, where a cluster of four zinc finger proteins is located at the amino terminus of the protein and a cluster of three Zn finger proteins is located at the carboxyl terminus (see Remade, et al. (1999) EMBO Journal 18(18):5073-5084). Each cluster of zinc fingers in these proteins is able to bind to a unique target sequence and the spacing between the two target sequences can comprise many nucleotides.


DNA Binding Domain:


In certain aspects, the DNA-binding domain of a Gene Writer™ polypeptide described herein is selected, designed, or constructed for binding to a desired host DNA target sequence.


In some embodiments, a Gene Writer polypeptide comprises a modification to a DNA-binding domain, e.g., relative to the wild-type polypeptide. In some embodiments, the DNA-binding domain comprises an addition, deletion, replacement, or modification to the amino acid sequence of the original DNA-binding domain. In some embodiments, the DNA-binding domain is modified to include a heterologous functional domain that binds specifically to a target nucleic acid (e.g., DNA) sequence of interest. In some embodiments, the functional domain replaces at least a portion (e.g., the entirety of) the prior DNA-binding domain of the polypeptide. In some embodiments, the functional domain comprises a zinc finger (e.g., a zinc finger that specifically binds to the target nucleic acid (e.g., DNA) sequence of interest. In some embodiments, the functional domain comprises a Cas domain (e.g., a Cas domain that specifically binds to the target nucleic acid (e.g., DNA) sequence of interest. In embodiments, the Cas domain comprises a Cas9 or a mutant or variant thereof (e.g., as described herein). In embodiments, the Cas domain is associated with a guide RNA (gRNA), e.g., as described herein. In embodiments, the Cas domain is directed to a target nucleic acid (e.g., DNA) sequence of interest by the gRNA. In embodiments, the Cas domain is encoded in the same nucleic acid (e.g., RNA) molecule as the gRNA. In embodiments, the Cas domain is encoded in a different nucleic acid (e.g., RNA) molecule from the gRNA.


In certain embodiments, the DNA-binding domain of the polypeptide is a heterologous DNA-binding protein or domain relative to a native retrotransposon sequence. In some embodiments the heterologous DNA binding element is a zinc-finger element or a TAL effector element, e.g., a zinc-finger or TAL polypeptide or functional fragment thereof. In some embodiments the heterologous DNA binding element is a sequence-guided DNA binding element, such as Cas9, Cpf1, or other CRISPR-related protein that has been altered to have no endonuclease activity. In some embodiments the heterologous DNA binding element retains endonuclease activity. In some embodiments, the heterologous DNA binding element retains partial endonuclease activity to cleave ssDNA, e.g., possesses nickase activity. In some embodiments the heterologous DNA binding element replaces the endonuclease element of the polypeptide. In specific embodiments, the heterologous DNA-binding domain can be any one or more of Cas9, TAL domain, ZF domain, Myb domain, combinations thereof, or multiples thereof. In certain embodiments, the heterologous DNA-binding domain is a DNA binding domain of a retrotransposon or virus described in Table 1 or Table 3. A person having ordinary skill in the art is capable of identifying DNA binding domains based upon homology to other known DNA binding domains using tools as Basic Local Alignment Search Tool (BLAST). In still other embodiments, DNA-binding domains are modified, for example by site-specific mutation, increasing or decreasing DNA-binding elements (for example, number and/or specificity of zinc fingers), etc., to alter DNA-binding specificity and affinity. In some embodiments the DNA binding domain is altered from its natural sequence to have altered codon usage, e.g. improved for human cells


In some embodiments, the DNA binding domain comprises a meganuclease domain (e.g., as described herein, e.g., in the endonuclease domain section), or a functional fragment thereof. In some embodiments, the meganuclease domain possesses endonuclease activity, e.g., double-strand cleavage and/or nickase activity. In other embodiments, the meganuclease domain has reduced activity, e.g., lacks endonuclease activity, e.g., the meganuclease is catalytically inactive. In some embodiments, a catalytically inactive meganuclease is used as a DNA binding domain, e.g., as described in Fonfara et al. Nucleic Acids Res 40(2):847-860 (2012), incorporated herein by reference in its entirety. In embodiments, the DNA binding domain comprises one or more modifications relative to a wild-type DNA binding domain, e.g., a modification via directed evolution, e.g., phage-assisted continuous evolution (PACE).


In certain aspects of the present invention, the host DNA-binding site integrated into by the Gene Writer™ system can be in a gene, in an intron, in an exon, an ORF, outside of a coding region of any gene, in a regulatory region of a gene, or outside of a regulatory region of a gene. In other aspects, the polypeptide may bind to one or more than one host DNA sequence.


In some embodiments, a Gene Writing system is used to edit a target locus in multiple alleles. In some embodiments, a Gene Writing system is designed to edit a specific allele. For example, a Gene Writing polypeptide may be directed to a specific sequence that is only present on one allele, e.g., comprises a template RNA with homology to a target allele, e.g., a gRNA or annealing domain, but not to a second cognate allele. In some embodiments, a Gene Writing system can alter a haplotype-specific allele. In some embodiments, a Gene Writing system that targets a specific allele preferentially targets that allele, e.g., has at least a 2, 4, 6, 8, or 10-fold preference for a target allele.


In certain embodiments, a Gene Writer™ gene editor system RNA further comprises an intracellular localization sequence, e.g., a nuclear localization sequence. The nuclear localization sequence may be an RNA sequence that promotes the import of the RNA into the nucleus. In certain embodiments the nuclear localization signal is located on the template RNA. In certain embodiments, the retrotransposase polypeptide is encoded on a first RNA, and the template RNA is a second, separate, RNA, and the nuclear localization signal is located on the template RNA and not on an RNA encoding the retrotransposase polypeptide. While not wishing to be bound by theory, in some embodiments, the RNA encoding the retrotransposase is targeted primarily to the cytoplasm to promote its translation, while the template RNA is targeted primarily to the nucleus to promote its retrotransposition into the genome. In some embodiments the nuclear localization signal is at the 3′ end, 5′ end, or in an internal region of the template RNA. In some embodiments the nuclear localization signal is 3′ of the heterologous sequence (e.g., is directly 3′ of the heterologous sequence) or is 5′ of the heterologous sequence (e.g., is directly 5′ of the heterologous sequence). In some embodiments the nuclear localization signal is placed outside of the 5′ UTR or outside of the 3′ UTR of the template RNA. In some embodiments the nuclear localization signal is placed between the 5′ UTR and the 3′ UTR, wherein optionally the nuclear localization signal is not transcribed with the transgene (e.g., the nuclear localization signal is an anti-sense orientation or is downstream of a transcriptional termination signal or polyadenylation signal). In some embodiments the nuclear localization sequence is situated inside of an intron. In some embodiments a plurality of the same or different nuclear localization signals are in the RNA, e.g., in the template RNA. In some embodiments the nuclear localization signal is less than 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 or 1000 bp in legnth. Various RNA nuclear localization sequences can be used. For example, Lubelsky and Ulitsky, Nature 555 (107-111), 2018 describe RNA sequences which drive RNA localization into the nucleus. In some embodiments, the nuclear localization signal is a SINE-derived nuclear RNA localization (SIRLOIN) signal. In some embodiments the nuclear localization signal binds a nuclear-enriched protein. In some embodiments the nuclear localization signal binds the HNRNPK protein. In some embodiments the nuclear localization signal is rich in pyrimidines, e.g., is a C/T rich, C/U rich, C rich, T rich, or U rich region. In some embodiments the nuclear localization signal is derived from a long non-coding RNA. In some embodiments the nuclear localization signal is derived from MALATI long non-coding RNA or is the 600 nucleotide M region of MALAT1 (described in Miyagawa et al., RNA 18, (738-751), 2012). In some embodiments the nuclear localization signal is derived from BORG long non-coding RNA or is a AGCCC motif (described in Zhang et al., Molecular and Cellular Biology 34, 2318-2329 (2014). In some embodiments the nuclear localization sequence is described in Shukla et al., The EMBO Journal e98452 (2018). In some embodiments the nuclear localization signal is derived from a non-LTR retrotransposon, an LTR retrotransposon, retrovirus, or an endogenous retrovirus.


In some embodiments, a polypeptide described hereinherein comprises one or more (e.g., 2, 3, 4, 5) nuclear targeting sequences, for example a nuclear localization sequence (NLS). In some embodiments, the NLS is a bipartite NLS. In some embodiments, an NLS facilitates the import of a protein comprising an NLS into the cell nucleus. In some embodiments, the NLS is fused to the N-terminus of a Gene Writer described herein. In some embodiments, the NLS is fused to the C-terminus of the Gene Writer. In some embodiments, the NLS is fused to the N-terminus or the C-terminus of a Cas domain. In some embodiments, a linker sequence is disposed between the NLS and theneighboring domain of the Gene Writer.


In some embodiments, an NLS comprises the amino acid sequence MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 1592), PKKRKVEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 1593), RKSGKIAAIWKRPRKPKKKRKV (SEQ ID NO: 1594), KRTADGSEFESPKKKRKV (SEQ ID NO: 1595), KKTELQTTNAENKTKKL (SEQ ID NO: 1596), or KRGINDRNFWRGENGRKTR (SEQ ID NO: 1597), KRPAATKKAGQAKKKK (SEQ ID NO: 1598), or a functional fragment or variant thereof. Exemplary NLS sequences are also described in PCT/EP2000/011690, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In some embodiments, an NLS comprises an amino acid sequence as disclosed in Table 39. An NLS of this table may be utilized with one or more copies in a polypeptide in one or more locations in a polypeptide, e.g., 1, 2, 3 or more copies of an NLS in an N-terminal domain, between peptide domains, in a C-terminal domain, or in a combination of locations, in order to improve subcellular localization to the nucleus. Multiple unique sequences may be used within a single polypeptide. Sequences may be naturally monopartite or bipartite, e.g., having one or two stretches of basic amino acids, or may be used as chimeric bipartite sequences. Sequence references correspond to UniProt accession numbers, except where indicated as SeqNLS for sequences mined using a subcellular localization prediction algorithm (Lin et al BMC Bioinformat 13:157 (2012), incorporated herein by reference in its entirety).









TABLE 39







Exemplary nuclear localization signals for use in Gene Writing systems










SEQ ID



Sequence
NO:
Sequence References





AHFKISGEKRPSTDPGKKAK
3411
Q76IQ7


NPKKKKKKDP







AHRAKKMSKTHA
3412
P21827





ASPEYVNLPINGNG
3413
SeqNLS





CTKRPRW
3414
O88622, Q86W56, Q9QYM2, 002776





DKAKRVSRNKSEKKRR
3415
O15516, Q5RAK8, Q91YB2, Q91YB0,




Q8QGQ6, 008785, Q9WVS9, Q6YGZ4





EELRLKEELLKGIYA
3416
Q9QY16, Q9UHL0, Q2TBP1, Q9QY15





EEQLRRRKNSRLNNTG
3417
G5EFF5





EVLKVIRTGKRKKKAWKR
3418
SeqNLS


MVTKVC







HHHHHHHHHHHHQPH
3419
Q63934, G3V7L5, Q12837





HKKKHPDASVNFSEFSK
3420
P10103, Q4R844, P12682, B0CM99,




A9RA84, Q6YKA4, P09429, P63159,




Q08IE6, P63158, Q9YH06, B1MTB0





HKRTKK
3421
Q2R2D5





IINGRKLKLKKSRRRSSQTS
3422
SeqNLS


NNSFTSRRS







KAEQERRK
3423
Q8LH59





KEKRKRREELFIEQKKRK
3424
SeqNLS





KKGKDEWFSRGKKP
3425
P30999





KKGPSVQKRKKT
3426
Q6ZN17





KKKTVINDLLHYKKEK
3427
SeqNLS, P32354





KKNGGKGKNKPSAKIKK
3428
SeqNLS





KKPKWDDFKKKKK
3429
Q15397, Q8BKS9, Q562C7





KKRKKD
3430
SeqNLS, Q91Z62, Q1A730, Q969P5,




Q2KHT6, Q9CPU7





KKRRKRRRK
3431
SeqNLS





KKRRRRARK
3432
Q9UMS6, D4A702, Q91YE8





KKSKRGR
3433
Q9UBS0





KKSRKRGS
3434
B4FG96





KKSTALSRELGKIMRRR
3435
SeqNLS, P32354





KKSYQDPEIIAHSRPRK
3436
Q9U7C9





KKTGKNRKLKSKRVKTR
3437
Q9Z301, 054943, Q8K3T2





KKVSIAGQSGKLWRWKR
3438
Q6YUL8





KKYENVVIKRSPRKRGRPRK
3439
SeqNLS





KNKKRK
3440
SeqNLS





KPKKKR
3441
SeqNLS





KRAMKDDSHGNSTSPKRRK
3442
Q0E671





KRANSNLVAAYEKAKKK
3443
P23508





KRASEDTTSGSPPKKSSAGPKR
3444
Q9BZZ5, Q5R644





KRFKRRWMVRKMKTKK
3445
SeqNLS





KRGLNSSFETSPKKVK
3446
Q8IV63





KRGNSSIGPNDLSKRKQRKK
3447
SeqNLS





KRIHSVSLSQSQIDPSKKVK
3448
SeqNLS


RAK







KRKGKLKNKGSKRKK
3449
015381





KRRRRRRREKRKR
3450
Q96GM8





KRSNDRTYSPEEEKQRRA
3451
Q91ZF2





KRTVATNGDASGAHRAKK
3452
SeqNLS


MSK







KRVYNKGEDEQEHLPKGKK
3453
SeqNLS


R







KSGKAPRRRAVSMDNSNK
3454
Q9WVH4, 043524





KVNFLDMSLDDIIIYKELE
3455
Q9P127





KVQHRIAKKTTRRRR
3456
Q9DXE6





LSPSLSPL
3457
Q9Y261, P32182, P35583





MDSLLMNRRKFLYQFKNVR
1735
Q9GZX7


WAKGRRETYLC







MPQNEYIELHRKRYGYRLD
3458
SeqNLS


YHEKKRKKESREAHERSKK




AKKMIGLKAKLYHK







MVQLRPRASR
3459
SeqNLS





NNKLLAKRRKGGASPKDDP
3460
Q965G5


MDDIK







NYKRPMDGTYGPPAKRHEG
3461
O14497, A2BH40


E







PDTKRAKLDSSETTMVKKK
3462
SeqNLS





PEKRTKI
3463
SeqNLS





PGGRGKKK
3464
Q719N1, Q9UBP0, A2VDN5





PGKMDKGEHRQERRDRPY
3465
Q01844, Q61545





PKKGDKYDKTD
3466
Q45FA5





PKKKSRK
3467
O35914, Q01954





PKKNKPE
3468
Q22663





PKKRAKV
3469
P04295, P89438





PKPKKLKVE
3470
P55263, P55262, P55264, Q64640





PKRGRGR
3471
Q9FYS5, Q43386





PKRRLVDDA
3472
POC797





PKRRRTY
3473
SeqNLS





PLFKRR
3474
A8X6H4, Q9TXJO





PLRKAKR
3475
Q86WB0, Q5R8V9





PPAKRKCIF
3476
Q6AZ28, 075928, Q8C5D8





PPARRRRL
3477
Q8NAG6





PPKKKRKV
3478
Q3L6L5, P03070, P14999, P03071





PPNKRMKVKH
3479
Q8BN78





PPRIYPQLPSAPT
3480
POC799





PQRSPFPKSSVKR
3481
SeqNLS





PRPRKVPR
3482
POC799





PRRRVQRKR
3483
SeqNLS, Q5R448, Q5TAQ9





PRRVRLK
3484
Q58DJ0, P56477, Q13568





PSRKRPR
3485
Q62315, Q5F363, Q92833





PSSKKRKV
3486
SeqNLS





PTKKRVK
3487
P07664





QRPGPYDRP
3488
SeqNLS





RGKGGKGLGKGGAKRHRK
3489
SeqNLS





RKAGKGGGGHKTTKKRSA
3490
B4FG96


KDEKVP







RKIKLKRAK
3491
A1L3G9





RKIKRKRAK
3492
B9X187





RKKEAPGPREELRSRGR
3493
O35126, P54258, Q5IS70, P54259


RKKRKGK
3494
SeqNLS, Q29243, Q62165, Q28685,




018738, Q9TSZ6, Q14118





RKKRRQRRR
3495
P04326, P69697, P69698, P05907,




P20879, P04613, P19553, POC1J9,




P20893, P12506, P04612, Q73370,




POC1K0, P05906, P35965, P04609,




P04610, P04614, P04608, P05905





RKKSIPLSIKNLKRKHKRKK
3496
Q9C0C9


NKITR







RKLVKPKNTKMKTKLRTNP
3497
Q14190


Y







RKRLILSDKGQLDWKK
3498
SeqNLS, Q91Z62, Q1A730, Q2KHT6,




Q9CPU7





RKRLKSK
3499
Q13309





RKRRVRDNM
3500
Q8QPH4, Q809M7, A8C8X1, Q2VNC5,




Q38SQ0, O89749, Q6DNQ9, Q809L9,




Q0A429, Q20NV3, P16509, P16505,




Q6DNQ5, P16506, Q6XT06, P26118,




Q2ICQ2, Q2RCG8, Q0A2D0, Q0A2H9,




Q9IQ46, Q809M3, Q6J847, Q6J856,




B4URE4, A4GCM7, Q0A440, P26120,




P16511,





RKRSPKDKKEKDLDGAGKR
3501
Q7RTP6


RKT







RKRTPRVDGQTGENDMNK
3502
O94851


RRRK







RLPVRRRRRR
3503
P04499, P12541, P03269, P48313,




P03270





RLRFRKPKSK
3504
P69469





RQQRKR
3505
Q14980





RRDLNSSFETSPKKVK
3506
Q8K3G5





RRDRAKLR
3507
Q9SLB8





RRGDGRRR
3508
Q80WE1, Q5R9B4, Q06787, P35922





RRGRKRKAEKQ
3509
Q812D1, Q5XXA9, Q99JF8, Q8MJG1,




Q66T72, O75475





RRKKRR
3510
Q0VD86, Q58DS6, Q5R6G2, Q9ERI5,




Q6AYK2, Q6NYC1





RRKRSKSEDMDSVESKRRR
3511
Q7TT18





RRKRSR
3512
Q99PU7, D3ZHS6, Q92560, A2VDM8





RRPKGKTLQKRKPK
3513
Q6ZN17





RRRGFERFGPDNMGRKRK
3514
Q63014, Q9DBR0





RRRGKNKVAAQNCRK
3515
SeqNLS





RRRKRR
3516
Q5FVH8, Q6MZT1, Q08DH5, Q8BQP9





RRRQKQKGGASRRR
3517
SeqNLS





RRRREGPRARRRR
3518
P08313, P10231





RRTIRLKLVYDKCDRSCKIQ
3519
SeqNLS


KKNRNKCQYCRFHKCLSVG




MSHNAIRFGRMPRSEKAKL




KAE







RRVPQRKEVSRCRKCRK
3520
Q5RJN4, Q32L09, Q8CAK3, Q9NUL5





RVGGRRQAVECIEDLLNEP
3521
P03255


GQPLDLSCKRPRP







RVVKLRIAP
3522
P52639, Q8JMNO





RVVRRR
3523
P70278





SKRKTKISRKTR
3524
Q5RAY1, O00443





SYVKTVPNRTRTYIKL
3525
P21935





TGKNEAKKRKIA
3526
P52739, Q8K3J5, Q5RAU9





TLSPASSPSSVSCPVIPASTD
3527
SeqNLS


ESPGSALNI







VSKKQRTGKKIH
3528
P52739, Q8K3J5, Q5RAU9





SPKKKRKVE
3529






KRTAD GSEFE SPKKKRKVE
3530






PAAKRVKLD
3531






PKKKRKV
3532






MDSLLMNRRKFLYQFKNVR
1735



WAKGRRETYLC







SPKKKRKVEAS
3533






MAPKKKRKVGIHRGVP
3534









In some embodiments, the NLS is a bipartite NLS. A bipartite NLS typically comprises two basic amino acid clusters separated by a spacer sequence (which may be, e.g., about 10 amino acids in length). A monopartite NLS typically lacks a spacer. An example of a bipartite NLS is the nucleoplasmin NLS, having the sequence KR[PAATKKAGQA]KKKK (SEQ ID NO: 1598), wherein the spacer is bracketed. Another exemplary bipartite NLS has the sequence PKKKRKVEGADKRTADGSEFESPKKKRKV (SEQ ID NO: 1600). Exemplary NLSs are described in International Application WO2020051561, which is herein incorporated by reference in its entirety, including for its disclosures regarding nuclear localization sequences.


In certain embodiments, a Gene Writer™ gene editor system polypeptide further comprises an intracellular localization sequence, e.g., a nuclear localization sequence and/or a nucleolar localization sequence. The nuclear localization sequence and/or nucleolar localization sequence may be amino acid sequences that promote the import of the protein into the nucleus and/or nucleolus, where it can promote integration of heterologous sequyence into the genome. In certain embodiments, a Gene Writer™ gene editor system polypeptide (e.g., a retrotransposase, e.g., a polypeptide according to any of Tables 1 or 3 herein) further comprises a nucleolar localization sequence. In certain embodiments, the retrotransposase polypeptide is encoded on a first RNA, and the template RNA is a second, separate, RNA, and the nucleolar localization signal is encoded on the RNA encoding the retrotransposase polypeptide and not on the template RNA. In some embodiments, the nucleolar localization signal is located at the N-terminus, C-terminus, or in an internal region of the polypeptide. In some embodiments, a plurality of the same or different nucleolar localization signals are used. In some embodiments, the nuclear localization signal is less than 5, 10, 25, 50, 75, or 100 amino acids in length. Various polypeptide nucleolar localization signals can be used. For example, Yang et al., Journal of Biomedical Science 22, 33 (2015), describe a nuclear localization signal that also functions as a nucleolar localization signal. In some embodiments, the nucleolar localization signal may also be a nuclear localization signal. In some embodiments, the nucleolar localization signal may overlap with a nuclear localization signal. In some embodiments, the nucleolar localization signal may comprise a stretch of basic residues. In some embodiments, the nucleolar localization signal may be rich in arginine and lysine residues. In some embodiments, the nucleolar localization signal may be derived from a protein that is enriched in the nucleolus. In some embodiments, the nucleolar localization signal may be derived from a protein enriched at ribosomal RNA loci. In some embodiments, the nucleolar localization signal may be derived from a protein that binds rRNA. In some embodiments, the nucleolar localization signal may be derived from MSP58. In some embodiments, the nucleolar localization signal may be a monopartite motif. In some embodiments, the nucleolar localization signal may be a bipartite motif. In some embodiments, the nucleolar localization signal may consist of a multiple monopartite or bipartite motifs. In some embodiments, the nucleolar localization signal may consist of a mix of monopartite and bipartite motifs. In some embodiments, the nucleolar localization signal may be a dual bipartite motif. In some embodiments, the nucleolar localization motif may be a KRASSQALGTIPKRRSSSRFIKRKK (SEQ ID NO: 1530). In some embodiments, the nucleolar localization signal may be derived from nuclear factor-KB-inducing kinase. In some embodiments, the nucleolar localization signal may be an RKKRKKK motif (SEQ ID NO: 1531) (described in Birbach et al., Journal of Cell Science, 117 (3615-3624), 2004).


In some embodiments, a nucleic acid described herein (e.g., an RNA encoding a Gene Writer™ polypeptide, or a DNA encoding the RNA) comprises a microRNA binding site. In some embodiments, the microRNA binding site is used to increase the target-cell specificity of a Gene Writer™ system. For instance, the microRNA binding site can be chosen on the basis that is is recognized by a miRNA that is present in a non-target cell type, but that is not present (or is present at a reduced level relative to the non-target cell) in a target cell type. Thus, when the RNA encoding the Gene Writer™ polypeptide is present in a non-target cell, it would be bound by the miRNA, and when the RNA encoding the Gene Writer™ polypeptide is present in a target cell, it would not be bound by the miRNA (or bound but at reduced levels relative to the non-target cell). While not wishing to be bound by theory, binding of the miRNA to the RNA encoding the Gene Writer™ polypeptide may reduce production of the Gene Writer™ polypeptide, e.g., by degrading the mRNA encoding the polypeptide or by interfering with translation. Accordingly, in such embodiments the Gene Writer would add to/edit the genome of target cells more efficiently than it edits the genome of non-target cells, e.g., the heterologous object sequence would be inserted into the genome of target cells more efficiently than into the genome of non-target cells, or an insertion or deletion is produced more efficiently in target cells than in non-target cells. A system having a microRNA binding site in the RNA encoding the Gene Writer™ polypeptide (or encoded in the DNA encoding the RNA) may also be used in combination with a template RNA that is regulated by a second microRNA binding site, e.g., as described herein in the section entitled “Template RNA component of Gene Writer™ gene editor system.” In some embodiments, e.g., for liver indications, a miRNA is selected from Table 4 of WO2020014209, incorporated herein by reference.


In some embodiments, the DNA encoding a Gene Writer polypeptide comprises a promoter sequence, e.g., a tissue specific promoter sequence. In some embodiments, the tissue-specific promoter is used to increase the target-cell specificity of a Gene Writer™ system. For instance, the promoter can be chosen on the basis that it is active in a target cell type but not active in (or active at a lower level in) a non-target cell type. A system having a tissue-specific promoter sequence in the DNA of the polypeptide may also be used in combination with a microRNA binding site, e.g., in the template RNA or a nucleic acid encoding a Gene Writer™ protein, e.g., as described herein. A system having a tissue-specific promoter sequence in the DNA encoding the Gene Writer polypeptide may also be used in combination with a DNA encoding the RNA template driven by a tissue-specific promoter, e.g., to achieve higher levels of RNA template in target cells than in non-target cells. In some embodiments, e.g., for liver indications, a tissue-specific promoter is selected from Table 3 of WO2020014209, incorporated herein by reference.


A skilled artisan can, based on the Accession numbers and/or sequences provided in Tables 1 and 3, determine the nucleic acid and corresponding polypeptide sequences of each retrotransposon or virus, and domains thereof, e.g., by using routine sequence analysis tools as Basic Local Alignment Search Tool (BLAST) or CD-Search for conserved domain analysis. Other sequence analysis tools are known and can be found, e.g., at molbiol-tools.ca, for example, at molbiol-tools.ca/Motifs.htm.


Tables 1 and 3 herein provide the sequences of exemplary transposons or viruses, including the amino acid sequence(s) of the retrotransposase, reverse transcriptase, DNA-binding domain, and/or endonuclease domain; sequences of 5′ and 3′ untranslated regions to allow a polypeptide, e.g., the retrotransposase to bind the template RNA; and/or the full transposon nucleic acid sequence. In some embodiments, a 5′ UTR contained in or referenced by Tables 1 or 3 allows a polypeptide, e.g., the retrotransposase, to bind the template RNA. In some embodiments, a 3′ UTR contained in or referenced by Tables 1 or 3 allows a polypeptide, e.g., the retrotransposase, to bind the template RNA. Thus, in some embodiments, a polypeptide for use in any of the systems described herein can be a polypeptide of any of Tables 1 or 3 herein, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, the system further comprises one or both of a 5′ or 3′ untranslated region contained in or referenced by Tables 1 or 3 (or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto), e.g., from the same transposon as the polypeptide referred to in the preceding sentence, as indicated in the same row of the same table. In some embodiments, the system comprises one or both of a 5′ or 3′ untranslated region contained in or referenced by Tables 1 or 3, e.g., a segment of the full transposon sequence that encodes an RNA that is capable of binding a retrotransposase, and/or the sub-sequence provided in the column entitled Predicted 5′ UTR or Predicted 3′ UTR.


In some embodiments, a system or method described herein involves a 3′ UTR, 5′ UTR, or both from a retrotransposon of Table 3. In some embodiments, the 3′ UTR, 5′ UTR, or both, has a sequence comprising a portion of the full retrotransposon DNA sequence shown in column 5 of Table 3 of International Application PCT/US2019/048607, which is incorporated by reference herein in its entirety, including Table 3. In some embodiments, the nucleic acid sequence or amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequence in Table 3 of PCT/US2019/048607.


In some embodiments, a system or method described herein involves a nucleic acid sequence or amino acid sequence of a retrotransposon described in Table 1 or Table 2 of International Application PCT/US2019/048607, which is incorporated by reference herein in its entirety, including Tables 1 and 2. In some embodiments, the nucleic acid sequence or amino acid sequence has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of a retrotransposon described in said Table 1 or Table 2 of PCT/US2019/048607.


In some embodiments, a polypeptide for use in any of the systems described herein can be a molecular reconstruction or ancestral reconstruction based upon the aligned polypeptide sequence of multiple retrotransposons. In some embodiments, a 5′ or 3′ untranslated region for use in any of the systems described herein can be a molecular reconstruction based upon the aligned 5′ or 3′ untranslated region of multiple retrotransposons. A skilled artisan can, based on the Accession numbers provided herein, align polypeptides or nucleic acid sequences, e.g., by using routine sequence analysis tools as Basic Local Alignment Search Tool (BLAST) or CD-Search for conserved domain analysis. Molecular reconstructions can be created based upon sequence consensus, e.g. using approaches described in Ivics et al., Cell 1997, 501-510; Wagstaff et al., Molecular Biology and Evolution 2013, 88-99. In some embodiments, the retrotransposon from which the 5′ or 3′ untranslated region or polypeptide is derived is a young or a recently active mobile element, as assessed via phylogenetic methods such as those described in Boissinot et al., Molecular Biology and Evolution 2000, 915-928.


Thermostable Gene Writer™ Systems


While not wishing to be bound by theory, in some embodiments, retrotransposases that evolved in cold environments may not function as well at human body temperature. This application provides a number of thermostable Gene Writer™ systems, including proteins derived from avian retrotransposases. Exemplary avian transposase sequences in Table 3 include those of Taeniopygia guttata (zebra finch; transposon name R2-1_TG), Geospiza fortis (medium ground finch; transposon name R2-1_Gfo), Zonotrichia albicollis (white-throated sparrow; transposon name R2-1_ZA), and Tinamus guttatus (white-throated tinamou; transposon name R2-1_TGut).


Thermostability may be measured, e.g., by testing the ability of a Gene Writer™ to polymerize DNA in vitro at a high temperature (e.g., 37° C.) and a low temperature (e.g., 25° C.). Suitable conditions for assaying in vitro DNA polymerization activity (e.g., processivity) are described, e.g., in Bibillo and Eickbush, “High Processivity of the Reverse Transcriptase from a Non-long Terminal Repeat Retrotransposon” (2002) JBC 277, 34836-34845. In some embodiments, the thermostable Gene Writer™ polypeptide has an activity, e.g., a DNA polymerization activity, at 37° C. that is no less than 70%, 75%, 80%, 85%, 90%, or 95% of its activity at 25° C. under otherwise similar conditions.


In some embodiments, a Gene Writer™ polypeptide (e.g., a sequence of Table 1 or 3 or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto) is stable in a subject chosen from a mammal (e.g., human) or a bird. In some embodiments, a Gene Writer™ polypeptide described herein is functional at 37° C. In some embodiments, a Gene Writer™ polypeptide described herein has greater activity at 37° C. than it does at a lower temperature, e.g., at 30° C., 25° C., or 20° C. In some embodiments, a Gene Writer™ polypeptide described herein has greater activity in a human cell than in a zebrafish cell.


In some embodiments, a Gene Writer™ polypeptide is active in a human cell cultured at 37° C., e.g., using an assay of Example 6 or Example 7 of PCT/US2019/048607 which are hereby incorporated by reference.


In some embodiments, the assay comprises steps of: (1) introducing HEK293T cells into one or more wells of 6.4 mm diameter, at 10,000 cells/well, (2) incubating the cells at 37° C. for 24 hr, (3) providing a transfection mixture comprising 0.5 μl if FuGENE® HD transfection reagent and 80 ng DNA (wherein the DNA is a plasmid comprising, in order, (a) CMV promoter, (b) 100 bp of sequence homologous to the 100 bp upstream of the target site, (c) sequence encoding a 5′ untranslated region that binds the Gene Writer™ protein, (d) sequence encoding the Gene Writer™ protein, (e) sequence encoding a 3′ untranslated region that binds the Gene Writer™ protein (f) 100 bp of sequence homologous to the 100 bp downstream of the target site, and (g) BGH polyadenylation sequence) and 10 μl Opti-MEM and incubating for 15 min at room temperature, (4) adding the transfection mixture to the cells, (5) incubating the cells for 3 days, and (6) assaying integration of the exogenous sequence into a target locus (e.g., rDNA) in the cell genome, e.g., wherein one or more of the preceding steps are performed as described in Example 6 of PCT/US2019/048607 which is hereby incorporated by reference.


In some embodiments, the Gene Writer™ polypeptide results in insertion of the heterologous object sequence (e.g., the GFP gene) into the target locus (e.g., rDNA) at an average copy number of at least 0.01, 0.025, 0.05, 0.075, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 4, or 5 copies per genome. In some embodiments, a cell described herein (e.g., a cell comprising a heterologous sequence at a target insertion site) comprises the heterologous object sequence at an average copy number of at least 0.01, 0.025, 0.05, 0.075, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 4, or 5 copies per genome.


In some embodiments, a Gene Writer™ causes integration of a sequence in a target RNA with relatively few truncation events at the terminus. For instance, in some embodiments, a Gene Writer™ protein (e.g., of SEQ ID NO: 1016) results in about 25-100%, 50-100%, 60-100%, 70-100%, 75-95%, 80%-90%, or 86.17% of integrants into the target site being non-truncated, as measured by an assay described herein, e.g., an assay of Example 6 and FIG. 8 of PCT/US2019/048607 which are hereby incorporated by reference. In some embodiments, a Gene Writer™ protein (e.g., of SEQ ID NO: 1016) results in at least about 30%, 40%, 50%, 60%, 70%, 80%, or 90% of integrants into the target site being non-truncated, as measured by an assay described herein. In some embodiments, an integrant is classified as truncated versus non-truncated using an assay comprising amplification with a forward primer situated 565 bp from the end of the element (e.g., a wild-type transposon sequence, e.g., of Taeniopygia guttata) and a reverse primer situated in the genomic DNA of the target insertion site, e.g., rDNA. In some embodiments, the number of full-length integrants in the target insertion site is greater than the number of integrants truncated by 300-565 nucleotides in the target insertion site, e.g., the number of full-length integrants is at least 1.1×, 1.2×, 1.5×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10× the number of the truncated integrants, or the number of full-length integrants is at least 1.1×-10×, 2×-10×, 3×-10×, or 5×-10× the number of the truncated integrants.


In some embodiments, a system or method described herein results in insertion of the heterologous object sequence only at one target site in the genome of the target cell. Insertion can be measured, e.g., using a threshold of above 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, e.g., as described in Example 8 of PCT/US2019/048607 which is hereby incorporated by reference. In some embodiments, a system or method described herein results in insertion of the heterologous object sequence wherein less than 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 10%, 20%, 30%, 40%, or 50% of insertions are at a site other than the target site, e.g., using an assay described herein, e.g., an assay of Example 8 of PCT/US2019/048607.


In some embodiments, a system or method described herein results in “scarless” insertion of the heterologous object sequence, while in some embodiments, the target site can show deletions or duplications of endogenous DNA as a result of insertion of the heterologous sequence. The mechanisms of different retrotransposons could result in different patterns of duplications or deletions in the host genome occurring during retrotransposition at the target site. In some embodiments, the system results in a scarless insertion, with no duplications or deletions in the surrounding genomic DNA. In some embodiments, the system results in a deletion of less than 1, 2, 3, 4, 5, 10, 50, or 100 bp of genomic DNA upstream of the insertion. In some embodiments, the system results in a deletion of less than 1, 2, 3, 4, 5, 10, 50, or 100 bp of genomic DNA downstream of the insertion. In some embodiments, the system results in a duplication of less than 1, 2, 3, 4, 5, 10, 50, or 100 bp of genomic DNA upstream of the insertion. In some embodiments, the system results in a duplication of less than 1, 2, 3, 4, 5, 10, 50, or 100 bp of genomic DNA downstream of the insertion.


In some embodiments, a Gene Writer™ described herein, or a DNA-binding domain thereof, binds to its target site specifically, e.g., as measured using an assay of Example 21 of PCT/US2019/048607 which is hereby incorporated by reference. In some embodiments, the Gene Writer™ or DNA-binding domain thereof binds to its target site more strongly than to any other binding site in the human genome. For example, in some embodiments, in an assay of Example 21 of PCT/US2019/048607, the target site represents more than 50%, 60%, 70%, 80%, 90%, or 95% of binding events of the Gene Writer™ or DNA-binding domain thereof to human genomic DNA.


Genetically Engineered, e.g., Dimerized Gene Writer™ Systems


Some non-LTR retrotransposons utilize two subunits to complete retrotransposition (Christensen et al PNAS 2006). In some embodiments, a retrotransposase described herein comprises two connected subunits as a single polypeptide. For instance, two wild-type retrotransposases could be joined with a linker to form a covalently “dimerized” protein. In some embodiments, the nucleic acid coding for the retrotransposase codes for two retrotransposase subunits to be expressed as a single polypeptide. In some embodiments, the subunits are connected by a peptide linker, such as has been described herein in the section entitled “Linker” and, e.g., in Chen et al Adv Drug Deliv Rev 2013. In some embodiments, the two subunits in the polypeptide are connected by a rigid linker. In some embodiments, the rigid linker consists of the motif (EAAAK)n(SEQ ID NO: 1534). In other embodiments, the two subunits in the polypeptide are connected by a flexible linker. In some embodiments, the flexible linker consists of the motif (Gly)n. In some embodiments, the flexible linker consists of the motif (GGGGS)n(SEQ ID NO: 1535). In some embodiments, the rigid or flexible linker consists of 1, 2, 3, 4, 5, 10, 15, or more amino acids in length to enable retrotransposition. In some embodiments, the linker consists of a combination of rigid and flexible linker motifs.


Based on mechanism, not all functions are required from both retrotransposase subunits. In some embodiments, the fusion protein may consist of a fully functional subunit and a second subunit lacking one or more functional domains. In some embodiments, one subunit may lack reverse transcriptase functionality. In some embodiments, one subunit may lack the reverse transcriptase domain. In some embodiments, one subunit may possess only endonuclease activity. In some embodiments, one subunit may possess only an endonuclease domain. In some embodiments, the two subunits comprising the single polypeptide may provide complimentary functions.


In some embodiments, one subunit may lack endonuclease functionality. In some embodiments, one subunit may lack the endonuclease domain. In some embodiments, one subunit may possess only reverse transcriptase activity. In some embodiments, one subunit may possess only a reverse transcriptase domain. In some embodiments, one subunit may possess only DNA-dependent DNA synthesis functionality.


Linkers


In some embodiments, domains of the compositions and systems described herein (e.g., the endonuclease and reverse transcriptase domains of a polypeptide or the DNA binding domain and reverse transcriptase domains of a polypeptide) may be joined by a linker. A composition described herein comprising a linker element has the general form S1-L-S2, wherein S1 and S2 may be the same or different and represent two domain moieties (e.g., each a polypeptide or nucleic acid domain) associated with one another by the linker. In some embodiments, a linker may connect two polypeptides. In some embodiments, a linker may connect two nucleic acid molecules. In some embodiments, a linker may connect a polypeptide and a nucleic acid molecule. A linker may be a chemical bond, e.g., one or more covalent bonds or non-covalent bonds. A linker may be flexible, rigid, and/or cleavable. In some embodiments, the linker is a peptide linker. Generally, a peptide linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids in length, e.g., 2-50 amino acids in length, 2-30 amino acids in length.


The most commonly used flexible linkers have sequences consisting primarily of stretches of Gly and Ser residues (“GS” linker). Flexible linkers may be useful for joining domains that require a certain degree of movement or interaction and may include small, non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids. Incorporation of Ser or Thr can also maintain the stability of the linker in aqueous solutions by forming hydrogen bonds with the water molecules, and therefore reduce unfavorable interactions between the linker and the other moieties. Examples of such linkers include those having the structure [GGS]≥1 or [GGGS]≥1 (SEQ ID NO: 1536). Rigid linkers are useful to keep a fixed distance between domains and to maintain their independent functions. Rigid linkers may also be useful when a spatial separation of the domains is critical to preserve the stability or bioactivity of one or more components in the agent. Rigid linkers may have an alpha helix-structure or Pro-rich sequence, (XP)n, with X designating any amino acid, preferably Ala, Lys, or Glu. Cleavable linkers may release free functional domains in vivo. In some embodiments, linkers may be cleaved under specific conditions, such as the presence of reducing reagents or proteases. In vivo cleavable linkers may utilize the reversible nature of a disulfide bond. One example includes a thrombin-sensitive sequence (e.g., PRS) between the two Cys residues. In vitro thrombin treatment of CPRSC (SEQ ID NO: 1537) results in the cleavage of the thrombin-sensitive sequence, while the reversible disulfide linkage remains intact. Such linkers are known and described, e.g., in Chen et al. 2013. Fusion Protein Linkers: Property, Design and Functionality. Adv Drug Deliv Rev. 65(10): 1357-1369. In vivo cleavage of linkers in compositions described herein may also be carried out by proteases that are expressed in vivo under pathological conditions (e.g. cancer or inflammation), in specific cells or tissues, or constrained within certain cellular compartments. The specificity of many proteases offers slower cleavage of the linker in constrained compartments.


In some embodiments the amino acid linkers are (or are homologous to) the endogenous amino acids that exist between such domains in a native polypeptide. In some embodiments the endogenous amino acids that exist between such domains are substituted but the length is unchanged from the natural length. In some embodiments, additional amino acid residues are added to the naturally existing amino acid residues between domains.


In some embodiments, the amino acid linkers are designed computationally or screened to maximize protein function (Anad et al., FEBS Letters, 587:19, 2013).


Additional Domains:


The Gene Writer™ polypeptide comprises the functions necessary to bind a target DNA sequence and template nucleic acid (e.g., template RNA), nick the target site, and write (e.g., reverse transcribe) the template into DNA, resulting in a modification of the target site. In some embodiments, additional domains may be added to the polypeptide to enhance the efficiency of the process. In some embodiments, the Gene Writer™ polypeptide may contain an additional DNA ligation domain to join reverse transcribed DNA to the DNA of the target site. In some embodiments, the polypeptide may comprise a heterologous RNA-binding domain. In some embodiments, the polypeptide may comprise a domain having 5′ to 3′ exonuclease activity (e.g., wherein the 5′ to 3′ exonuclease activity increases repair of the alteration of the target site, e.g., in favor of alteration over the original genomic sequence). In some embodiments, the polypeptide may comprise a domain having 3′ to 5′ exonuclease activity, e.g., proof-reading activity. In some embodiments, the writing domain, e.g., RT domain, has 3′ to 5′ exonuclease activity, e.g., proof-reading activity.


In some embodiments, the polypeptide does not comprise an RNase H domain. In some embodiments, the polypeptide comprises an RNaseH domain endogenous to one of the other domains. In some embodiments, the polypeptide comprises an RNase H domain that is heterologous to the other domains. In some embodiments, the polypeptide comprises an inactivated endogenous RNaseH domain.


In some embodiments, a Gene Writer as described herein comprises a polypeptide associated with a guide RNA (gRNA). In certain embodiments, the gRNA is comprised in the template nucleic acid molecule. In other embodiments, the gRNA is separate from the template nucleic acid molecule. In some embodiments wherein the gRNA is comprised in the template nucleic acid molecule, the template nucleic acid molecule further comprises a gRNA spacer sequence (e.g., at or within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides of its 5′ end). In embodiments, the gRNA spacer comprises a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleic acid sequence comprised in the target nucleic acid molecule. In embodiments, the gRNA spacer directs Cas domain (e.g., Cas9) activity at the nucleic acid sequence comprised in the target nucleic acid molecule. In some embodiments wherein the gRNA is comprised in the template nucleic acid molecule, the template nucleic acid molecule further comprises a primer binding site (e.g., at or within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides of its 3′ end). In embodiments, the primer binding site comprises a nucleic acid sequence comprising at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleic acid sequence positioned at the 5′ end (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 nucleotides) of a nick site on the target nucleic acid molecule. In embodiments, binding of the primer binding site to the target nucleic acid molecule operates to prime TPRT.


Template Nucleic Acid Component of Gene Writer™ Gene Editor System

The Gene Writer™ systems described herein can modify a host target DNA site using a template nucleic acid sequence. In some embodiments, the Gene Writer™ systems described herein transcribe an RNA sequence template into host target DNA sites by target-primed reverse transcription (TPRT). By writing DNA sequence(s) via reverse transcription of the RNA sequence template directly into the host genome, the Gene Writer™ system can insert an object sequence into a target genome without the need for exogenous DNA sequences to be introduced into the host cell (unlike, for example, CRISPR systems), as well as eliminate an exogenous DNA insertion step. The Gene Writer™ system can also delete a sequence from the target genome or introduce a substitution using an object sequence. Therefore, the Gene Writer™ system provides a platform for the use of customized RNA sequence templates containing object sequences, e.g., sequences comprising heterologous gene coding and/or function information.


In some embodiments, a Gene Writer system comprises a template nucleic acid (e.g., RNA or DNA) molecule. In some embodiments, the template nucleic acid molecule comprises a 5′ homology region and/or a 3′ homology region. In some embodiments, the 5′ homology region comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence similarity with a nucleic acid sequence comprised in a target nucleic acid molecule. In embodiments, the nucleic acid sequence in the target nucleic acid molecule is within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides of (e.g., 5′ relative to) a target insertion site, e.g., for a heterologous object sequence, e.g., comprised in the template nucleic acid molecule.


In some embodiments, the 3′ homology region comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a nucleic acid sequence comprised in a target nucleic acid molecule. In embodiments, the nucleic acid sequence in the target nucleic acid molecule is within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides of (e.g., 3′ relative to) a target insertion site, e.g., for a heterologous object sequence, e.g., comprised in the template nucleic acid molecule. In some embodiments, the 5′ homology region is heterologous to the remainder of the template nucleic acid molecule. In some embodiments, the 3′ homology region is heterologous to the remainder of the template nucleic acid molecule.


In some embodiments, a template nucleic acid (e.g., template RNA) comprises a 3′ target homology domain. In some embodiments, a 3′ target homology domain is disposed 3′ of the heterologous object sequence and is complementary to a sequence adjacent to a site to be modified by a system described herein, or comprises no more than 1, 2, 3, 4, or 5 mismatches to a sequence complementary to the sequence adjacent to a site to be modified by the system/Gene Writer™. In some embodiments, the 3′ homology region binds within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of a nick site in the target nucleic acid molecule. In some embodiments, binding of the 3′ homology region to the target nucleic acid molecule permits initiation of target-primed reverse transcription (TPRT), e.g., with the 3′ homology region acting as a primer for TPRT. In some embodiments, the 3′ target homology domain anneals to the target site, which provides a binding site and the 3′ hydroxyl for the initiation of TPRT by a Gene Writer polypeptide. In some embodiments, the 3′ target homology domain is 3-5, 5-10, 10-30, 10-25, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11, 11-30, 11-25, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-30, 12-25, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-30, 13-25, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 13-14, 14-30, 14-25, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-30, 15-25, 15-20, 15-19, 15-18, 15-17, 15-16, 16-30, 16-25, 16-20, 16-19, 16-18, 16-17, 17-30, 17-25, 17-20, 17-19, 17-18, 18-30, 18-25, 18-20, 18-19, 19-30, 19-25, 19-20, 20-30, 20-25, or 25-30 nt in length, e.g., 10-17, 12-16, or 12-14 nt in length.


In some embodiments, a template nucleic acid (e.g., template RNA) comprises a heterologous object sequence. In some embodiments, the heterologous object sequence may be transcribed by the RT domain of a Gene Writer™ polypeptide, e.g., thereby introducing an alteration into a target site in genomic DNA. In some embodiments, the heterologous object sequence is at least 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 120, 140, 160, 180, 200, 500, or 1,000 nucleotides (nts) in length, or at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 kilobases in length. In some embodiments, the heterologous object sequence is no more than 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 120, 140, 160, 180, 200, 500, 1,000, or 2000 nucleotides (nts) in length, or no more than 20, 15, 10, 9, 8, 7, 6, 5, 4, or 3 kilobases in length. In some embodiments, the heterologous object sequence is 30-1000, 40-1000, 50-1000, 60-1000, 70-1000, 74-1000, 75-1000, 76-1000, 77-1000, 78-1000, 79-1000, 80-1000, 85-1000, 90-1000, 100-1000, 120-1000, 140-1000, 160-1000, 180-1000, 200-1000, 500-1000, 30-500, 40-500, 50-500, 60-500, 70-500, 74-500, 75-500, 76-500, 77-500, 78-500, 79-500, 80-500, 85-500, 90-500, 100-500, 120-500, 140-500, 160-500, 180-500, 200-500, 30-200, 40-200, 50-200, 60-200, 70-200, 74-200, 75-200, 76-200, 77-200, 78-200, 79-200, 80-200, 85-200, 90-200, 100-200, 120-200, 140-200, 160-200, 180-200, 30-100, 40-100, 50-100, 60-100, 70-100, 74-100, 75-100, 76-100, 77-100, 78-100, 79-100, 80-100, 85-100, or 90-100 nucleotides (nts) in length, or 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-15, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-15, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-20, 5-15, 5-10, 5-9, 5-8, 5-7, 5-6, 6-20, 6-15, 6-10, 6-9, 6-8, 6-7, 7-20, 7-15, 7-10, 7-9, 7-8, 8-20, 8-15, 8-10, 8-9, 9-20, 9-15, 9-10, 10-15, 10-20, or 15-20 kilobases in length. In some embodiments, the heterologous object sequence is 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, or 10-20 nt in length, e.g., 10-80, 10-50, or 10-20 nt in length, e.g., about 10-20 nt in length. In some embodiments, a template RNA comprises a sequence as listed in Table 43, or a sequence with at lest 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.


In certain embodiments, the template nucleic acid comprises a customized RNA sequence template which can be identified, designed, engineered and constructed to contain sequences altering or specifying host genome function, for example by introducing a heterologous coding region into a genome; affecting or causing exon structure/alternative splicing; causing disruption of an endogenous gene; causing transcriptional activation of an endogenous gene; causing epigenetic regulation of an endogenous DNA; causing up- or down-regulation of operably liked genes, etc. In certain embodiments, a customized RNA sequence template can be engineered to contain sequences coding for exons and/or transgenes, provide for binding sites to transcription factor activators, repressors, enhancers, etc., and combinations of thereof. In other embodiments, the coding sequence can be further customized with splice acceptor sites, poly-A tails. In certain embodiments the RNA sequence can contain sequences coding for an RNA sequence template homologous to the RLE retrotransposase, be engineered to contain heterologous coding sequences, or combinations thereof.


The template nucleic acid (e.g., template RNA) may have some homology to the target DNA. In some embodiments, the template nucleic acid (e.g., template RNA) 3′ target homology domain may serve as an annealing region to the target DNA, such that the target DNA is positioned to prime the reverse transcription of the template nucleic acid (e.g., template RNA). In some embodiments the template nucleic acid (e.g., template RNA) has at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 175, 200 or more bases of exact homology to the target DNA at the 3′ end of the RNA. In some embodiments the template nucleic acid (e.g., template RNA) has at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 175, 180, or 200 or more bases of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% homology to the target DNA, e.g., at the 5′ end of the template nucleic acid (e.g., template RNA). In some embodiments the template nucleic acid (e.g., template RNA) has a 3′ region of at least 10, 15, 20, 25, 30, 40, 50, 60, 80, 100, 120, 140, 160, 180, 200 or more bases of at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% homology to the 3′ sequence of a non-LTR retrotransposon, e.g., a non-LTR retrotransposon described herein, e.g. a non-LTR retrotransposon in Table 1 or 3.


The template nucleic acid (e.g., template RNA) component of a Gene Writer™ genome editing system described herein typically is able to bind the Gene Writer™ genome editing protein of the system. In some embodiments the template nucleic acid (e.g., template RNA) has a 3′ region that is capable of binding a Gene Writer™ genome editing protein. The binding region, e.g., 3′ region, may be a structured RNA region, e.g., having at least 1, 2 or 3 hairpin loops, capable of binding the Gene Writer™ genome editing protein of the system. The binding region may associate the template nucleic acid (e.g., template RNA) with any of the polypeptide modules. In some embodiments, the binding region of the template nucleic acid (e.g., template RNA) may associate with an RNA-binding domain in the polypeptide. In some embodiments, the binding region of the template nucleic acid (e.g., template RNA) may associate with the reverse transcription domain of the polypeptide (e.g., specifically bind to the RT domain). For example, where the reverse transcription domain is derived from a non-LTR retrotransposon, the template nucleic acid (e.g., template RNA) may contain a binding region derived from a non-LTR retrotransposon, e.g., a 3′ UTR from a non-LTR retrotransposon. In some embodiments, the template nucleic acid (e.g., template RNA) may associate with the DNA binding domain of the polypeptide, e.g., a gRNA associating with a Cas9-derived DNA binding domain. In some embodiments, the binding region may also provide DNA target recognition, e.g., a gRNA hybridizing to the target DNA sequence and binding the polypeptide, e.g., a Cas9 domain. In some embodiments, the template nucleic acid (e.g., template RNA) may associate with multiple components of the polypeptide, e.g., DNA binding domain and reverse transcription domain. For example, the template nucleic acid (e.g., template RNA) may comprise a gRNA region that associates with a Cas9-derived DNA binding domain and a 3′ UTR from a non-LTR retrotransposon that associated with a non-LTR retrotransposon-derived reverse transcription domain.


In some embodiments the template RNA has a poly-A tail at the 3′ end. In some embodiments the template RNA does not have a poly-A tail at the 3′ end. In some embodiments the template nucleic acid (e.g., template RNA) has a 5′ region of at least 10, 15, 20, 25, 30, 40, 50, 60, 80, 100, 120, 140, 160, 180, 200 or more bases of at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater homology to the 5′ sequence of a non-LTR retrotransposon, e.g., a non-LTR retrotransposon described herein.


The template nucleic acid (e.g., template RNA) of the system typically comprises an object sequence (e.g., a heterologous object sequence) for insertion into a target DNA. The object sequence may be coding or non-coding.


In some embodiments a system or method described herein comprises a single template nucleic acid (e.g., template RNA). In some embodiments a system or method described herein comprises a plurality of template nucleic acids (e.g., template RNAs). For example, a system described herein comprises a first RNA comprising (e.g., from 5′ to 3′) a sequence that binds the Gene Writer™ polypeptide (e.g., the DNA-binding domain and/or the endonuclease domain, e.g., a gRNA) and a sequence that binds a target site (e.g., a second strand of a site in a target genome), and a second RNA (e.g., a template RNA) comprising (e.g., from 5′ to 3′) optionally a sequence that binds the Gene Writer™ polypeptide (e.g., that specifically binds the RT domain), a heterologous object sequence, and a 3′ target homology domain. In some embodiments, when the system comprises a plurality of nucleic acids, each nucleic acid comprises a conjugating domain. In some embodiments, a conjugating domain enables association of nucleic acid molecules, e.g., by hybridization of complementary sequences. For example, in some embodiments a first RNA comprises a first conjugating domain and a second RNA comprises a second conjugating domain, and the first and second conjugating domains are capable of hybridizing to one another, e.g., under stringent conditions. In some embodiments, the stringent conditions for hybridization include hybridization in 4× sodium chloride/sodium citrate (SSC), at about 65° C., followed by a wash in 1×SSC, at about 65° C.


In some embodiments, the object sequence may contain an open reading frame. In some embodiments the template nucleic acid (e.g., template RNA) has a Kozak sequence. In some embodiments the template RNA has an internal ribosome entry site. In some embodiments the template RNA has a self-cleaving peptide such as a T2A or P2A site. In some embodiments the template RNA has a start codon. In some embodiments the template RNA has a splice acceptor site. In some embodiments the template RNA has a splice donor site. Exemplary splice acceptor and splice donor sites are described in WO2016044416, incorporated herein by reference in its entirety. Exemplary splice acceptor site sequences are known to those of skill in the art and include, by way of example only, CTGACCCTTCTCTCTCTCCCCCAGAG (SEQ ID NO: 1601) (from human HBB gene) and TTTCTCTCCCACAAG (SEQ ID NO: 1602) (from human immunoglobulin-gamma gene). In some embodiments the template RNA has a microRNA binding site downstream of the stop codon. In some embodiments the template RNA has a polyA tail downstream of the stop codon of an open reading frame. In some embodiments the template RNA comprises one or more exons. In some embodiments the template RNA comprises one or more introns. In some embodiments the template RNA comprises a eukaryotic transcriptional terminator. In some embodiments the template RNA comprises an enhanced translation element or a translation enhancing element. In some embodiments the RNA comprises the human T-cell leukemia virus (HTLV-1) R region. In some embodiments the RNA comprises a posttranscriptional regulatory element that enhances nuclear export, such as that of Hepatitis B Virus (HPRE) or Woodchuck Hepatitis Virus (WPRE).


In some embodiments, a nucleic acid described herein (e.g., a template RNA or a DNA encoding a template RNA) comprises a microRNA binding site. In some embodiments, the microRNA binding site is used to increase the target-cell specificity of a Gene Writer™ system. For instance, the microRNA binding site can be chosen on the basis that is is recognized by a miRNA that is present in a non-target cell type, but that is not present (or is present at a reduced level relative to the non-target cell) in a target cell type. Thus, when the template RNA is present in a non-target cell, it would be bound by the miRNA, and when the template RNA is present in a target cell, it would not be bound by the miRNA (or bound but at reduced levels relative to the non-target cell). While not wishing to be bound by theory, binding of the miRNA to the template RNA may interfere with its activity, e.g., may interfere with insertion of the heterologous object sequence into the genome. Accordingly, the system would edit the genome of target cells more efficiently than it edits the genome of non-target cells, e.g., the heterologous object sequence would be inserted into the genome of target cells more efficiently than into the genome of non-target cells, or an insertion or deletion is produced more efficiently in target cells than in non-target cells. A system having a microRNA binding site in the template RNA (or DNA encoding it) may also be used in combination with a nucleic acid encoding a Gene Writer™ polypeptide, wherein expression of the Gene Writer™ polypeptide is regulated by a second microRNA binding site, e.g., as described herein, e.g., in the section entitled “Polypeptide component of Gene Writer™ gene editor system”. In some embodiments, e.g., for liver indications, a miRNA is selected from Table 4 of WO2020014209, incorporated herein by reference.


In some embodiments, the object sequence may contain a non-coding sequence. For example, the template nucleic acid (e.g., template RNA) may comprise a regulatory element, e.g., a promoter or enhancer sequence or miRNA binding site. In some embodiments, integration of the object sequence at a target site will result in upregulation of an endogenous gene. In some embodiments, integration of the object sequence at a target site will result in downregulation of an endogenous gene. In some embodiments the template nucleic acid (e.g., template RNA) comprises a tissue specific promoter or enhancer, each of which may be unidirectional or bidirectional. In some embodiments the promoter is an RNA polymerase I promoter, RNA polymerase II promoter, or RNA polymerase III promoter. In some embodiments the promoter comprises a TATA element. In some embodiments the promoter comprises a B recognition element. In some embodiments the promoter has one or more binding sites for transcription factors.


In some embodiments, a nucleic acid described herein (e.g., a template RNA or a DNA encoding a template RNA) comprises a promoter sequence, e.g., a tissue specific promoter sequence. In some embodiments, the tissue-specific promoter is used to increase the target-cell specificity of a Gene Writer™ system. For instance, the promoter can be chosen on the basis that it is active in a target cell type but not active in (or active at a lower level in) a non-target cell type. Thus, even if the promoter integrated into the genome of a non-target cell, it would not drive expression (or only drive low level expression) of an integrated gene. A system having a tissue-specific promoter sequence in the template RNA may also be used in combination with a microRNA binding site, e.g., in the template RNA or a nucleic acid encoding a Gene Writer™ protein, e.g., as described herein. A system having a tissue-specific promoter sequence in the template RNA may also be used in combination with a DNA encoding a Gene Writer™ polypeptide, driven by a tissue-specific promoter, e.g., to achieve higher levels of Gene Writer™ protein in target cells than in non-target cells. In some embodiments, e.g., for liver indications, a tissue-specific promoter is selected from Table 3 of WO2020014209, incorporated herein by reference.


In some embodiments, a Gene Writer system, e.g., DNA encoding a Gene Writer polypeptide, DNA encoding a template RNA, or DNA or RNA encoding a heterologous object sequence, is designed such that one or more elements is operably linked to a tissue-specific promoter, e.g., a promoter that is active in T-cells. In further embodiments, the T-cell active promoter is inactive in other cell types, e.g., B-cells, NK cells. In some embodiments, the T-cell active promoter is derived from a promoter for a gene encoding a component of the T-cell receptor, e.g., TRAC, TRBC, TRGC, TRDC. In some embodiments, the T-cell active promoter is derived from a promoter for a gene encoding a component of a T-cell-specific cluster of differentiation protein, e.g., CD3, e.g., CD3D, CD3E, CD3G, CD3Z. In some embodiments, T-cell-specific promoters in Gene Writer systems are discovered by comparing publicly available gene expression data across cell types and selecting promoters from the genes with enhanced expression in T-cells. In some embodiments, promoters may be selecting depending on the desired expression breadth, e.g., promoters that are active in T-cells only, promoters that are active in NK cells only, promoters that are active in both T-cells and NK cells.


In some embodiments the template RNA comprises a microRNA sequence, a siRNA sequence, a guide RNA sequence, a piwi RNA sequence.


In some embodiments the template nucleic acid (e.g., template RNA) comprises a site that coordinates epigenetic modification. In some embodiments the template nucleic acid (e.g., template RNA) comprises a chromatin insulator. For example, the template nucleic acid (e.g., template RNA) comprises a CTCF site or a site targeted for DNA methylation.


In some embodiments the template nucleic acid (e.g., template RNA) comprises a gene expression unit composed of at least one regulatory region operably linked to an effector sequence. The effector sequence may be a sequence that is transcribed into RNA (e.g., a coding sequence or a non-coding sequence such as a sequence encoding a micro RNA).


In some embodiments the object sequence of the template nucleic acid (e.g., template RNA) is inserted into a target genome in an endogenous intron. In some embodiments the object sequence of the template nucleic acid (e.g., template RNA) is inserted into a target genome and thereby acts as a new exon. In some embodiments the insertion of the object sequence into the target genome results in replacement of a natural exon or the skipping of a natural exon.


In some embodiments, the object sequence of the template nucleic acid (e.g., template RNA) is inserted into the target genome in a genomic safe harbor site, such as AAVS1, CCR5, ROSA26, or albumin locus. In some embodiments, a Gene Writer is used to integrate a CAR into the T-cell receptor a constant (TRAC) locus (Eyquem et al Nature 543, 113-117 (2017)). In some embodiments, a Gene Writer is used to integrate a CAR into a T-cell receptor R constant (TRBC) locus. Many other safe harbors have been identified by computational approaches (Pellenz et al Hum Gen Ther 30, 814-828 (2019)) and could be used for Gene Writer-mediated integration. In some embodiments, the object sequence of the template nucleic acid (e.g., template RNA) is added to the genome in an intergenic or intragenic region. In some embodiments, the object sequence of the template nucleic acid (e.g., template RNA) is added to the genome 5′ or 3′ within 0.1 kb, 0.25 kb, 0.5 kb, 0.75, kb, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 7.5 kb, 10 kb, 15 kb, 20 kb, 25 kb, 50, 75 kb, or 100 kb of an endogenous active gene. In some embodiments, the object sequence of the template nucleic acid (e.g., template RNA) is added to the genome 5′ or 3′ within 0.1 kb, 0.25 kb, 0.5 kb, 0.75, kb, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 7.5 kb, 10 kb, 15 kb, 20 kb, 25 kb, 50, 75 kb, or 100 kb of an endogenous promoter or enhancer. In some embodiments, the object sequence of the template nucleic acid (e.g., template RNA) can be, e.g., 50-50,000 base pairs (e.g., between 50-40,000 bp, between 500-30,000 bp between 500-20,000 bp, between 100-15,000 bp, between 500-10,000 bp, between 50-10,000 bp, between 50-5,000 bp.


The template nucleic acid (e.g., template RNA) can be designed to result in insertions, mutations, or deletions at the target DNA locus. In some embodiments, the template nucleic acid (e.g., template RNA) may be designed to cause an insertion in the target DNA. For example, the template nucleic acid (e.g., template RNA) may contain a heterologous sequence, wherein the reverse transcription will result in insertion of the heterologous sequence into the target DNA. In other embodiments, the RNA template may be designed to write a deletion into the target DNA. For example, the template nucleic acid (e.g., template RNA) may match the target DNA upstream and downstream of the desired deletion, wherein the reverse transcription will result in the copying of the upstream and downstream sequences from the template nucleic acid (e.g., template RNA) without the intervening sequence, e.g., causing deletion of the intervening sequence. In other embodiments, the template nucleic acid (e.g., template RNA) may be designed to write an edit into the target DNA. For example, the template RNA may match the target DNA sequence with the exception of one or more nucleotides, wherein the reverse transcription will result in the copying of these edits into the target DNA, e.g., resulting in mutations, e.g., transition or transversion mutations.


In some embodiments, the template possesses one or more sequences aiding in association of the template with the Gene Writer™ polypeptide. In some embodiments, these sequences may be derived from retrotransposon UTRs. In some embodiments, the UTRs may be located flanking the desired insertion sequence. In some embodiments, a sequence with target site homology may be located outside of one or both UTRs. In some embodiments, the sequence with target site homology can anneal to the target sequence to prime reverse transcription. In some embodiments, the 5′ and/or 3′ UTR may be located terminal to the target site homology sequence, e.g., such that target primed reverse transcription excludes reverse transcription of the 5′ and/or 3′ UTR. In some embodiments, the Gene Writer™ system may result in the insertion of a desired payload without any additional sequence (e.g. gene expression unit without UTRs used to bind the Gene Writer™ protein).


Alternative orientations of the template RNA motifs can be employed, e.g., to limit target site integration to the desired genetic payload. In some embodiments, the polypeptide association domains may be located 5′ of the desired template sequence. For example, the heterologous object sequence may be located downstream of the 5′ UTR and 3′ UTR, giving the 5′-3′ orientation 5′UTR-3′UTR-(heterologous object sequence). In other embodiments, only the 3′ UTR is added upstream of the heterologous object sequence. For example, giving the 5′-3′ orientation 3′UTR-(heterologous object sequence). In certain embodiments, the polypeptide coding region and the heterologous object sequence may be encoded on the same molecule, but where the 5′ UTR (e.g., 5′ UTR from R2 retrotransposon) occurs between the two regions, e.g., giving the 5′-3′ orientation (polypeptide coding sequence)-5′UTR-(heterologous object sequence).


In some embodiments, the template nucleic acid, e.g., template RNA, may comprise a gRNA (e.g., pegRNA). In some embodiments, the template nucleic acid, e.g., template RNA, may bind to the Gene Writer™ polypeptide by interaction of a gRNA portion of the template nucleic acid with a template nucleic acid binding domain, e.g., a RNA binding domain (e.g., a heterologous RNA binding domain). In some embodiments, the heterologous RNA binding domain is a CRISPR/Cas protein, e.g., Cas9.


In some embodiments, the region of the template nucleic acid, e.g., template RNA, comprising the gRNA adopts an underwound ribbon-like structure of gRNA bound to target DNA (e.g., as described in Mulepati et al. Science 19 Sep. 2014: Vol. 345, Issue 6203, pp. 1479-1484). Without wishing to be bound by theory, this non-canonical structure is thought to be facilitated by rotation of every sixth nucleotide out of the RNA-DNA hybrid. Thus, in some embodiments, the region of the template nucleic acid, e.g., template RNA, comprising the gRNA may tolerate increased mismatching with the target site at some interval, e.g., every sixth base. In some embodiments, the region of the template nucleic acid, e.g., template RNA, comprising the gRNA comprising homology to the target site may possess wobble positions at a regular interval, e.g., every sixth base, that do not need to base pair with the target site.


gRNAs with Inducible Activity


In some embodiments, a template nucleic acid, e.g., template RNA, comprises a gRNA with inducible activity. Inducible activity may be achieved by the template nucleic acid, e.g., template RNA, further comprising (in addition to the gRNA) a blocking domain, wherein the sequence of a portion of or all of the blocking domain is at least partially complementary to a portion or all of the gRNA. The blocking domain is thus capable of hybridizing or substantially hybridizing to a portion of or all of the gRNA. In some embodiments, the blocking domain and inducibly active gRNA are disposed on the template nucleic acid, e.g., template RNA, such that the gRNA can adopt a first conformation where the blocking domain is hybridized or substantially hybridized to the gRNA, and a second conformationwhere the blocking domain is not hybridized or or not substantially hybridized to the gRNA. In some embodiments, in the first conformation the gRNA is unable to bind to the Gene Writer polypeptide (e.g., the template nucleic acid binding domain, DNA binding domain, or endonuclease domain (e.g., a CRISPR/Cas protein)) or binds with substantially decreased affinity compared to an otherwise similar template RNA lacking the blocking domain. In some embodiments, in the second conformation the gRNA is able to bind to the Gene Writer polypeptide (e.g., the template nucleic acid binding domain, DNA binding domain, or endonuclease domain (e.g., a CRISPR/Cas protein)). In some embodiments, whether the gRNA is in the first or second conformation can influence whether the DNA binding or endonuclease activities of the Gene Writer polypeptide (e.g., of the CRISPR/Cas protein the Gene Writer polypeptide comprises) are active. In some embodiments, hybridization of the gRNA to the blocking domain can be disrupted using an opener molecule. In some embodiments, an opener molecule comprises an agent that binds to a portion or all of the gRNA or blocking domain and inhibits hybridization of the gRNA to the blocking domain. In some embodiments, the opener molecule comprises a nucleic acid, e.g., comprising a sequence that is partially or wholly complementary to the gRNA, blocking domain, or both. By choosing or designing an appropriate opener molecule, providing the opener molecule can promote a change in the conformation of the gRNA such that it can associate with a CRISPR/Cas protein and provide the associated functions of the CRISPR/Cas protein (e.g., DNA binding and/or endonuclease activity). Without wishing to be bound by theory, providing the opener molecule at a selected time and/or location may allow for spatial and temporal control of the activity of the gRNA, CRISPR/Cas protein, or Gene Writer system comprising the same. In some embodiments, a Gene Writer may comprise a Cas protein as listed in Table 40 or Table 37 or a functional fragment thereof, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto.









TABLE 40







CRISPR/Cas Proteins, Species, and Mutations













SEQ





Parental
ID

Nickase


Variant
Host
NO:
Protein Sequence
Mutation





Nme2Cas9

Neisseria

3262
MAAFKPNPINYILGLDIGIASVGWAMVEIDEEENPIRLID
N611A




meningitidis


LGVRVFERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLL






RARRLLKREGVLQAADFDENGLIKSLPNTPWQLRAAALDR






KLTPLEWSAVLGDYSHTFSRKDLQAELILLFEKQKEFGNP






HVSGGLKEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEP






KAAKNTYTAERFIWLTKLNNLRILEQGSERPLTDTERATL






MDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEAS






TLMEMKAYHAISRALEKEGLKDKKSPLNLSSELQDEIGTA






FSLFKTDEDITGRLKDRVQPEILEALLKHISFDKFVQISL






KALRRIVPLMEQGKRYDEACAEIYGDHYGKKNTEEKIYLP






PIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIET






AREVGKSFKDRKEIEKRQEENRKDREKAAAKFREYFPNFV






GEPKSKDILKLRLYEQQHGKCLYSGKEINLVRLNEKGYVE






IDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNG






KDNSREWQEFKARVETSRFPRSKKQRILLQKFDEDGFKEC






NLNDTRYVNRFLCQFVADHILLTGKGKRRVFASNGQITNL






LRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFV






RYKEMNAFDGKTIDKETGKVLHQKTHFPQPWEFFAQEVMI






RVFGKPDGKPEFEEADTPEKLRTLLAEKLSSRPEAVHEYV






TPLFVSRAPNRKMSGAHKDTLRSAKRFVKHNEKISVKRVW






LTEIKLADLENMVNYKNGREIELYEALKARLEAYGGNAKQ






AFDPKDNPFYKKGGQLVKAVRVEKTQESGVLLNKKNAYTI






ADNGDMVRVDVFCKVDKKGKNQYFIVPIYAWQVAENILPD






IDCKGYRIDDSYTFCFSLHKYDLIAFQKDEKSKVEFAYYI






NCDSSNGRFYLAWHDKGSKEQQFRISTQNLVLIQKYQVNE






LGKEIRPCRLKKRPPVR






PpnCas9

Pasteurella

3263
MQNNPLNYILGLDLGIASIGWAVVEIDEESSPIRLIDVGV
N605A




pneumotropica


RTFERAEVAKTGESLALSRRLARSSRRLIKRRAERLKKAK






RLLKAEKILHSIDEKLPINVWQLRVKGLKEKLERQEWAAV






LLHLSKHRGYLSQRKNEGKSDNKELGALLSGIASNHQMLQ






SSEYRTPAEIAVKKFQVEEGHIRNQRGSYTHTFSRLDLLA






EMELLFQRQAELGNSYTSTTLLENLTALLMWQKPALAGDA






ILKMLGKCTFEPSEYKAAKNSYSAERFVWLTKLNNLRILE






NGTERALNDNERFALLEQPYEKSKLTYAQVRAMLALSDNA






IFKGVRYLGEDKKTVESKTTLIEMKFYHQIRKTLGSAELK






KEWNELKGNSDLLDEIGTAFSLYKTDDDICRYLEGKLPER






VLNALLENLNFDKFIQLSLKALHQILPLMLQGQRYDEAVS






AIYGDHYGKKSTETTRLLPTIPADEIRNPVVLRTLTQARK






VINAVVRLYGSPARIHIETAREVGKSYQDRKKLEKQQEDN






RKQRESAVKKFKEMFPHFVGEPKGKDILKMRLYELQQAKC






LYSGKSLELHRLLEKGYVEVDHALPFSRTWDDSFNNKVLV






LANENQNKGNLTPYEWLDGKNNSERWQHFVVRVQTSGFSY






AKKQRILNHKLDEKGFIERNLNDTRYVARFLCNFIADNML






LVGKGKRNVFASNGQITALLRHRWGLQKVREQNDRHHALD






AVVVACSTVAMQQKITRFVRYNEGNVFSGERIDRETGEII






PLHFPSPWAFFKENVEIRIFSENPKLELENRLPDYPQYNH






EWVQPLFVSRMPTRKMTGQGHMETVKSAKRLNEGLSVLKV






PLTQLKLSDLERMVNRDREIALYESLKARLEQFGNDPAKA






FAEPFYKKGGALVKAVRLEQTQKSGVLVRDGNGVADNASM






VRVDVFTKGGKYFLVPIYTWQVAKGILPNRAATQGKDEND






WDIMDEMATFQFSLCQNDLIKLVTKKKTIFGYFNGLNRAT






SNINIKEHDLDKSKGKLGIYLEVGVKLAISLEKYQVDELG






KNIRPCRPTKRQHVR






SauCas9

Staphylococcus

3264
MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEAN
N580A




aureus


VENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDH






SELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHN






VNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKK






DGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDT






YIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYF






PEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEK






FQUIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGK






PEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQS






SEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI






NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTL






VDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAR






EKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYL






IEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIP






RSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKIS






YETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKD






FINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGF






TSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKK






LDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQI






KHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTL






IVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKL






KLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKI






KYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDN






GVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQA






EFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDIT






YREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYE






VKSKKHPQIIKKG






SauCas9-

Staphylococcus

3265
MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEAN
N580A


KKH

aureus


VENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDH






SELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHN






VNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKK






DGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDT






YIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYF






PEELRSVKYAYNADLYNALNVYHDIKDITARKEIIENAEL






LDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLK






GYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKK






VDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKK






YGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEE






IIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLL






NNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRT






PFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLL






EERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVN






NLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALI






IANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETE






QEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDT






LYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKL






LMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTK






YSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKL






SLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCY






EEAKKLKKISNQAEFIASFYKNDLIKINGELYRVIGVNND






LLNRIEVNMIDITYREYLENMNDKRPPHIIKTIASKTQSI






KKYSTDILGNLYEVKSKKHPQIIKKG






SauriCas9

Staphylococcus

3266
MQENQQKQNYILGLDIGITSVGYGLIDSKTREVIDAGVRL
N588A




auricularis


FPEADSENNSNRRSKRGARRLKRRRIHRLNRVKDLLADYQ






MIDLNNVPKSTDPYTIRVKGLREPLTKEEFAIALLHIAKR






RGLHNISVSMGDEEQDNELSTKQQLQKNAQQLQDKYVCEL






QLERLTNINKVRGEKNRFKTEDFVKEVKQLCETQRQYHNI






DDQFIQQYIDLVSTRREYFEGPGNGSPYGWDGDLLKWYEK






LMGRCTYFPEELRSVKYAYSADLFNALNDLNNLVVTRDDN






PKLEYYEKYHIIENVFKQKKNPTLKQIAKEIGVQDYDIRG






YRITKSGKPQFTSFKLYHDLKNIFEQAKYLEDVEMLDEIA






KILTIYQDEISIKKALDQLPELLTESEKSQIAQLTGYTGT






HRLSLKCIHIVIDELWESPENQMEIFTRLNLKPKKVEMSE






IDSIPTTLVDEFILSPVVKRAFIQSIKVINAVINRFGLPE






DIIIELAREKNSKDRRKFINKLQKQNEATRKKIEQLLAKY






GNTNAKYMIEKIKLHDMQEGKCLYSLEAIPLEDLLSNPTH






YEVDHIIPRSVSFDNSLNNKVLVKQSENSKKGNRTPYQYL






SSNESKISYNQFKQHILNLSKAKDRISKKKRDMLLEERDI






NKFEVQKEFINRNLVDTRYATRELSNLLKTYFSTHDYAVK






VKTINGGFTNHLRKVWDFKKHRNHGYKHHAEDALVIANAD






FLFKTHKALRRTDKILEQPGLEVNDTTVKVDTEEKYQELF






ETPKQVKNIKQFRDFKYSHRVDKKPNRQLINDTLYSTREI






DGETYVVQTLKDLYAKDNEKVKKLFTERPQKILMYQHDPK






TFEKLMTILNQYAEAKNPLAAYYEDKGEYVTKYAKKGNGP






AIHKIKYIDKKLGSYLDVSNKYPETQNKLVKLSLKSFRFD






IYKCEQGYKMVSIGYLDVLKKDNYYYIPKDKYEAEKQKKK






IKESDLFVGSFYYNDLIMYEDELFRVIGVNSDINNLVELN






MVDITYKDFCEVNNVTGEKRIKKTIGKRVVLIEKYTTDIL






GNLYKTPLPKKPQLIFKRGEL






SauriCas9-

Staphylococcus

3267
MQENQQKQNYILGLDIGITSVGYGLIDSKTREVIDAGVRL
N588A


KKH

auricularis


FPEADSENNSNRRSKRGARRLKRRRIHRLNRVKDLLADYQ






MIDLNNVPKSTDPYTIRVKGLREPLTKEEFAIALLHIAKR






RGLHNISVSMGDEEQDNELSTKQQLQKNAQQLQDKYVCEL






QLERLTNINKVRGEKNRFKTEDFVKEVKQLCETQRQYHNI






DDQFIQQYIDLVSTRREYFEGPGNGSPYGWDGDLLKWYEK






LMGRCTYFPEELRSVKYAYSADLFNALNDLNNLVVTRDDN






PKLEYYEKYHIIENVFKQKKNPTLKQIAKEIGVQDYDIRG






YRITKSGKPQFTSFKLYHDLKNIFEQAKYLEDVEMLDEIA






KILTIYQDEISIKKALDQLPELLTESEKSQIAQLTGYTGT






HRLSLKCIHIVIDELWESPENQMEIFTRLNLKPKKVEMSE






IDSIPTTLVDEFILSPVVKRAFIQSIKVINAVINRFGLPE






DIIIELAREKNSKDRRKFINKLQKQNEATRKKIEQLLAKY






GNTNAKYMIEKIKLHDMQEGKCLYSLEAIPLEDLLSNPTH






YEVDHIIPRSVSFDNSLNNKVLVKQSENSKKGNRTPYQYL






SSNESKISYNQFKQHILNLSKAKDRISKKKRDMLLEERDI






NKFEVQKEFINRNLVDTRYATRELSNLLKTYFSTHDYAVK






VKTINGGFTNHLRKVWDFKKHRNHGYKHHAEDALVIANAD






FLFKTHKALRRTDKILEQPGLEVNDTTVKVDTEEKYQELF






ETPKQVKNIKQFRDFKYSHRVDKKPNRKLINDTLYSTREI






DGETYVVQTLKDLYAKDNEKVKKLFTERPQKILMYQHDPK






TFEKLMTILNQYAEAKNPLAAYYEDKGEYVTKYAKKGNGP






AIHKIKYIDKKLGSYLDVSNKYPETQNKLVKLSLKSFRFD






IYKCEQGYKMVSIGYLDVLKKDNYYYIPKDKYEAEKQKKK






IKESDLFVGSFYKNDLIMYEDELFRVIGVNSDINNLVELN






MVDITYKDFCEVNNVTGEKHIKKTIGKRVVLIEKYTTDIL






GNLYKTPLPKKPQLIFKRGEL






ScaCas9-

Streptococcus

3268
MEKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTNR
N872A


Sc++

canis


KSIKKNLMGALLFDSGETAEATRLKRTARRRYTRRKNRIR






YLQEIFANEMAKLDDSFFQRLEESFLVEEDKKNERHPIFG






NLADEVAYHRNYPTIYHLRKKLADSPEKADLRLIYLALAH






IIKFRGHFLIEGKLNAENSDVAKLFYQLIQTYNQLFEESP






LDEIEVDAKGILSARLSKSKRLEKLIAVFPNEKKNGLFGN






IIALALGLTPNFKSNFDLTEDAKLQLSKDTYDDDLDELLG






QIGDQYADLFSAAKNLSDAILLSDILRSNSEVTKAPLSAS






MVKRYDEHHQDLALLKTLVRQQFPEKYAEIFKDDTKNGYA






GYVGADKKLRKRSGKLATEEEFYKFIKPILEKMDGAEELL






AKLNRDDLLRKQRTFDNGSIPHQIHLKELHAILRRQEEFY






PFLKENREKIEKILTFRIPYYVGPLARGNSRFAWLTRKSE






EAITPWNFEEVVDKGASAQSFIERMTNFDEQLPNKKVLPK






HSLLYEYFTVYNELTKVKYVTERMRKPEFLSGEQKKAIVD






LLFKTNRKVTVKQLKEDYFKKIECFDSVEIIGVEDRFNAS






LGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE






MIEERLKTYAHLFDDKVMKQLKRRHYTGWGRLSRKMINGI






RDKQSGKTILDFLKSDGFSNRNFMQLIHDDSLTFKEEIEK






AQVSGQGDSLHEQIADLAGSPAIKKGILQTVKIVDELVKV






MGHKPENIVIEMARENQTTTKGLQQSRERKKRIEEGIKEL






ESQILKENPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN






RLSDYDVDHIVPQSFIKDDSIDNKVLTRSVENRGKSDNVP






SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSEA






DKAGFIKRQLVETRQITKHVARILDSRMNTKRDKNDKPIR






EVKVITLKSKLVSDFRKDFQLYKVRDINNYHHAHDAYLNA






VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK






ATAKRFFYSNIMNFFKTEVKLANGEIRKRPLIETNGETGE






VVWNKEKDFATVRKVLAMPQVNIVKKTEVQTGGFSKESIL






SKRESAKLIPRKKGWDTRKYGGFGSPTVAYSILVVAKVEK






GKAKKLKSVKVLVGITIMEKGSYEKDPIGFLEAKGYKDIK






KELIFKLPKYSLFELENGRRRMLASAKELQKANELVLPQH






LVRLLYYTQNISATTGSNNLGYIEQHREEFKEIFEKIIDF






SEKYILKNKVNSNLKSSFDEQFAVSDSILLSNSFVSLLKY






TSFGASGGFTFLDLDVKQGRLRYQTVTEVLDATLIYQSIT






GLYETRTDLSQLGGD






SpyCas9

Streptococcus

3269
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR
N863A




pyogenes


HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC






YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG






NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH






MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP






INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN






LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA






QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS






MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA






GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR






KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKI






EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE






VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV






YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT






VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI






IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA






HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL






DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL






HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIV






IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP






VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH






IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK






NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ






LVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS






KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK






YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS






NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF






ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI






ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSV






KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK






YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLAS






HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV






ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA






PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRI






DLSQLGGD






SpyCas9-

Streptococcus

3270
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR
N863A


NG

pyogenes


HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC






YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG






NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH






MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP






INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN






LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA






QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS






MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA






GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR






KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKI






EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE






VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV






YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT






VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI






IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA






HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL






DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL






HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIV






IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP






VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH






IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK






NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ






LVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS






KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK






YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS






NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF






ATVRKVLSMPQVNIVKKTEVQTGGFSKESIRPKRNSDKLI






ARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSV






KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK






YSLFELENGRKRMLASARFLQKGNELALPSKYVNFLYLAS






HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV






ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA






PRAFKYFDTTIDRKVYRSTKEVLDATLIHQSITGLYETRI






DLSQLGGD






SpyCas9-

Streptococcus

3271
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR
N863A


SpRY

pyogenes


HSIKKNLIGALLFDSGETAERTRLKRTARRRYTRRKNRIC






YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG






NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH






MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP






INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN






LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA






QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS






MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA






GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR






KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKI






EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE






VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV






YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT






VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI






IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA






HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL






DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL






HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIV






IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP






VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH






IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK






NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ






LVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS






KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK






YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS






NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF






ATVRKVLSMPQVNIVKKTEVQTGGFSKESIRPKRNSDKLI






ARKKDWDPKKYGGFLWPTVAYSVLVVAKVEKGKSKKLKSV






KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK






YSLFELENGRKRMLASAKQLQKGNELALPSKYVNFLYLAS






HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV






ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTRLGA






PRAFKYFDTTIDPKQYRSTKEVLDATLIHQSITGLYETRI






DLSQLGGD



St1Cas9

Streptococcus

3272
MSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQA
N622A




thermophilus


ENNLVRRTNRQGRRLARRKKHRRVRLNRLFEESGLITDFT






KISINLNPYQLRVKGLTDELSNEELFIALKNMVKHRGISY






LDDASDDGNSSVGDYAQIVKENSKQLETKTPGQIQLERYQ






TYGQLRGDFTVEKDGKKHRLINVFPTSAYRSEALRILQTQ






QEFNPQITDEFINRYLEILTGKRKYYHGPGNEKSRTDYGR






YRTSGETLDNIFGILIGKCTFYPDEFRAAKASYTAQEFNL






LNDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLF






KYIAKLLSCDVADIKGYRIDKSGKAEIHTFEAYRKMKTLE






TLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFADGS






FSQKQVDELVQFRKANSSIFGKGWHNFSVKLMMELIPELY






ETSEEQMTILTRLGKQKTTSSSNKTKYIDEKLLTEEIYNP






VVAKSVRQAIKIVNAAIKEYGDFDNIVIEMARETNEDDEK






KAIQKIQKANKDEKDAAMLKAANQYNGKAELPHSVFHGHK






QLATKIRLWHQQGERCLYTGKTISIHDLINNSNQFEVDHI






LPLSITFDDSLANKVLVYATANQEKGQRTPYQALDSMDDA






WSFRELKAFVRESKTLSNKKKEYLLTEEDISKFDVRKKFI






ERNLVDTRYASRVVLNALQEHFRAHKIDTKVSVVRGQFTS






QLRRHWGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKNT






LVSYSEDQLLDIETGELISDDEYKESVFKAPYQHFVDTLK






SKEFEDSILFSYQVDSKFNRKISDATIYATRQAKVGKDKA






DETYVLGKIKDIYTQDGYDAFMKIYKKDKSKFLMYRHDPQ






TFEKVIEPILENYPNKQINEKGKEVPCNPFLKYKEEHGYI






RKYSKKGNGPEIKSLKYYDSKLGNHIDITPKDSNNKVVLQ






SVSPWRADVYFNKTTGKYEILGLKYADLQFEKGTGTYKIS






QEKYNDIKKKEGVDSDSEFKFTLYKNDLLLVKDTETKEQQ






LFRFLSRTMPKQKHYVELKPYDKQKFEGGEALIKVLGNVA






NSGQCKKGLGKSNISIYKVRTDVLGNQHIIKNEGDKPKLD






F






BlatCas9

Brevibacillus

3273
MAYTMGIDVGIASCGWAIVDLERQRIIDIGVRTFEKAENP
N607A




laterosporus


KNGEALAVPRREARSSRRRLRRKKHRIERLKHMFVRNGLA






VDIQHLEQTLRSQNEIDVWQLRVDGLDRMLTQKEWLRVLI






HLAQRRGFQSNRKTDGSSEDGQVLVNVTENDRLMEEKDYR






TVAEMMVKDEKFSDHKRNKNGNYHGVVSRSSLLVEIHTLF






ETQRQHHNSLASKDFELEYVNIWSAQRPVATKDQIEKMIG






TCTFLPKEKRAPKASWHFQYFMLLQTINHIRITNVQGTRS






LNKEEIEQVVNMALTKSKVSYHDTRKILDLSEEYQFVGLD






YGKEDEKKKVESKETIIKLDDYHKLNKIFNEVELAKGETW






EADDYDTVAYALTFFKDDEDIRDYLQNKYKDSKNRLVKNL






ANKEYTNELIGKVSTLSFRKVGHLSLKALRKIIPFLEQGM






TYDKACQAAGFDFQGISKKKRSVVLPVIDQISNPVVNRAL






TQTRKVINALIKKYGSPETIHIETARELSKTFDERKNITK






DYKENRDKNEHAKKHLSELGIINPTGLDIVKYKLWCEQQG






RCMYSNQPISFERLKESGYTEVDHIIPYSRSMNDSYNNRV






LVMTRENREKGNQTPFEYMGNDTQRWYEFEQRVTTNPQIK






KEKRQNLLLKGFTNRRELEMLERNLNDTRYITKYLSHFIS






TNLEFSPSDKKKKVVNTSGRITSHLRSRWGLEKNRGQNDL






HHAMDAIVIAVTSDSFIQQVTNYYKRKERRELNGDDKFPL






PWKFFREEVIARLSPNPKEQIEALPNHFYSEDELADLQPI






FVSRMPKRSITGEAHQAQFRRVVGKTKEGKNITAKKTALV






DISYDKNGDFNMYGRETDPATYEAIKERYLEFGGNVKKAF






STDLHKPKKDGTKGPLIKSVRIMENKTLVHPVNKPNDLIF






IRQNPKKKISLKKRIESHSISDSKEVQEIHAYYKGVDSST






AAIEFIIHDGSYYAKGVGVQNLDCFEKYQVDILGNYFKVK






GEKRLELETSDSNHKGKDVNSIKSTSR






cCas9-

Staphylococcus

3274
MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEAN
N580A


v16

aureus


VENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDH






SELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHN






VNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKK






DGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDT






YIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYF






PEELRSVKYAYNADLYNALNVYHDIKDITARKEIIENAEL






LDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLK






GYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKK






VDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKK






YGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEE






IIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLL






NNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRT






PFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLL






EERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVN






NLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALI






IANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETE






QEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLINDT






LYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKL






LMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTK






YSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKL






SLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCY






EEAKKLKKISNQAEFIASFYKNDLIKINGELYRVIGVNSD






KNNLIEVNMIDITYREYLENMNDKRPPHIIKTIASKTQSI






KKYSTDILGNLYEVKSKKHPQIIKKG






cCas9-

Staphylococcus

3275
MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEAN
N580A


v17

aureus


VENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDH






SELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHN






VNEVEEDTGNEFIDTYIDLLETRRTYYEGPGEGSPFGWKD






IKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNL






VITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILV






NEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENA






ELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISN






LKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVP






KKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAII






KKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERI






EEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLED






LLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGN






RTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEY






LLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFR






VNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDA






LIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIE






TEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRKLIN






DTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPE






KLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYL






TKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVV






KLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSK






CYEEAKKLKKISNQAEFIASFYKNDLIKINGELYRVIGVN






NSTRNIVELNMIDITYREYLENMNDKRPPHIIKTIASKTQ






SIKKYSTDILGNLYEVKSKKHPQIIKKG






cCas9-

Staphylococcus

3276
MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEAN
N580A


v21

aureus


VENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDH






SELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHN






VNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKK






DGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDT






YIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYF






PEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEK






FQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGK






PEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQS






SEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI






NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTL






VDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAR






EKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYL






IEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIP






RSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKIS






YETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKD






FINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGF






TSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKK






LDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQI






KHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTL






IVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKL






KLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKI






KYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDN






GVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQA






EFIASFYKNDLIKINGELYRVIGVNSDDRNIIELNMIDIT






YREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYE






VKSKKHPQIIKKG






cCas9-

Staphylococcus

3277
MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEAN
N580A


v42

aureus


VENNEGRRSKRGARRLKRRRRHRIQRVKKLLFDYNLLTDH






SELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHN






VNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKK






DGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDT






YIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYF






PEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEK






FQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGK






PEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQS






SEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI






NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTL






VDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAR






EKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYL






IEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIP






RSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKIS






YETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKD






FINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGF






TSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKK






LDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQI






KHIKDFKDYKYSHRVDKKPNRKLINDTLYSTRKDDKGNTL






IVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKL






KLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKI






KYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDN






GVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQA






EFIASFYKNDLIKINGELYRVIGVNNNRLNKIELNMIDIT






YREYLENMNDKRPPHIIKTIASKTQSIKKYSTDILGNLYE






VKSKKHPQIIKKG






CdiCas9

Corynebacterium

3278
MKYHVGIDVGTFSVGLAAIEVDDAGMPIKTLSLVSHIHDS
H573A




diphtheriae


GLDPDEIKSAVTRLASSGIARRTRRLYRRKRRRLQQLDKF
(Alternate)





IQRQGWPVIELEDYSDPLYPWKVRAELAASYIADEKERGE






KLSVALRHIARHRGWRNPYAKVSSLYLPDGPSDAFKAIRE






EIKRASGQPVPETATVGQMVTLCELGTLKLRGEGGVLSAR






LQQSDYAREIQEICRMQEIGQELYRKIIDVVFAAESPKGS






ASSRVGKDPLQPGKNRALKASDAFQRYRIAALIGNLRVRV






DGEKRILSVEEKNLVFDHLVNLTPKKEPEWVTIAEILGID






RGQLIGTATMTDDGERAGARPPTHDTNRSIVNSRIAPLVD






WWKTASALEQHAMVKALSNAEVDDFDSPEGAKVQAFFADL






DDDVHAKLDSLHLPVGRAAYSEDTLVRLTRRMLSDGVDLY






TARLQEFGIEPSWTPPTPRIGEPVGNPAVDRVLKTVSRWL






ESATKTWGAPERVIIEHVREGFVTEKRAREMDGDMRRRAA






RNAKLFQEMQEKLNVQGKPSRADLWRYQSVQRQNCQCAYC






GSPITFSNSEMDHIVPRAGQGSTNTRENLVAVCHRCNQSK






GNTPFAIWAKNTSIEGVSVKEAVERTRHWVTDTGMRSTDF






KKFTKAVVERFQRATMDEEIDARSMESVAWMANELRSRVA






QHFASHGTTVRVYRGSLTAEARRASGISGKLKFFDGVGKS






RLDRRHHAIDAAVIAFTSDYVAETLAVRSNLKQSQAHRQE






APQWREFTGKDAEHRAAWRVWCQKMEKLSALLTEDLRDDR






VVVMSNVRLRLGNGSAHKETIGKLSKVKLSSQLSVSDIDK






ASSEALWCALTREPGFDPKEGLPANPERHIRVNGTHVYAG






DNIGLFPVSAGSIALRGGYAELGSSFHHARVYKITSGKKP






AFAMLRVYTIDLLPYRNQDLFSVELKPQTMSMRQAEKKLR






DALATGNAEYLGWLVVDDELVVDTSKIATDQVKAVEAELG






TIRRWRVDGFFSPSKLRLRPLQMSKEGIKKESAPELSKII






DRPGWLPAVNKLFSDGNVTVVRRDSLGRVRLESTAHLPVT






WKVQ






CjeCas9

Campylobacter

3279
MARILAFDIGISSIGWAFSENDELKDCGVRIFTKVENPKT
N582A




jejuni


GESLALPRRLARSARKRLARRKARLNHLKHLIANEFKLNY






EDYQSFDESLAKAYKGSLISPYELRFRALNELLSKQDFAR






VILHIAKRRGYDDIKNSDDKEKGAILKAIKQNEEKLANYQ






SVGEYLYKEYFQKFKENSKEFTNVRNKKESYERCIAQSFL






KDELKLIFKKQREFGFSFSKKFEEEVLSVAFYKRALKDFS






HLVGNCSFFTDEKRAPKNSPLAFMFVALTRIINLLNNLKN






TEGILYTKDDLNALLNEVLKNGTLTYKQTKKLLGLSDDYE






FKGEKGTYFIEFKKYKEFIKALGEHNLSQDDLNEIAKDIT






LIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNISFKA






LKLVTPLMLEGKKYDEACNELNLKVAINEDKKDFLPAFNE






TYYKDEVTNPVVLRAIKEYRKVLNALLKKYGKVHKINIEL






AREVGKNHSQRAKIEKEQNENYKAKKDAELECEKLGLKIN






SKNILKLRLFKEQKEFCAYSGEKIKISDLQDEKMLEIDHI






YPYSRSFDDSYMNKVLVFTKQNQEKLNQTPFEAFGNDSAK






WQKIEVLAKNLPTKKQKRILDKNYKDKEQKNFKDRNLNDT






RYIARLVLNYTKDYLDFLPLSDDENTKLNDTQKGSKVHVE






AKSGMLTSALRHTWGFSAKDRNNHLHHAIDAVIIAYANNS






IVKAFSDFKKEQESNSAELYAKKISELDYKNKRKFFEPFS






GFRQKVLDKIDEIFVSKPERKKPSGALHEETFRKEEEFYQ






SYGGKEGVLKALELGKIRKVNGKIVKNGDMFRVDIFKHKK






TNKFYAVPIYTMDFALKVLPNKAVARSKKGEIKDWILMDE






NYEFCFSLYKDSLILIQTKDMQEPEFVYYNAFTSSTVSLI






VSKHDNKFETLSKNQKILFKNANEKEVIAKSIGIQNLKVF






EKYIVSALGEVTKAEFRQREDFKK






GeoCas9

Geobacillus

3280
MRYKIGLDIGITSVGWAVMNLDIPRIEDLGVRIFDRAENP
N605A




stearother


QTGESLALPRRLARSARRRLRRRKHRLERIRRLVIREGIL





mophilus


TKEELDKLFEEKHEIDVWQLRVEALDRKLNNDELARVLLH






LAKRRGFKSNRKSERSNKENSTMLKHIEENRAILSSYRTV






GEMIVKDPKFALHKRNKGENYTNTIARDDLEREIRLIFSK






QREFGNMSCTEEFENEYITIWASQRPVASKDDIEKKVGFC






TFEPKEKRAPKATYTFQSFIAWEHINKLRLISPSGARGLT






DEERRLLYEQAFQKNKITYHDIRTLLHLPDDTYFKGIVYD






RGESRKQNENIRFLELDAYHQIRKAVDKVYGKGKSSSFLP






IDFDTFGYALTLFKDDADIHSYLRNEYEQNGKRMPNLANK






VYDNELIEELLNLSFTKFGHLSLKALRSILPYMEQGEVYS






SACERAGYTFTGPKKKQKTMLLPNIPPIANPVVMRALTQA






RKVVNAIIKKYGSPVSIHIELARDLSQTFDERRKTKKEQD






ENRKKNETAIRQLMEYGLTLNPTGHDIVKFKLWSEQNGRC






AYSLQPIEIERLLEPGYVEVDHVIPYSRSLDDSYTNKVLV






LTRENREKGNRIPAEYLGVGTERWQQFETFVLTNKQFSKK






KRDRLLRLHYDENEETEFKNRNLNDTRYISRFFANFIREH






LKFAESDDKQKVYTVNGRVTAHLRSRWEFNKNREESDLHH






AVDAVIVACTTPSDIAKVTAFYQRREQNKELAKKTEPHFP






QPWPHFADELRARLSKHPKESIKALNLGNYDDQKLESLQP






VFVSRMPKRSVTGAAHQETLRRYVGIDERSGKIQTVVKTK






LSEIKLDASGHFPMYGKESDPRTYEAIRQRLLEHNNDPKK






AFQEPLYKPKKNGEPGPVIRTVKIIDTKNQVIPLNDGKTV






AYNSNIVRVDVFEKDGKYYCVPVYTMDIMKGILPNKAIEP






NKPYSEWKEMTEDYTFRFSLYPNDLIRIELPREKTVKTAA






GEEINVKDVFVYYKTIDSANGGLELISHDHRFSLRGVGSR






TLKRFEKYQVDVLGNIYKVRGEKRVGLASSAHSKPGKTIR






PLQSTRD






iSpyMac

Streptococcus

3281
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR
N863A


Cas9
spp.

HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC






YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG






NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH






MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP






INASGVDAKAILSARLSKSRKLENLIAQLPGEKKNGLFGN






LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA






QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS






MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA






GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLKREDLLR






KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKI






EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE






VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV






YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT






VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI






IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA






HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL






DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL






HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIV






IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP






VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH






IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK






NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ






LVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS






KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK






YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS






NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF






ATVRKVLSMPQVNIVKKTEIQTVGQNGGLFDDNPKSPLEV






TPSKLVPLKKELNPKKYGGYQKPTTAYPVLLITDTKQLIP






ISVMNKKQFEQNPVKFLRDRGYQQVGKNDFIKLPKYTLVD






IGDGIKRLWASSKEIHKGNQLVVSKKSQILLYHAHHLDSD






LSNDYLQNHNQQFDVLFNEIISFSKKCKLGKEHIQKIENV






YSNKKNSASIEELAESFIKLLGFTQLGATSPFNFLGVKLN






QKQYKGKKDYILPCTEGTLIRQSITGLYETRVDLSKIGED






SGGSGGSKRTADGSEFES






NmeCas9

Neisseria

3282
MAAFKPNSINYILGLDIGIASVGWAMVEIDEEENPIRLID
N611A




meningitidis


LGVRVFERAEVPKTGDSLAMARRLARSVRRLTRRRAHRLL






RTRRLLKREGVLQAANFDENGLIKSLPNTPWQLRAAALDR






KLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLK






GVAGNAHALQTGDFRTPAELALNKFEKESGHIRNQRSDYS






HTFSRKDLQAELILLFEKQKEFGNPHVSGGLKEGIETLLM






TQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWL






TKLNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQA






RKLLGLEDTAFFKGLRYGKDNAEASTLMEMKAYHAISRAL






EKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRLK






DRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKR






YDEACAEIYGDHYGKKNTEEKIYLPPIPADEIRNPVVLRA






LSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRKEIE






KRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYE






QQHGKCLYSGKEINLGRLNEKGYVEIDHALPFSRTWDDSF






NNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFKARVE






TSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQF






VADRMRLTGKGKKRVFASNGQITNLLRGFWGLRKVRAEND






RHHALDAVVVACSTVAMQQKITRFVRYKEMNAFDGKTIDK






ETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEA






DTLEKLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSG






QGHMETVKSAKRLDEGVSVLRVPLTQLKLKDLEKMVNRER






EPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQV






KAVRVEQVQKTGVWVRNHNGIADNATMVRVDVFEKGDKYY






LVPIYSWQVAKGILPDRAVVQGKDEEDWQLIDDSFNFKFS






LHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHK






IGKNGILEGIGVKTALSFQKYQIDELGKEIRPCRLKKRPP






VR






ScaCas9

Streptococcus

3283
MEKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTNR
N872A




canis


KSIKKNLMGALLFDSGETAEATRLKRTARRRYTRRKNRIR






YLQEIFANEMAKLDDSFFQRLEESFLVEEDKKNERHPIFG






NLADEVAYHRNYPTIYHLRKKLADSPEKADLRLIYLALAH






IIKFRGHFLIEGKLNAENSDVAKLFYQLIQTYNQLFEESP






LDEIEVDAKGILSARLSKSKRLEKLIAVFPNEKKNGLFGN






IIALALGLTPNFKSNFDLTEDAKLQLSKDTYDDDLDELLG






QIGDQYADLFSAAKNLSDAILLSDILRSNSEVTKAPLSAS






MVKRYDEHHQDLALLKTLVRQQFPEKYAEIFKDDTKNGYA






GYVGIGIKHRKRTTKLATQEEFYKFIKPILEKMDGAEELL






AKLNRDDLLRKQRTFDNGSIPHQIHLKELHAILRRQEEFY






PFLKENREKIEKILTFRIPYYVGPLARGNSRFAWLTRKSE






EAITPWNFEEVVDKGASAQSFIERMTNFDEQLPNKKVLPK






HSLLYEYFTVYNELTKVKYVTERMRKPEFLSGEQKKAIVD






LLFKTNRKVTVKQLKEDYFKKIECFDSVEIIGVEDRFNAS






LGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE






MIEERLKTYAHLFDDKVMKQLKRRHYTGWGRLSRKMINGI






RDKQSGKTILDFLKSDGFSNRNFMQLIHDDSLTFKEEIEK






AQVSGQGDSLHEQIADLAGSPAIKKGILQTVKIVDELVKV






MGHKPENIVIEMARENQTTTKGLQQSRERKKRIEEGIKEL






ESQILKENPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN






RLSDYDVDHIVPQSFIKDDSIDNKVLTRSVENRGKSDNVP






SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSEA






DKAGFIKRQLVETRQITKHVARILDSRMNTKRDKNDKPIR






EVKVITLKSKLVSDFRKDFQLYKVRDINNYHHAHDAYLNA






VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK






ATAKRFFYSNIMNFFKTEVKLANGEIRKRPLIETNGETGE






VVWNKEKDFATVRKVLAMPQVNIVKKTEVQTGGFSKESIL






SKRESAKLIPRKKGWDTRKYGGFGSPTVAYSILVVAKVEK






GKAKKLKSVKVLVGITIMEKGSYEKDPIGFLEAKGYKDIK






KELIFKLPKYSLFELENGRRRMLASATELQKANELVLPQH






LVRLLYYTQNISATTGSNNLGYIEQHREEFKEIFEKIIDF






SEKYILKNKVNSNLKSSFDEQFAVSDSILLSNSFVSLLKY






TSFGASGGFTFLDLDVKQGRLRYQTVTEVLDATLIYQSIT






GLYETRTDLSQLGGD






ScaCas9-

Streptococcus

3284
MEKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTNR
N872A


HiFi-

canis


KSIKKNLMGALLFDSGETAEATRLKRTARRRYTRRKNRIR



Sc++


YLQEIFANEMAKLDDSFFQRLEESFLVEEDKKNERHPIFG






NLADEVAYHRNYPTIYHLRKKLADSPEKADLRLIYLALAH






IIKFRGHFLIEGKLNAENSDVAKLFYQLIQTYNQLFEESP






LDEIEVDAKGILSARLSKSKRLEKLIAVFPNEKKNGLFGN






IIALALGLTPNFKSNFDLTEDAKLQLSKDTYDDDLDELLG






QIGDQYADLFSAAKNLSDAILLSDILRSNSEVTKAPLSAS






MVKRYDEHHQDLALLKTLVRQQFPEKYAEIFKDDTKNGYA






GYVGADKKLRKRSGKLATEEEFYKFIKPILEKMDGAEELL






AKLNRDDLLRKQRTFDNGSIPHQIHLKELHAILRRQEEFY






PFLKENREKIEKILTFRIPYYVGPLARGNSRFAWLTRKSE






EAITPWNFEEVVDKGASAQSFIERMTNFDEQLPNKKVLPK






HSLLYEYFTVYNELTKVKYVTERMRKPEFLSGEQKKAIVD






LLFKTNRKVTVKQLKEDYFKKIECFDSVEIIGVEDRFNAS






LGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE






MIEERLKTYAHLFDDKVMKQLKRRHYTGWGRLSRKMINGI






RDKQSGKTILDFLKSDGFSNANFMQLIHDDSLTFKEEIEK






AQVSGQGDSLHEQIADLAGSPAIKKGILQTVKIVDELVKV






MGHKPENIVIEMARENQTTTKGLQQSRERKKRTEEGIKEL






ESQILKENPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN






RLSDYDVDHIVPQSFIKDDSIDNKVLTRSVENRGKSDNVP






SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSEA






DKAGFIKRQLVETRQITKHVARILDSRMNTKRDKNDKPIR






EVKVITLKSKLVSDFRKDFQLYKVRDINNYHHAHDAYLNA






VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGK






ATAKRFFYSNIMNFFKTEVKLANGEIRKRPLIETNGETGE






VVWNKEKDFATVRKVLAMPQVNIVKKTEVQTGGFSKESIL






SKRESAKLIPRKKGWDTRKYGGFGSPTVAYSILVVAKVEK






GKAKKLKSVKVLVGITIMEKGSYEKDPIGFLEAKGYKDIK






KELIFKLPKYSLFELENGRRRMLASAKELQKANELVLPQH






LVRLLYYTQNISATTGSNNLGYIEQHREEFKEIFEKIIDF






SEKYILKNKVNSNLKSSFDEQFAVSDSILLSNSFVSLLKY






TSFGASGGFTFLDLDVKQGRLRYQTVTEVLDATLIYQSIT






GLYETRTDLSQLGGD






SpyCas9-

Streptococcus

3285
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR
N863A


3var-

pyogenes


HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC



NRRH


YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG






NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH






MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP






INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN






LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA






QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS






MVKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA






GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR






KQRTFDNGIIPHQIHLGELHAILRRQGDFYPFLKDNREKI






EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE






VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV






YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT






VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI






IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA






HLFDDKVMKQLKRLRYTGWGRLSRKLINGIRDKQSGKTIL






DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL






HEHIANLAGSPAIKKGILQTVKVVDELVKVMGGHKPENIV






IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP






VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH






IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK






NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ






LVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS






KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK






YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS






NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF






ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKGNSDKLI






ARKKDWDPKKYGGFNSPTAAYSVLVVAKVEKGKSKKLKSV






KELLGITIMERSSFEKNPIGFLEAKGYKEVKKDLIIKLPK






YSLFELENGRKRMLASAGVLHKGNELALPSKYVNFLYLAS






HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV






ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGV






PAAFKYFDTTIDKKRYTSTKEVLDATLIHQSITGLYETRI






DLSQLGGD






SpyCas9-

Streptococcus

3286
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR
N863A


3var-

pyogenes


HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC



NRTH


YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG






NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH






MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP






INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN






LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA






QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS






MVKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA






GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR






KQRTFDNGIIPHQIHLGELHAILRRQGDFYPFLKDNREKI






EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE






VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV






YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT






VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI






IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA






HLFDDKVMKQLKRLRYTGWGRLSRKLINGIRDKQSGKTIL






DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL






HEHIANLAGSPAIKKGILQTVKVVDELVKVMGGHKPENIV






IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP






VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH






IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK






NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ






LVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS






KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK






YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS






NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF






ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKGNSDKLI






ARKKDWDPKKYGGFNSPTVAYSVLVVAKVEKGKSKKLKSV






KELLGITIMERSSFEKNPIGFLEAKGYKEVKKDLIIKLPK






YSLFELENGRKRMLASASVLHKGNELALPSKYVNFLYLAS






HYEKLKGSSEDNKQKQLFVEQHKHYLDEIIEQISEFSKRV






ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA






SAAFKYFDTTIGRKLYTSTKEVLDATLIHQSITGLYETRI






DLSQLGGD






SpyCas9-

Streptococcus

3287
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR
N863A


3var-

pyogenes


HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC



NRCH


YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG






NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH






MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP






INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN






LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA






QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS






MVKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA






GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR






KQRTFDNGIIPHQIHLGELHAILRRQGDFYPFLKDNREKI






EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE






VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV






YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT






VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI






IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA






HLFDDKVMKQLKRLRYTGWGRLSRKLINGIRDKQSGKTIL






DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL






HEHIANLAGSPAIKKGILQTVKVVDELVKVMGGHKPENIV






IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP






VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH






IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK






NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ






LVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS






KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK






YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS






NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF






ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKGNSDKLI






ARKKDWDPKKYGGFNSPTVAYSVLVVAKVEKGKSKKLKSV






KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK






YSLFELENGRKRMLASAGVLQKGNELALPSKYVNFLYLAS






HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV






ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA






PAAFKYFDTTINRKQYNTTKEVLDATLIRQSITGLYETRI






DLSQLGGD






SpyCas9-

Streptococcus

3269
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR
N863A


HF1

pyogenes


HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC






YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG






NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH






MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP






INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN






LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA






QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS






MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA






GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR






KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKI






EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE






VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV






YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT






VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI






IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA






HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL






DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL






HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIV






IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP






VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH






IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK






NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ






LVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS






KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK






YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS






NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF






ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI






ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSV






KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK






YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLAS






HYEKLKGSPNLGAPAAFKYFDTTIDRKRYTSTKEVLDATL






IHQSITGLYETRIDLSQLGGD






SpyCas9-

Streptococcus

3288
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR
N863A


QQR1

pyogenes


HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC






YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG






NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH






MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP






INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN






LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA






QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS






MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA






GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR






KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKI






EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE






VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV






YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT






VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI






IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA






HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL






DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL






HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIV






IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP






VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH






IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK






NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ






LVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS






KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK






YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS






NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF






ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI






ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSV






KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK






YSLFELENGRKRMLASARELQKGNELALPSKYVNFLYLAS






HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV






ILADAQLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA






PAAFKYFDTTFKQKQYRSTKEVLDATLIHQSITGLYETRI






DLSQLGGD






SpyCas9-

Streptococcus

3289
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR
N863A


SpG

pyogenes


HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC






YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG






NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH






MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP






INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN






LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA






QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS






MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA






GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR






KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKI






EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE






VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV






YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT






VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI






IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA






HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL






DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL






HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIV






IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP






VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH






IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK






NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ






LVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS






KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK






YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS






NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF






ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI






ARKKDWDPKKYGGFLWPTVAYSVLVVAKVEKGKSKKLKSV






KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK






YSLFELENGRKRMLASAKQLQKGNELALPSKYVNFLYLAS






HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV






ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA






PAAFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRI






DLSQLGGD






SpyCas9-

Streptococcus

3290
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR
N863A


VOR

pyogenes


HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC






YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG






NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH






MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP






INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN






LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA






QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS






MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA






GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR






KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKI






EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE






VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV






YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT






VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI






IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA






HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL






DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL






HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIV






IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP






VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH






IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK






NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ






LVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS






KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK






YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS






NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF






ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI






ARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSV






KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK






YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLAS






HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV






ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA






PAAFKYFDTTIDRKQYRSTKEVLDATLIHQSITGLYETRI






DLSQLGGD






SpyCas9-

Streptococcus

3291
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR
N863A


VRER

pyogenes


HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC






YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG






NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH






MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP






INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN






LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA






QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS






MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA






GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR






KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKI






EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE






VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV






YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVT






VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI






IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA






HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL






DFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL






HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIV






IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP






VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH






IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK






NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ






LVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS






KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK






YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS






NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF






ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI






ARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSV






KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK






YSLFELENGRKRMLASARELQKGNELALPSKYVNFLYLAS






HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV






ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA






PAAFKYFDTTIDRKEYRSTKEVLDATLIHQSITGLYETRI






DLSQLGGD






SpyCas9-

Streptococcus

3292
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR
N863A


xCas

pyogenes


HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC






YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG






NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH






MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP






INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN






LIALSLGLTPNFKSNFDLAEDTKLQLSKDTYDDDLDNLLA






QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS






MIKLYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA






GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR






KQRTFDNGIIPHQIHLGELHAILRRQEDFYPFLKDNREKI






EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEK






VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV






YNELTKVKYVTEGMRKPAFLSGDQKKAIVDLLFKTNRKVT






VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI






IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA






HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL






DFLKSDGFANRNFIQLIHDDSLTFKEDIQKAQVSGQGDSL






HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIV






IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP






VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH






IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK






NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ






LVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS






KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK






YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS






NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF






ATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLI






ARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSV






KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK






YSLFELENGRKRMLASAGVLQKGNELALPSKYVNFLYLAS






HYEKLKGSPNLGAPAAFKYFDTTIDRKRYTSTKEVLDATL






IHQSITGLYETRIDLSQLGGD






SpyCas9-

Streptococcus

3293
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR
N863A


xCas-NG

pyogenes


HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRIC






YLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFG






NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAH






MIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP






INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN






LIALSLGLTPNFKSNFDLAEDTKLQLSKDTYDDDLDNLLA






QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS






MIKLYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYA






GYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR






KQRTFDNGIIPHQIHLGELHAILRRQEDFYPFLKDNREKI






EKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEK






VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV






YNELTKVKYVTEGMRKPAFLSGDQKKAIVDLLFKTNRKVT






VKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI






IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA






HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTIL






DFLKSDGFANRNFIQLIHDDSLTFKEDIQKAQVSGQGDSL






HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIV






IEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP






VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDH






IVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMK






NYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ






LVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS






KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK






YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYS






NIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDF






ATVRKVLSMPQVNIVKKTEVQTGGFSKESIRPKRNSDKLI






ARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSV






KELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK






YSLFELENGRKRMLASARFLQKGNELALPSKYVNFLYLAS






HYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRV






ILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA






PRAFKYFDTTIDRKVYRSTKEVLDATLIHQSITGLYETRI






DLSQLGGD






St1Cas9-

Streptococcus

3294
MSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQA
N622A


CNRZ1066

thermophilus


ENNLVRRTNRQGRRLARRKKHRRVRLNRLFEESGLITDFT






KISINLNPYQLRVKGLTDELSNEELFIALKNMVKHRGISY






LDDASDDGNSSVGDYAQIVKENSKQLETKTPGQIQLERYQ






TYGQLRGDFTVEKDGKKHRLINVFPTSAYRSEALRILQTQ






QEFNPQITDEFINRYLEILTGKRKYYHGPGNEKSRTDYGR






YRTSGETLDNIFGILIGKCTFYPDEFRAAKASYTAQEFNL






LNDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLF






KYIAKLLSCDVADIKGYRIDKSGKAEIHTFEAYRKMKTLE






TLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFADGS






FSQKQVDELVQFRKANSSIFGKGWHNFSVKLMMELIPELY






ETSEEQMTILTRLGKQKTTSSSNKTKYIDEKLLTEEIYNP






VVAKSVRQAIKIVNAAIKEYGDFDNIVIEMARETNEDDEK






KAIQKIQKANKDEKDAAMLKAANQYNGKAELPHSVFHGHK






QLATKIRLWHQQGERCLYTGKTISIHDLINNSNQFEVDHI






LPLSITFDDSLANKVLVYATANQEKGQRTPYQALDSMDDA






WSFRELKAFVRESKTLSNKKKEYLLTEEDISKFDVRKKFI






ERNLVDTRYASRVVLNALQEHFRAHKIDTKVSVVRGQFTS






QLRRHWGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKNT






LVSYSEEQLLDIETGELISDDEYKESVFKAPYQHFVDTLK






SKEFEDSILFSYQVDSKFNRKISDATIYATRQAKVGKDKK






DETYVLGKIKDIYTQDGYDAFMKIYKKDKSKFLMYRHDPQ






TFEKVIEPILENYPNKQMNEKGKEVPCNPFLKYKEEHGYI






RKYSKKGNGPEIKSLKYYDSKLLGNPIDITPENSKNKVVL






QSLKPWRTDVYFNKATGKYEILGLKYADLQFEKGTGTYKI






SQEKYNDIKKKEGVDSDSEFKFTLYKNDLLLVKDTETKEQ






QLFRFLSRTLPKQKHYVELKPYDKQKFEGGEALIKVLGNV






ANGGQCIKGLAKSNISIYKVRTDVLGNQHIIKNEGDKPKL






DF






St1Cas9-

Streptococcus

3295
MSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQA
N622A


LMG1831

thermophilus


ENNLVRRTNRQGRRLARRKKHRRVRLNRLFEESGLITDFT






KISINLNPYQLRVKGLTDELSNEELFIALKNMVKHRGISY






LDDASDDGNSSVGDYAQIVKENSKQLETKTPGQIQLERYQ






TYGQLRGDFTVEKDGKKHRLINVFPTSAYRSEALRILQTQ






QEFNPQITDEFINRYLEILTGKRKYYHGPGNEKSRTDYGR






YRTSGETLDNIFGILIGKCTFYPDEFRAAKASYTAQEFNL






LNDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLF






KYIAKLLSCDVADIKGYRIDKSGKAEIHTFEAYRKMKTLE






TLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFADGS






FSQKQVDELVQFRKANSSIFGKGWHNFSVKLMMELIPELY






ETSEEQMTILTRLGKQKTTSSSNKTKYIDEKLLTEEIYNP






VVAKSVRQAIKIVNAAIKEYGDFDNIVIEMARETNEDDEK






KAIQKIQKANKDEKDAAMLKAANQYNGKAELPHSVFHGHK






QLATKIRLWHQQGERCLYTGKTISIHDLINNSNQFEVDHI






LPLSITFDDSLANKVLVYATANQEKGQRTPYQALDSMDDA






WSFRELKAFVRESKTLSNKKKEYLLTEEDISKFDVRKKFI






ERNLVDTRYASRVVLNALQEHFRAHKIDTKVSVVRGQFTS






QLRRHWGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKNT






LVSYSEEQLLDIETGELISDDEYKESVFKAPYQHFVDTLK






SKEFEDSILFSYQVDSKFNRKISDATIYATRQAKVGKDKK






DETYVLGKIKDIYTQDGYDAFMKIYKKDKSKFLMYRHDPQ






TFEKVIEPILENYPNKQMNEKGKEVPCNPFLKYKEEHGYI






RKYSKKGNGPEIKSLKYYDSKLLGNPIDITPENSKNKVVL






QSLKPWRTDVYFNKNTGKYEILGLKYADLQFEKKTGTYKI






SQEKYNGIMKEEGVDSDSEFKFTLYKNDLLLVKDTETKEQ






QLFRFLSRTMPNVKYYVELKPYSKDKFEKNESLIEILGSA






DKSGRCIKGLGKSNISIYKVRTDVLGNQHIIKNEGDKPKL






DF






St1Cas9-

Streptococcus

3296
MSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQA
N622A


MTH17C

thermophilus


ENNLVRRTNRQGRRLARRKKHRRVRLNRLFEESGLITDFT



L396


KISINLNPYQLRVKGLTDELSNEELFIALKNMVKHRGISY






LDDASDDGNSSVGDYAQIVKENSKQLETKTPGQIQLERYQ






TYGQLRGDFTVEKDGKKHRLINVFPTSAYRSEALRILQTQ






QEFNPQITDEFINRYLEILTGKRKYYHGPGNEKSRTDYGR






YRTSGETLDNIFGILIGKCTFYPDEFRAAKASYTAQEFNL






LNDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLF






KYIAKLLSCDVADIKGYRIDKSGKAEIHTFEAYRKMKTLE






TLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFADGS






FSQKQVDELVQFRKANSSIFGKGWHNFSVKLMMELIPELY






ETSEEQMTILTRLGKQKTTSSSNKTKYIDEKLLTEEIYNP






VVAKSVRQAIKIVNAAIKEYGDFDNIVIEMARETNEDDEK






KAIQKIQKANKDEKDAAMLKAANQYNGKAELPHSVFHGHK






QLATKIRLWHQQGERCLYTGKTISIHDLINNSNQFEVDHI






LPLSITFDDSLANKVLVYATANQEKGQRTPYQALDSMDDA






WSFRELKAFVRESKTLSNKKKEYLLTEEDISKFDVRKKFI






ERNLVDTRYASRVVLNALQEHFRAHKIDTKVSVVRGQFTS






QLRRHWGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKNT






LVSYSEDQLLDIETGELISDDEYKESVFKAPYQHFVDTLK






SKEFEDSILFSYQVDSKFNRKISDATIYATRQAKVGKDKA






DETYVLGKIKDIYTQDGYDAFMKIYKKDKSKFLMYRHDPQ






TFEKVIEPILENYPNKQINEKGKEVPCNPFLKYKEEHGYI






RKYSKKGNGPEIKSLKYYDSKLGNHIDITPKDSNNKVVLQ






SLKPWRTDVYFNKNTGKYEILGLKYSDMQFEKGTGKYSIS






KEQYENIKVREGVDENSEFKFTLYKNDLLLLKDSENGEQI






LLRFTSRNDTSKHYVELKPYNRQKFEGSEYLIKSLGTVAK






GGQCIKGLGKSNISIYKVRTDVLGNQHIIKNEGDKPKLDF






St1Cas9-

Streptococcus

3297
MSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQA
N622A


TH1477

thermophilus


ENNLVRRTNRQGRRLARRKKHRRVRLNRLFEESGLITDFT






KISINLNPYQLRVKGLTDELSNEELFIALKNMVKHRGISY






LDDASDDGNSSVGDYAQIVKENSKQLETKTPGQIQLERYQ






TYGQLRGDFTVEKDGKKHRLINVFPTSAYRSEALRILQTQ






QEFNPQITDEFINRYLEILTGKRKYYHGPGNEKSRTDYGR






YRTSGETLDNIFGILIGKCTFYPDEFRAAKASYTAQEFNL






LNDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLF






KYIAKLLSCDVADIKGYRIDKSGKAEIHTFEAYRKMKTLE






TLDIEQMDRETLDKLAYVLTLNTEREGIQEALEHEFADGS






FSQKQVDELVQFRKANSSIFGKGWHNFSVKLMMELIPELY






ETSEEQMTILTRLGKQKTTSSSNKTKYIDEKLLTEEIYNP






VVAKSVRQAIKIVNAAIKEYGDFDNIVIEMARETNEDDEK






KAIQKIQKANKDEKDAAMLKAANQYNGKAELPHSVFHGHK






QLATKIRLWHQQGERCLYTGKTISIHDLINNSNQFEVDHI






LPLSITFDDSLANKVLVYATANQEKGQRTPYQALDSMDDA






WSFRELKAFVRESKTLSNKKKEYLLTEEDISKFDVRKKFI






ERNLVDTRYASRVVLNALQEHFRAHKIDTKVSVVRGQFTS






QLRRHWGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKNT






LVSYSEDQLLDIETGELISDDEYKESVFKAPYQHFVDTLK






SKEFEDSILFSYQVDSKFNRKISDATIYATRQAKVGKDKA






DETYVLGKIKDIYTQDGYDAFMKIYKKDKSKFLMYRHDPQ






TFEKVIEPILENYPNKQINEKGKEVPCNPFLKYKEEHGYI






RKYSKKGNGPEIKSLKYYDSKLGNHIDITPKDSNNKVVLQ






SLKPWRTDVYFNKNTGKYEILGLKYSDMQFEKGTGKYSIS






KEQYENIKVREGVDENSEFKFTLYKNDLLLLKDSENGEQI






LLRFTSRNDTSKHYVELKPYNRQKFEGSEYLIKSLGTVVK






GGRCIKGLGKSNISIYKVRTDVLGNQHIIKNEGDKPKLDF









Table 9B provides parameters to define the necessary components for designing gRNA and/or Template RNAs to apply Cas variants listed in Table 3A for Gene Writing. Tier indicates preferred Cas variants if they are available for use at a given locus. The cut site indicates the validated or predicted protospacer adjacent motif(PAM) requirements, validated or predicted location of cut site (relative to the most upstream base of the PAM site). The gRNA for a given enzyme can be assembled by concatenating the crRNA, Tetraloop, and tracrRNA sequences, and further adding a 5′ spacer of a length within Spacer (min) and Spacer (max) that matches a protospacer at a target site. Further, the predicted location of the ssDNA nick at the target is important for designing the 3′ region of a Template RNA that needs to anneal to the sequence immediately 5′ of the nick in order to initiate target primed reverse transcription.









TABLE 9B







parameters to define the necessary components for designing gRNA and/or Template


RNAs to apply Cas variants listed in Table 9A for Gene Writing
























SEQ


SEQ






Spacer
Spacer

ID
Tetra

ID


Variant
PAM(s)
Cut
Tier
(min)
(max)
crRNA
NO:
loop
tracrRNA
NO:




















Nme2Cas9
NNNNC
−3
1
22
24
GTTG
3535
GAA
CGAAATGA
3536



C




TAGC

A
GAACCGTT









TCCCT


GCTACAAT









TTCTC


AAGGCCGT









ATTTC


CTGAAAAG









G


ATGTGCCG












CAACGCTC












TGCCCCTT












AAAGCTTC












TGCTTTAA












GGGGCATC












GTTTA






PpnCas9
NNNNR

1
21
24
GTTG
3537
GAA
GCGAAATG
3538



TT




TAGC

A
AAAAACGT









TCCCT


TGTTACAA









TTTTC


TAAGAGAT









ATTTC


GAATTTCT









GC


CGCAAAGC












TCTGCCTC












TTGAAATT












TCGGTTTC












AAGAGGCA












TCTTTTT






SauCas9
NNGRR;
−3
1
21
23
GTTTT
3539
GAA
CAGAATCT
3540



NNGRRT




AGTA

A
ACTAAAAC









CTCT


AAGGCAAA









G


ATGCCGTG












TTTATCTC












GTCAACTT












GTTGGCGA












GA






SauCas9-
NNNRR;
−3
1
21
21
GTTTT
3541
GAA
ATTACAGA
3542


KKH
NNNRRT




AGTA

A
ATCTACTA









CTCT


AAACAAGG









GTAA


CAAAATGC









T


CGTGTTTA












TCTCGTCA












ACTTGTTG












GCGAGA






SauriCas9
NNGG
−3
1
21
21
GTTTT
3539
GAA
CAGAATCT
3543








AGTA

A
ACTAAAAC









CTCT


AAGGCAAA









G


ATGCCGTG












TTTATCTC












GTCAACTT












GTTGGCGA












GATTTTT






SauriCas9-
NNRG
−3
1
21
21
GTTTT
3539
GAA
CAGAATCT
3543


KKH





AGTA

A
ACTAAAAC









CTCT


AAGGCAAA









G


ATGCCGTG












TTTATCTC












GTCAACTT












GTTGGCGA












GATTTTT






ScaCas9-
NNG
3
1
20
20
GTTTT
3544
GAA
TAGCAAGT
3545


Sc++





AGAG

A
TAAAATAA









CTA


GGCTAGTC












CGTTATCA












ACTTGAAA












AAGTGGCA












CCGAGTCG












GTGC






SpyCas9
NGG
3
1
20
20
GTTTT
3544
GAA
TAGCAAGT
3545








AGAG

A
TAAAATAA









CTA


GGCTAGTC












CGTTATCA












ACTTGAAA












AAGTGGCA












CCGAGTCG












GTGC






SpyCas9-
NG
−3
1
20
20
GTTT
3546
GAA
CAGCATAG
3547


NG
(NGG = N




AAGA

A
CAAGTTTA




GA = NGT




GCTA


AATAAGGC




> NGC)




TGCT


TAGTCCGT









G


TATCAACT












TGAAAAAG












TGGCACCG












AGTCGGTG












C






SpyCas9-
NRN > N
−3
1
20
20
GTTTT
3544
GAA
TAGCAAGT
3545


SpRY
YN




AGAG

A
TAAAATAA









CTA


GGCTAGTC












CGTTATCA












ACTTGAAA












AAGTGGCA












CCGAGTCG












GTGC






St1Cas9
NNAGA
−3
1
20
20
GTCTT
3548
GTA
CAGAAGCT
3549



AW > NN




TGTA

C
ACAAAGAT




AGGAW




CTCT


AAGGCTTC




= NNGG




G


ATGCCGAA




AAW







ATCAACAC












CCTGTCAT












TTTATGGC












AGGGTGTT












TT






BlatCas9
NNNNC
−3
1
19
23
GCTA
3550
GAA
GGTAAGTT
3551



NAA > N




TAGT

A
GCTATAGT




NNNCN




TCCTT


AAGGGCAA




DD > NN




ACT


CAGACCCG




NNC







AGGCGTTG












GGGATCGC












CTAGCCCG












TGTTTACG












GGCTCTCC












CCATATTC












AAAATAAT












GACAGACG












AGCACCTT












GGAGCATT












TATCTCCG












AGGTGCT






cCas9-
NNVAC
−3
2
21
21
GUCU
3552
GAA
CAGAAUCU
3553


v16
T; NNVA




UAGU

A
ACUAAGAC




TGM; NN




ACUC


AAGGCAAA




VATT; N




UG


AUGCCGUG




NVGCT;







UUUAUCUC




NNVGT







GUCAACUU




G; NNVG







GUUGGCGA




TT







GAUUUUUU












U






cCas9-
NNVRR
−3
2
21
21
GUCU
3552
GAA
CAGAAUCU
3553


v17
N




UAGU

A
ACUAAGAC









ACUC


AAGGCAAA









UG


AUGCCGUG












UUUAUCUC












GUCAACUU












GUUGGCGA












GAUUUUUU












U






cCas9-
NNVAC
−3
2
21
21
GUCU
3552
GAA
CAGAAUCU
3553


v21
T; NNVA




UAGU

A
ACUAAGAC




TGM; NN




ACUC


AAGGCAAA




VATT; N




UG


AUGCCGUG




NVGCT;







UUUAUCUC




NNVGT







GUCAACUU




G; NNVG







GUUGGCGA




TT







GAUUUUUU












U






cCas9-
NNVRR
−3
2
21
21
GUCU
3552
GAA
CAGAAUCU
3553


v42
N




UAGU

A
ACUAAGAC









ACUC


AAGGCAAA









UG


AUGCCGUG












UUUAUCUC












GUCAACUU












GUUGGCGA












GAUUUUUU












U






CdiCas9
NNRHH

2
22
22
ACUG
3554
GAA
CUGAACCU
3555



HY; NNR




GGGU

A
CAGUAAGC




AAAY




UCAG


AUUGGCUC












GUUUCCAA












UGUUGAUU












GCUCCGCC












GGUGCUCC












UUAUUUUU












AAGGGCGC












CGGC






CjeCas9
NNNNR
−3
2
21
23
GTTTT
3556
GAA
AGGGACTA
3557



YAC




AGTC

A
AAATAAAG









CCT


AGTTTGCG












GGACTCTG












CGGGGTTA












CAATCCCC












TAAAACCG












CTTTTTT






GeoCas9
NNNNC

2
21
23
GUCA
3558
GAA
UCAGGGUU
3559



RAA




UAGU

A
ACUAUGAU









UCCC


AAGGGCUU









CUGA


UCUGCCUA












AGGCAGAC












UGACCCGC












GGCGUUGG












GGAUCGCC












UGUCGCCC












GCUUUUGG












CGGGCAUU












CCCCAUCC












UU






iSpyMacCas9
NAAN
−3
2
19
21
GTTTT
3544
GAA
TAGCAAGT
3545








AGAG

A
TAAAATAA









CTA


GGCTAGTC












CGTTATCA












ACTTGAAA












AAGTGGCA












CCGAGTCG












GTGC






NmeCas9
NNNNG
−3
2
20
24
GTTG
3535
GAA
CGAAATGA
3536



AYT; NN




TAGC

A
GAACCGTT




NNGYTT;




TCCCT


GCTACAAT




NNNNG




TTCTC


AAGGCCGT




AYA; NN




ATTTC


CTGAAAAG




NNGTCT




G


ATGTGCCG












CAACGCTC












TGCCCCTT












AAAGCTTC












TGCTTTAA












GGGGCATC












GTTTA






ScaCas9
NNG
−3
2
20
20
GTTTT
3544
GAA
TAGCAAGT
3545








AGAG

A
TAAAATAA









CTA


GGCTAGTC












CGTTATCA












ACTTGAAA












AAGTGGCA












CCGAGTCG












GTGC






ScaCas9-
NNG
−3
2
20
20
GTTTT
3544
GAA
TAGCAAGT
3545


HiFi-





AGAG

A
TAAAATAA



Sc++





CTA


GGCTAGTC












CGTTATCA












ACTTGAAA












AAGTGGCA












CCGAGTCG












GTGC






SpyCas9-
NRRH
−3
2
20
20
GTTT
3546
GAA
CAGCATAG
3547


3var-





AAGA

A
CAAGTTTA



NRRH





GCTA


AATAAGGC









TGCT


TAGTCCGT









G


TATCAACT












TGAAAAAG












TGGCACCG












AGTCGGTG












C






SpyCas9-
NRTH
−3
2
20
20
GTTT
3546
GAA
CAGCATAG
3547


3var-





AAGA

A
CAAGTTTA



NRTH





GCTA


AATAAGGC









TGCT


TAGTCCGT









G


TATCAACT












TGAAAAAG












TGGCACCG












AGTCGGTG












C






SpyCas9-
NRCH
−3
2
20
20
GTTT
3546
GAA
CAGCATAG
3547


3var-





AAGA

A
CAAGTTTA



NRCH





GCTA


AATAAGGC









TGCT


TAGTCCGT









G


TATCAACT












TGAAAAAG












TGGCACCG












AGTCGGTG












C






SpyCas9-
NGG
−3
2
20
20
GTTTT
3544
GAA
TAGCAAGT
3545


HF1





AGAG

A
TAAAATAA









CTA


GGCTAGTC












CGTTATCA












ACTTGAAA












AAGTGGCA












CCGAGTCG












GTGC






SpyCas9-
NAAG
−3
2
20
20
GTTTT
3544
GAA
TAGCAAGT
3545


QQR1





AGAG

A
TAAAATAA









CTA


GGCTAGTC












CGTTATCA












ACTTGAAA












AAGTGGCA












CCGAGTCG












GTGC






SpyCas9-
NGN
−3
2
20
20
GTTTT
3544
GAA
TAGCAAGT
3545


SpG





AGAG

A
TAAAATAA









CTA


GGCTAGTC












CGTTATCA












ACTTGAAA












AAGTGGCA












CCGAGTCG












GTGC






SpyCas9-
NGAN
−3
2
20
20
GTTTT
3544
GAA
TAGCAAGT
3545


VOR





AGAG

A
TAAAATAA









CTA


GGCTAGTC












CGTTATCA












ACTTGAAA












AAGTGGCA












CCGAGTCG












GTGC






SpyCas9-
NGCG
−3
2
20
20
GTTTT
3544
GAA
TAGCAAGT
3545


VRER





AGAG

A
TAAAATAA









CTA


GGCTAGTC












CGTTATCA












ACTTGAAA












AAGTGGCA












CCGAGTCG












GTGC






SpyCas9-
NG; GAA;
−3
2
20
20
GTTT
3546
GAA
CAGCATAG
3547


xCas
GAT




AAGA

A
CAAGTTTA









GCTA


AATAAGGC









TGCT


TAGTCCGT









G


TATCAACT












TGAAAAAG












TGGCACCG












AGTCGGTG












C






SpyCas9-
NG
−3
2
20
20
GTTT
3546
GAA
CAGCATAG
3547


xCas-





AAGA

A
CAAGTTTA



NG





GCTA


AATAAGGC









TGCT


TAGTCCGT









G


TATCAACT












TGAAAAAG












TGGCACCG












AGTCGGTG












C






St1Cas9-
NNACA
−3
2
20
20
GTCTT
3548
GTA
CAGAAGCT
3549


CNRZ1066
A




TGTA

C
ACAAAGAT









CTCT


AAGGCTTC









G


ATGCCGAA












ATCAACAC












CCTGTCAT












TTTATGGC












AGGGTGTT












TT






St1Cas9-
NNGCA
−3
2
20
20
GTCTT
3548
GTA
CAGAAGCT
3549


LMG1831
A




TGTA

C
ACAAAGAT









CTCT


AAGGCTTC









G


ATGCCGAA












ATCAACAC












CCTGTCAT












TTTATGGC












AGGGTGTT












TT






St1Cas9-
NNAAA
−3
2
20
20
GTCTT
3548
GTA
CAGAAGCT
3549


MTH17
A




TGTA

C
ACAAAGAT



CL396





CTCT


AAGGCTTC









G


ATGCCGAA












ATCAACAC












CCTGTCAT












TTTATGGC












AGGGTGTT












TT






St1Cas9-
NNGAA
−3
2
20
20
GTCTT
3548
GTA
CAGAAGCT
3549


TH1477
A




TGTA

C
ACAAAGAT









CTCT


AAGGCTTC









G


ATGCCGAA












ATCAACAC












CCTGTCAT












TTTATGGC












AGGGTGTT












TT









In some embodiments, the opener molecule is exogenous to the cell comprising the Gene Writer polypeptide and or template nucleic acid. In some embodiments, the opener molecule comprises an endogenous agent (e.g., endogenous to the cell comprising the Gene Writer polypeptide and or template nucleic acid comprising the gRNA and blocking domain). For example, an inducible gRNA, blocking domain, and opener molecule may be chosen such that the opener molecule is an endogenous agent expressed in a target cell or tissue, e.g., thereby ensuring activity of a Gene Writer system in the target cell or tissue. As a further example, an inducible gRNA, blocking domain, and opener molecule may be chosen such that the opener molecule is absent or not substantially expressed in one or more non-target cells or tissues, e.g., thereby ensuring that activity of a Gene Writer system does not occur or substantially occur in the one or more non-target cells or tissues, or occurs at a reduced level compared to a target cell or tissue. Exemplary blocking domains, opener molecules, and uses thereof are described in PCT App. Publication WO2020044039A1, which is incorporated herein by reference in its entirety. In some embodiments, the template nucleic acid, e.g., template RNA, may comprise one or more UTRs (e.g. from an R2-type retrotransposon) and a gRNA. In some embodiments, the UTR facilitates interaction of the template nucleic acid (e.g., template RNA) with the writing domain, e.g., reverse transcriptase domain, of the Gene Writer polypeptide. In some embodiments, the gRNA facilitates interaction with the template nucleic acid binding domain (e.g., RNA binding domain) of the polypeptide. In some embodiments, the gRNA directs the polypeptide to the matching target sequence, e.g., in a target cell genome. In some embodiments, the template nucleic acid may contain only the reverse transcriptase binding motif (e.g. 3′ UTR from R2) and the gRNA may be provided as a second nucleic acid molecule (e.g., second RNA molecule) for target site recognition. In some embodiments, the template nucleic acid containing the RT-binding motif may exist on the same molecule as the gRNA, but be processed into two RNA molecules by cleavage activity (e.g. ribozyme).


In some embodiments, a template RNA may be customized to correct a given mutation in the genomic DNA of a target cell (e.g., ex vivo or in vivo, e.g., in a target tissue or organ, e.g., in a subject). For example, the mutation may be a disease-associated mutation relative to the wild-type sequence. Without wishing to be bound by theory, sets of empirical parameters help ensure optimal initial in silico designs of template RNAs or portions thereof. As a non-limiting illustrative example, for a selected mutation, the following design parameters may be employed. In some embodiments, design is initiated by acquiring approximately 500 bp (e.g., up to 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or 700 bp, and optionally at least 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 bp) flanking sequence on either side of the mutation to serve as the target region. In some embodiments, a template nucleic acid comprises a gRNA. Methodology for designing gRNAs is known to those of skill in the art. In some embodiments, a gRNA comprises a sequence (e.g., a CRISPR spacer) that binds a target site. In some embodiments, the sequence (e.g., a CRISPR spacer) that binds a target site for use in targeting a template nucleic acid to a target region is selected by considering the particular Gene Writer polypeptide (e.g., endonuclease domain or writing domain, e.g., comprising a CRISPR/Cas domain) being used (e.g., for Cas9, a protospacer-adjacent motif (PAM) of NGG immediately 3′ of a 20 nt gRNA binding region). In some embodiments, the CRISPR spacer is selected by ranking first by whether the PAM will be disrupted by the Gene Writing induced edit. In some embodiments, disruption of the PAM may increase edit efficiency. In some embodiments, the PAM can be disrupted by also introducing (e.g., as part of or in addition to another modification to a target site in genomic DNA) a silent mutation (e.g., a mutation that does not alter an amino acid residue encoded by the target nucleic acid sequence, if any) in the target site during Gene Writing. In some embodiments, the CRISPR spacer is selected by ranking sequences by the proximity of their corresponding genomic site to the desired edit location. In some embodiments, the gRNA comprises a gRNA scaffold. In some embodiments, the gRNA scaffold used may be a standard scaffold (e.g., for Cas9, 5′-GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAA AGTGGGACCGAGTCGGTCC-3′ (SEQ ID NO: 1591)), or may contain one or more nucleotide substitutions. In some embodiments, the heterologous object sequence has at least 90% identity, e.g., at least 90%, 95%, 98%, 99%, or 100% identity, or comprises no more than 1, 2, 3, 4, or 5 positions of non-identity to the target site 3′ of the first strand nick (e.g., immediately 3′ of the first strand nick or up to 1, 2, 3, 4, or 5 nucleotides 3′ of the first strand nick), with the exception of any insertion, substitution, or deletion that may be written into the target site by the Gene Writer. In some embodiments, the 3′ target homology domain contains at least 90% identity, e.g., at least 90%, 95%, 98%, 99%, or 100% identity, or comprises no more than 1, 2, 3, 4, or 5 positions of non-identity to the target site 5′ of the first strand nick (e.g., immediately 5′ of the first strand nick or up to 1, 2, 3, 4, or 5 nucleotides 3′ of the first strand nick).


Methods and Compositions for Modified RNA (e.g., gRNA or Template RNA)


In some embodiments, an RNA component of the system (e.g., a template RNA or a gRNA) comprises one or more nucleotide modifications. In some embodiments, the modification pattern of a gRNA can significantly affect in vivo activity compared to unmodified or end-modified guides (e.g., as shown in FIG. 1D from Finn et al. Cell Rep 22(9):2227-2235 (2018); incorporated herein by reference in its entirety). Without wishing to be bound by theory, this process may be due, at least in part, to a stabilization of the RNA conferred by the modifications. Non-limiting examples of such modifications may include 2′-O-methyl (2′-O-Me), 2′-O-(2-methoxyethyl) (2′-O-MOE), 2′-fluoro (2′-F), phosphorothioate (PS) bond between nucleotides, G-C substitutions, and inverted abasic linkages between nucleotides and equivalents thereof.


In some embodiments, the template RNA (e.g., at the portion thereof that binds a target site) or the guide RNA comprises a 5′ terminus region. In some embodiments, the template RNA or the guide RNA does not comprise a 5′ terminus region. In some embodiments, the 5′ terminus region comprises a CRISPR spacer region, e.g., as described with respect to sgRNA in Briner A E et al, Molecular Cell 56: 333-339 (2014) (incorporated herein by reference in its entirety; applicable herein, e.g., to all guide RNAs). In some embodiments, the 5′ terminus region comprises a 5′ end modification. In some embodiments, a 5′ terminus region with or without a spacer region may be associated with a crRNA, trRNA, sgRNA and/or dgRNA. The CRISPR spacer region can, in some instances, comprise a guide region, guide domain, or targeting domain. In some embodiments, a target domain or target sequence may comprise a sequence of nucleic acid to which the guide region/domain directs a nuclease for cleavage. In some embodiments, a spyCas9 protein may be directed by a guide region/domain to a target sequence of a target nucleic acid molecule by the nucleotides present in the CRISPR spacer region.


In some embodiments, the template RNAs (e.g., at the portion thereof that binds a target site) or guide RNAs described herein comprises any of the sequences shown in Table 4 of WO2018107028A1, incorporated herein by reference in its entirety. In some embodiments, where a sequence shows a guide and/or spacer region, the composition may comprise this region or not. In some embodiments, a guide RNA comprises one or more of the modifications of any of the sequences shown in Table 4 of WO2018107028A1, e.g., as identified therein by a SEQ ID NO. In embodiments, the nucleotides may be the same or different, and/or the modification pattern shown may be the same or similar to a modification pattern of a guide sequence as shown in Table 4 of WO2018107028A1. In some embodiments, a modification pattern includes the relative position and identity of modifications of the gRNA or a region of the gRNA (e.g. 5′ terminus region, lower stem region, bulge region, upper stem region, nexus region, hairpin 1 region, hairpin 2 region, 3′ terminus region). In some embodiments, the modification pattern contains at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the modifications of any one of the sequences shown in the sequence column of Table 4 of WO2018107028A1, and/or over one or more regions of the sequence. In some embodiments, the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the modification pattern of any one of the sequences shown in the sequence column of Table 4 of WO2018107028A1. In some embodiments, the modification pattern is at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical over one or more regions of the sequence shown in Table 4 of WO2018107028A1, e.g., in a 5′terminus region, lower stem region, bulge region, upper stem region, nexus region, hairpin 1 region, hairpin 2 region, and/or 3′ terminus region. In some embodiments, the modification pattern is least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the modification pattern of a sequence over the 5′ terminus region. In some embodiments, the modification pattern is least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the lower stem. In some embodiments, the modification pattern is least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the bulge. In some embodiments, the modification pattern is least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the upper stem. In some embodiments, the modification pattern is least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the nexus. In some embodiments, the modification pattern is least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the hairpin 1. In some embodiments, the modification pattern is least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the hairpin 2. In some embodiments, the modification pattern is least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the 3′terminus. In some embodiments, the modification pattern differs from the modification pattern of a sequence of Table 4 of WO2018107028A1, or a region (e.g. 5′ terminus, lower stem, bulge, upper stem, nexus, hairpin 1, hairpin 2, 3′ terminus) of such a sequence, e.g., at 0, 1, 2, 3, 4, 5, 6, or more nucleotides. In some embodiments, the gRNA comprises modifications that differ from the modifications of a sequence of Table 4 of WO2018107028A1, e.g., at 0, 1, 2, 3, 4, 5, 6, or more nucleotides. In some embodiments, the gRNA comprises modifications that differ from modifications of a region (e.g. 5′terminus, lower stem, bulge, upper stem, nexus, hairpin 1, hairpin 2, 3′ terminus) of a sequence of Table 4 of WO2018107028A1, e.g., at 0, 1, 2, 3, 4, 5, 6, or more nucleotides.


In some embodiments, the template RNAs (e.g., at the portion thereof that binds a target site) or the gRNA comprises a 2′-O-methyl (2′-O-Me) modified nucleotide. In some embodiments, the gRNA comprises a 2′-O-(2-methoxy ethyl) (2′-O-moe) modified nucleotide. In some embodiments, the gRNA comprises a 2′-fluoro (2′-F) modified nucleotide. In some embodiments, the gRNA comprises a phosphorothioate (PS) bond between nucleotides. In some embodiments, the gRNA comprises a 5′ end modification, a 3′ end modification, or 5′ and 3′ end modifications. In some embodiments, the 5′ end modification comprises a phosphorothioate (PS) bond between nucleotides. In some embodiments, the 5′ end modification comprises a 2′-O-methyl (2′-O-Me), 2′-O-(2-methoxy ethyl) (2′-O-MOE), and/or 2′-fluoro (2′-F) modified nucleotide. In some embodiments, the 5′ end modification comprises at least one phosphorothioate (PS) bond and one or more of a 2′-O-methyl (2′-O-Me), 2′-O-(2-methoxyethyl) (2′-O-MOE), and/or 2′-fluoro (2′-F) modified nucleotide. The end modification may comprise a phosphorothioate (PS), 2′-O-methyl (2′-O-Me), 2′-O-(2-methoxyethyl) (2′-O-MOE), and/or 2′-fluoro (2′-F) modification. Equivalent end modifications are also encompassed by embodiments described herein. In some embodiments, the template RNA or gRNA comprises an end modification in combination with a modification of one or more regions of the template RNA or gRNA. Additional exemplary modifications and methods for protecting RNA, e.g., gRNA, and formulae thereof, are described in WO2018126176A1, which is incorporated herein by reference in its entirety.


In some embodiments, structure-guided and systematic approaches are used to introduce modifications (e.g., 2′-OMe-RNA, 2′-F-RNA, and PS modifications) to a template RNA or guide RNA, for example, as described in Mir et al. Nat Commun 9:2641 (2018) (incorporated by reference herein in its entirety). In some embodiments, the incorporation of 2′-F-RNAs increases thermal and nuclease stability of RNA:RNA or RNA:DNA duplexes, e.g., while minimally interfering with C3′-endo sugar puckering. In some embodiments, 2′-F may be better tolerated than 2′-OMe at positions where the 2′-OH is important for RNA:DNA duplex stability. In some embodiments, a crRNA comprises one or more modifications that do not reduce Cas9 activity, e.g., C10, C20, or C21 (fully modified), e.g., as dscribed in Supplementary Table 1 of Mir et al. Nat Commun 9:2641 (2018), incorporated herein by reference in its entirety. In some embodiments, a tracrRNA comprises one or more modifications that do not reduce Cas9 activity, e.g., T2, T6, T7, or T8 (fully modified) of Supplementary Table 1 of Mir et al. Nat Commun 9:2641 (2018). In some embodiments, a crRNA comprises one or more modifications (e.g., as described herein) may be paired with a tracrRNA comprising one or more modifications, e.g., C20 and T2. In some embodiments, a gRNA comprises a chimera, e.g., of a crRNA and a tracrRNA (e.g., Jinek et al. Science 337(6096):816-821 (2012)). In embodiments, modifications from the crRNA and tracrRNA are mapped onto the single-guide chimera, e.g., to produce a modified gRNA with enhanced stability.


In some embodiments, gRNA molecules may be modified by the addition or subtraction of the naturally occurring structural components, e.g., hairpins. In some embodiments, a gRNA may comprise a gRNA with one or more 3′ hairpin elements deleted, e.g., as described in WO2018106727, incorporated herein by reference in its entirety. In some embodiments, a gRNA may contain an added hairpin structure, e.g., an added hairpin structure in the spacer region, which was shown to increase specificity of a CRISPR-Cas system in the teachings of Kocak et al. Nat Biotechnol 37(6):657-666 (2019). Additional modifications, including examples of shortened gRNA and specific modifications improving in vivo activity, can be found in US20190316121, incorporated herein by reference in its entirety.


In some embodiments, structure-guided and systematic approaches (e.g., as described in Mir et al. Nat Commun 9:2641 (2018); incorporated herein by reference in its entirety) are employed to find modifications for the template RNA. In embodiments, the modifications are identified with the inclusion or exclusion of a guide region of the template RNA. In some embodiments, a structure of polypeptide bound to template RNA is used to determine non-protein-contacted nucleotides of the RNA that may then be selected for modifications, e.g., with lower risk of disrupting the association of the RNA with the polypeptide. Secondary structures in a template RNA can also be predicted in silico by software tools, e.g., the RNAstructure tool available at rna.urmc.rochester.edu/RNAstructureWeb (Bellaousov et al. Nucleic Acids Res 41:W471-W474 (2013); incorporated by reference herein in its entirety), e.g., to determine secondary structures for selecting modifications, e.g., hairpins, stems, and/or bulges.


Further included here are compositions and methods for the assembly of full or partial template RNA molecules (e.g., Gene Writing template RNA molecules optionally comprising a gRNA, or separate gRNA molecules). In some embodiments, RNA molecules may be assembled by the connection of two or more (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) RNA segments with each other. In an aspect, the disclosure provides methods for producing nucleic acid molecules, the methods comprising contacting two or more linear RNA segments with each other under conditions that allow for the 5′ terminus of a first RNA segment to be covalently linked with the 3′ terminus of a second RNA segment. In some embodiments, the joined molecule may be contacted with a third RNA segment under conditions that allow for the 5′ terminus of the joined molecule to be covalently linked with the 3′ terminus of the third RNA segment. In embodiments, the method further comprises joining a fourth, fifth, or additional RNA segments to the elongated molecule. This form of assembly may, in some instances, allow for rapid and efficient assembly of RNA molecules.


The present disclosure also provides compositions and methods for the connection (e.g., covalent connection) of crRNA molecules and tracrRNA molecules. In some embodiments, guide RNA molecules with specificity for different target sites can be generated using a single tracrRNA molecule/segment connected to a target site specific crRNA molecule/segment (e.g., as shown in FIG. 10 of US20160102322A1; incorporated herein by reference in its entirety). For example, FIG. 10 of US20160102322A1 shows four tubes with different crRNA molecules with crRNA molecule 3 being connected to a tracrRNA molecule to form a guide RNA molecule, thereby depicting an exemplary connection of two RNA segments to form a product RNA molecule.


The disclosure also provides compositions and methods for the production of template RNA molecules with specificity for a Gene Writer polypeptide and/or a genomic target site. In an aspect, the method comprises: (1) identification of the target site and desired modification thereto, (2) production of RNA segments including an upstream homology segment, a heterologous object sequence segment, a Gene Writer polypeptide binding motif, and a gRNA segment, and/or (3) connection of the four or more segments into at least one molecule, e.g., into a single RNA molecule. In some embodiments, some or all of the template RNA segments comprised in (2) are assembled into a template RNA molecule, e.g., one, two, three, or four of the listed components. In some embodiments, the segments comprised in (2) may be produced in further segmented molecules, e.g., split into at least 2, at least 3, at least 4, or at least 5 or more sub-segments, e.g., that are subsequently assembled, e.g., by one or more methods described herein.


In some embodiments, RNA segments may be produced by chemical synthesis. In some embodiments, RNA segments may be produced by in vitro transcription of a nucleic acid template, e.g., by providing an RNA polymerase to act on a cognate promoter of a DNA template to produce an RNA transcript. In some embodiments, in vitro transcription is performed using, e.g., a T7, T3, or SP6 RNA polymerase, or a derivative thereof, acting on a DNA, e.g., dsDNA, ssDNA, linear DNA, plasmid DNA, linear DNA amplicon, linearized plasmid DNA, e.g., encoding the RNA segment, e.g., under transcriptional control of a cognate promoter, e.g., a T7, T3, or SP6 promoter. In some embodiments, a combination of chemical synthesis and in vitro transcription is used to generate the RNA segments for assembly. In embodiments, the gRNA, upstream target homology, and Gene Writer polypeptide binding segments are produced by chemical synthesis and the heterologous object sequence segment is produced by in vitro transcription. Without wishing to be bound by theory, in vitro transcription may be better suited for the production of longer RNA molecules. In some embodiments, reaction temperature for in vitro transcription may be lowered, e.g., be less than 37° C. (e.g., between 0-10 C, 10-20 C, or 20-30 C), to result in a higher proportion of full-length transcripts (Krieg Nucleic Acids Res 18:6463 (1990)). In some embodiments, a protocol for improved synthesis of long transcripts is employed to synthesize a long template RNA, e.g., a template RNA greater than 5 kb, such as the use of e.g., T7 RiboMAX Express, which can generate 27 kb transcripts in vitro (Thiel et al. J Gen Virol 82(6):1273-1281 (2001)). In some embodiments, modifications to RNA molecules as described herein may be incorporated during synthesis of RNA segments (e.g., through the inclusion of modified nucleotides or alternative binding chemistries), following synthesis of RNA segments through chemical or enzymatic processes, following assembly of one or more RNA segments, or a combination thereof.


In some embodiments, an mRNA of the system (e.g., an mRNA encoding a Gene Writer polypeptide) is synthesized in vitro using T7 polymerase-mediated DNA-dependent RNA transcription from a linearized DNA template, where UTP is optionally substituted with 1-methylpseudoUTP. In some embodiments, the transcript incorporates 5′ and 3′ UTRs, e.g., GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC (SEQ ID NO: 1603) and UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGA (SEQ ID NO: 1604), or functional fragments or variants thereof, and optionally includes a poly-A tail, which can be encoded in the DNA template or added enzymatically following transcription. In some embodiments, a donor methyl group, e.g., S-adenosylmethionine, is added to a methylated capped RNA with cap 0 structure to yield a cap 1 structure that increases mRNA translation efficiency (Richner et al. Cell 168(6): P1114-1125 (2017)).


In some embodiments, the transcript from a T7 promoter starts with a GGG motif. In some embodiments, a transcript from a T7 promoter does not start with a GGG motif. It has been shown that a GGG motif at the transcriptional start, despite providing superior yield, may lead to T7 RNAP synthesizing a ladder of poly(G) products as a result of slippage of the transcript on the three C residues in the template strand from +1 to +3 (Imburgio et al. Biochemistry 39(34):10419-10430 (2000). For tuning transcription levels and altering the transcription start site nucleotides to fit alternative 5′ UTRs, the teachings of Davidson et al. Pac Symp Biocomput 433-443 (2010) describe T7 promoter variants, and the methods of discovery thereof, that fulfill both of these traits.


In some embodiments, RNA segments may be connected to each other by covalent coupling. In some embodiments, an RNA ligase, e.g., T4 RNA ligase, may be used to connect two or more RNA segments to each other. When a reagent such as an RNA ligase is used, a 5′ terminus is typically linked to a 3′ terminus. In some embodiments, if two segments are connected, then there are two possible linear constructs that can be formed (i.e., (1) 5′-Segment 1-Segment 2-3′ and (2) 5′-Segment 2-Segment 1-3′). In some embodiments, intramolecular circularization can also occur. Both of these issues can be addressed, for example, by blocking one 5′ terminus or one 3′ terminus so that RNA ligase cannot ligate the terminus to another terminus. In embodiments, if a construct of 5′-Segment 1-Segment 2-3′ is desired, then placing a blocking group on either the 5′ end of Segment 1 or the 3′ end of Segment 2 may result in the formation of only the correct linear ligation product and/or prevent intramolecular circularization. Compositions and methods for the covalent connection of two nucleic acid (e.g., RNA) segments are disclosed, for example, in US20160102322A1 (incorporated herein by reference in its entirety), along with methods including the use of an RNA ligase to directionally ligate two single-stranded RNA segments to each other.


One example of an end blocker that may be used in conjunction with, for example, T4 RNA ligase, is a dideoxy terminator. T4 RNA ligase typically catalyzes the ATP-dependent ligation of phosphodiester bonds between 5′-phosphate and 3′-hydroxyl termini. In some embodiments, when T4 RNA ligase is used, suitable termini must be present on the termini being ligated. One means for blocking T4 RNA ligase on a terminus comprises failing to have the correct terminus format. Generally, termini of RNA segments with a 5-hydroxyl or a 3′-phosphate will not act as substrates for T4 RNA ligase.


Additional exemplary methods that may be used to connect RNA segments is by click chemistry (e.g., as described in U.S. Pat. Nos. 7,375,234 and 7,070,941, and US Patent Publication No. 2013/0046084, the entire disclosures of which are incorporated herein by reference). For example, one exemplary click chemistry reaction is between an alkyne group and an azide group (see FIG. 11 of US20160102322A1, which is incorporated herein by reference in its entirety). Any click reaction may potentially be used to link RNA segments (e.g., Cu-azide-alkyne, strain-promoted-azide-alkyne, staudinger ligation, tetrazine ligation, photo-induced tetrazole-alkene, thiol-ene, NHS esters, epoxides, isocyanates, and aldehyde-aminooxy). In some embodiments, ligation of RNA molecules using a click chemistry reaction is advantageous because click chemistry reactions are fast, modular, efficient, often do not produce toxic waste products, can be done with water as a solvent, and/or can be set up to be stereospecific.


In some embodiments, RNA segments may be connected using an Azide-Alkyne Huisgen Cycloaddition. reaction, which is typically a 1,3-dipolar cycloaddition between an azide and a terminal or internal alkyne to give a 1,2,3-triazole for the ligation of RNA segments. Without wishing to be bound by theory, one advantage of this ligation method may be that this reaction can initiated by the addition of required Cu(I) ions. Other exemplary mechanisms by which RNA segments may be connected include, without limitatoin, the use of halogens (F-, Br-, I-)/alkynes addition reactions, carbonyls/sulfhydryls/maleimide, and carboxyl/amine linkages. For example, one RNA molecule may be modified with thiol at 3′ (using disulfide amidite and universal support or disulfide modified support), and the other RNA molecule may be modified with acrydite at 5′ (using acrylic phosphoramidite), then the two RNA molecules can be connected by a Michael addition reaction. This strategy can also be applied to connecting multiple RNA molecules stepwise. Also provided are methods for linking more than two (e.g., three, four, five, six, etc.) RNA molecules to each other. Without wishing to be bound by theory, this may be useful when a desired RNA molecule is longer than about 40 nucleotides, e.g., such that chemical synthesis efficiency degrades, e.g., as noted in US20160102322A1 (incorporated herein by reference in its entirety).


By way of illustration, a tracrRNA is typically around 80 nucleotides in length. Such RNA molecules may be produced, for example, by processes such as in vitro transcription or chemical synthesis. In some embodiments, when chemical synthesis is used to produce such RNA molecules, they may be produced as a single synthesis product or by linking two or more synthesized RNA segments to each other. In embodiments, when three or more RNA segments are connected to each other, different methods may be used to link the individual segments together. Also, the RNA segments may be connected to each other in one pot (e.g., a container, vessel, well, tube, plate, or other receptacle), all at the same time, or in one pot at different times or in different pots at different times. In a non-limiting example, to assemble RNA Segments 1, 2 and 3 in numerical order, RNA Segments 1 and 2 may first be connected, 5′ to 3′, to each other. The reaction product may then be purified for reaction mixture components (e.g., by chromatography), then placed in a second pot, for connection of the 3′ terminus with the 5′ terminus of RNA Segment 3. The final reaction product may then be connected to the 5′ terminus of RNA Segment 3.


In another non-limiting example, RNA Segment 1 (about 30 nucleotides) is the target locus recognition sequence of a crRNA and a portion of Hairpin Region 1. RNA Segment 2 (about 35 nucleotides) contains the remainder of Hairpin Region 1 and some of the linear tracrRNA between Hairpin Region 1 and Hairpin Region 2. RNA Segment 3 (about 35 nucleotides) contains the remainder of the linear tracrRNA between Hairpin Region 1 and Hairpin Region 2 and all of Hairpin Region 2. In this example, RNA Segments 2 and 3 are linked, 5′ to 3′, using click chemistry. Further, the 5′ and 3′ end termini of the reaction product are both phosphorylated. The reaction product is then contacted with RNA Segment 1, having a 3′ terminal hydroxyl group, and T4 RNA ligase to produce a guide RNA molecule.


A number of additional linking chemistries may be used to connect RNA segments according to method of the invention. Some of these chemistries are set out in Table 6 of US20160102322A1, which is incorporated herein by reference in its entirety.


Template Nucleic Acid Composition


In some embodiments, the template nucleic acid is a template RNA. In some embodiments, the template RNA comprises one or more modified nucleotides. For example, in some embodiments, the template RNA comprises one or more deoxyribonucleotides. In some embodiments, regions of the template RNA are replaced by DNA nucleotides, e.g., to enhance stability of the molecule. For example, the 3′ end of the template may comprise DNA nucleotides, while the rest of the template comprises RNA nucleotides that can be reverse transcribed. For instance, in some embodiments, the heterologous object sequence is primarily or wholly made up of RNA nucleotides (e.g., at least 90%, 95%, 98%, or 99% RNA nucleotides). In some embodiments, one or both of the 3′ UTR and the 3′ target homology domain are primarily or wholly made up of DNA nucleotides (e.g., at least 90%, 95%, 98%, or 99% DNA nucleotides). In other embodiments, the template region for writing into the genome may comprise DNA nucleotides. In some embodiments, the DNA nucleotides in the template are copied into the genome by a domain capable of DNA-dependent DNA polymerase activity. In some embodiments, the DNA-dependent DNA polymerase activity is provided by a DNA polymerase domain in the polypeptide. In some embodiments, the DNA-dependent DNA polymerase activity is provided by a reverse transcriptase domain that is also capable of DNA-dependent DNA polymerization, e.g., second strand synthesis. In some embodiments, the DNA-dependent DNA polymerase activity is provided by a DNA polymerase. In some embodiments, the DNA-dependent DNA polymerase activity provided by a DNA polymerase domain in the polypeptide is not capable of reverse transcription activity. In some embodiments, the template molecule is composed of only DNA nucleotides. In some embodiments, the DNA template is polymerized into the genome by a DNA polymerase. In some embodiments, the template composed of DNA nucleotides comprises modified DNA nucleotides. In some embodiments, the template composed of DNA nucleotides comprises a modified backbone.


The nucleotides comprising the template of the Gene Writer™ system can be natural or modified bases, or a combination thereof. For example, the template may contain pseudouridine, dihydrouridine, inosine, 7-methylguanosine, or other modified bases. In some embodiments, the template may contain locked nucleic acid nucleotides. In some embodiments, the modified bases used in the template do not inhibit the reverse transcription of the template. In some embodiments, the modified bases used in the template may improve reverse transcription, e.g., specificity or fidelity.


Additional Functional Characteristics for Gene Writers™

A Gene Writer as described herein may, in some instances, be characterized by one or more functional measurements or characteristics. In some embodiments, the DNA binding domain has one or more of the functional characteristics described below. In some embodiments, the RNA binding domain has one or more of the functional characteristics described below. In some embodiments, the endonuclease domain has one or more of the functional characteristics described below. In some embodiments, the reverse transcriptase domain has one or more of the functional characteristics described below. In some embodiments, the template (e.g., template RNA) has one or more of the functional characteristics described below. In some embodiments, the target site bound by the Gene Writer has one or more of the functional characteristics described below.


Gene Writer Polypeptide


DNA Binding Domain


In some embodiments, the DNA binding domain is capable of binding to a target sequence (e.g., a dsDNA target sequence) with greater affinity than a reference DNA binding domain. In some embodiments, the reference DNA binding domain is a DNA binding domain from R2_BM of B. mori. In some embodiments, the DNA binding domain is capable of binding to a target sequence (e.g., a dsDNA target sequence) with an affinity between 100 pM-10 nM (e.g., between 100 pM-1 nM or 1 nM-10 nM).


In some embodiments, the affinity of a DNA binding domain for its target sequence (e.g., dsDNA target sequence) is measured in vitro, e.g., by thermophoresis, e.g., as described in Asmari et al. Methods 146:107-119 (2018) (incorporated by reference herein in its entirety).


In embodiments, the DNA binding domain is capable of binding to its target sequence (e.g., dsDNA target sequence), e.g, with an affinity between 100 pM-10 nM (e.g., between 100 pM-1 nM or 1 nM-10 nM) in the presence of a molar excess of scrambled sequence competitor dsDNA, e.g., of about 100-fold molar excess.


In some embodiments, the DNA binding domain is found associated with its target sequence (e.g., dsDNA target sequence) more frequently than any other sequence in the genome of a target cell, e.g., human target cell, e.g., as measured by ChIP-seq (e.g., in HEK293T cells), e.g., as described in He and Pu (2010) Curr. Protoc Mol Biol Chapter 21 (incorporated herein by reference in its entirety). In some embodiments, the DNA binding domain is found associated with its target sequence (e.g., dsDNA target sequence) at least about 5-fold or 10-fold, more frequently than any other sequence in the genome of a target cell, e.g., as measured by ChIP-seq (e.g., in HEK293T cells), e.g., as described in He and Pu (2010), supra.


RNA Binding Domain


In some embodiments, the RNA binding domain is capable of binding to a template RNA with greater affinity than a reference RNA binding domain. In some embodiments, the reference RNA binding domain is an RNA binding domain from R2_BM of B. mori. In some embodiments, the RNA binding domain is capable of binding to a template RNA with an affinity between 100 pM-10 nM (e.g., between 100 pM-1 nM or 1 nM-10 nM). In some embodiments, the affinity of a RNA binding domain for its template RNA is measured in vitro, e.g., by thermophoresis, e.g., as described in Asmari et al. Methods 146:107-119 (2018) (incorporated by reference herein in its entirety). In some embodiments, the affinity of a RNA binding domain for its template RNA is measured in cells (e.g., by FRET or CLIP-Seq).


In some embodiments, the RNA binding domain is associated with the template RNA in vitro at a frequency at least about 5-fold or 10-fold higher than with a scrambled RNA. In some embodiments, the frequency of association between the RNA binding domain and the template RNA or scrambled RNA is measured by CLIP-seq, e.g., as described in Lin and Miles (2019) Nucleic Acids Res 47(11):5490-5501 (incorporated by reference herein in its entirety). In some embodiments, the RNA binding domain is associated with the template RNA in cells (e.g., in HEK293T cells) at a frequency at least about 5-fold or 10-fold higher than with a scrambled RNA. In some embodiments, the frequency of association between the RNA binding domain and the template RNA or scrambled RNA is measured by CLIP-seq, e.g., as described in Lin and Miles (2019), supra.


Endonuclease Domain


In some embodiments, the endonuclease domain is associated with the target dsDNA in vitro at a frequency at least about 5-fold or 10-fold higher than with a scrambled dsDNA. In some embodiments, the endonuclease domain is associated with the target dsDNA in vitro at a frequency at least about 5-fold or 10-fold higher than with a scrambled dsDNA, e.g., in a cell (e.g., a HEK293T cell). In some embodiments, the frequency of association between the endonuclease domain and the target DNA or scrambled DNA is measured by ChIP-seq, e.g., as described in He and Pu (2010) Curr. Protoc Mol Biol Chapter 21 (incorporated by reference herein in its entirety).


In some embodiments, the endonuclease domain can catalyze the formation of a nick at a target sequence, e.g., to an increase of at least about 5-fold or 10-fold relative to a non-target sequence (e.g., relative to any other genomic sequence in the genome of the target cell). In some embodiments, the level of nick formation is determined using NickSeq, e.g., as described in Elacqua et al. (2019) bioRxiv doi.org/10.1101/867937 (incorporated herein by reference in its entirety).


In some embodiments, the endonuclease domain is capable of nicking DNA in vitro. In embodiments, the nick results in an exposed base. In embodiments, the exposed base can be detected using a nuclease sensitivity assay, e.g., as described in Chaudhry and Weinfeld (1995) Nucleic Acids Res 23(19):3805-3809 (incorporated by reference herein in its entirety). In embodiments, the level of exposed bases (e.g., detected by the nuclease sensitivity assay) is increased by at least 10%, 50%, or more relative to a reference endonuclease domain. In some embodiments, the reference endonuclease domain is an endonuclease domain from R2_BM of B. mori.


In some embodiments, the endonuclease domain is capable of nicking DNA in a cell. In embodiments, the endonuclease domain is capable of nicking DNA in a HEK293T cell. In embodiments, an unrepaired nick that undergoes replication in the absence of Rad51 results in increased NHEJ rates at the site of the nick, which can be detected, e.g., by using a Rad51 inhibition assay, e.g., as described in Bothmer et al. (2017) Nat Commun 8:13905 (incorporated by reference herein in its entirety). In embodiments, NHEJ rates are increased above 0-5%. In embodiments, NHEJ rates are increased to 20-70% (e.g., between 30%-60% or 40-50%), e.g., upon Rad51 inhibition.


In some embodiments, the endonuclease domain releases the target after cleavage. In some embodiments, release of the target is indicated indirectly by assessing for multiple turnovers by the enzyme, e.g., as described in Yourik at al. RNA 25(1):35-44 (2019) (incorporated herein by reference in its entirety) and shown in FIG. 2. In some embodiments, the kexp of an endonuclease domain is 1×10−3-1×10−5 min−1 as measured by such methods.


In some embodiments, the endonuclease domain has a catalytic efficiency (kcat/Km) greater than about 1×108 s−1 M−1 in vitro. In embodiments, the endonuclease domain has a catalytic efficiency greater than about 1×105, 1×106, 1×107, or 1×108, s−1 M−1 in vitro. In embodiments, catalytic efficiency is determined as described in Chen et al. (2018) Science 360(6387):436-439 (incorporated herein by reference in its entirety). In some embodiments, the endonuclease domain has a catalytic efficiency (kcat/Km) greater than about 1×108 s−1 M−1 in cells. In embodiments, the endonuclease domain has a catalytic efficiency greater than about 1×105, 1×106, 1×107, or 1×108 s−1 M−1 in cells.


Reverse Transcriptase Domain


In some embodiments, the reverse transcriptase domain has a lower probability of premature termination rate (Poff) in vitro relative to a reference reverse transcriptase domain. In some embodiments, the reference reverse transcriptase domain is a reverse transcriptase domain from R2_BM of B. mori or a viral reverse transcriptase domain, e.g., the RT domain from M-MLV.


In some embodiments, the reverse transcriptase domain has a lower probability of premature termination rate (Poff) in vitro of less than about 5×10−3/nt, 5×10−4/nt, or 5×10−6/nt, e.g., as measured on a 1094 nt RNA. In embodiments, the in vitro premature termination rate is determined as described in Bibillo and Eickbush (2002) J Biol Chem 277(38):34836-34845 (incorporated by reference herein its entirety).


In some embodiments, the reverse transcriptase domain is able to complete at least about 30% or 50% of integrations in cells. The percent of complete integrations can be measured by dividing the number of substantially full-length integration events (e.g., genomic sites that comprise at least 98% of the expected integrated sequence) by the number of total (including substantially full-length and partial) integration events in a population of cells. In embodiments, the integrations in cells is determined (e.g., across the integration site) using long-read amplicon sequencing, e.g., as described in Karst et al. (2020) bioRxiv doi.org/10.1101/645903 (incorporated by reference herein its in entirety).


In embodiments, quantifying integrations in cells comprises counting the fraction of integrations that contain at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the DNA sequence corresponding to the template RNA (e.g., a template RNA having a length of at least 0.05, 0.1, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 3, 4, or 5 kb, e.g., a length between 0.5-0.6, 0.6-0.7, 0.7-0.8, 0.8-0.9, 1.0-1.2, 1.2-1.4, 1.4-1.6, 1.6-1.8, 1.8-2.0, 2-3, 3-4, or 4-5 kb).


In some embodiments, the reverse transcriptase domain is capable of polymerizing dNTPs in vitro. In embodiments, the reverse transciptase domain is capable of polymerizing dNTPs in vitro at a rate between 0.1-50 nt/sec (e.g., between 0.1-1, 1-10, or 10-50 nt/sec). In embodiments, polymerization of dNTPs by the reverse transcriptase domain is measured by a single-molecule assay, e.g., as described in Schwartz and Quake (2009) PNAS 106(48):20294-20299 (incorporated by reference in its entirety).


In some embodiments, the reverse transcriptase domain has an in vitro error rate (e.g., misincorporation of nucleotides) of between 1×10−3-1×10−4 or 1×10−4-1×10−5 substitutions/nt, e.g., as described in Yasukawa et al. (2017) Biochem Biophys Res Commun 492(2):147-153 (incorporated herein by reference in its entirety). In some embodiments, the reverse transcriptase domain has an error rate (e.g., misincorporation of nucleotides) in cells (e.g., HEK293T cells) of between 1×10−3-1×10−4 or 1×10−4-1×10−5 substitutions/nt, e.g., by long-read amplicon sequencing, e.g., as described in Karst et al. (2020) bioRxiv doi.org/10.1101/645903 (incorporated by reference herein in its entirety).


In some embodiments, the reverse transcriptase domain is capable of performing reverse transcription of a target RNA in vitro. In some embodiments, the reverse transcriptase requires a primer of at least 3 nt to initiate reverse transcription of a template. In some embodiments, reverse transcription of the target RNA is determined by detection of cDNA from the target RNA (e.g., when provided with a ssDNA primer, e.g., which anneals to the target with at least 3, 4, 5, 6, 7, 8, 9, or 10 nt at the 3′ end), e.g., as described in Bibillo and Eickbush (2002) J Biol Chem 277(38):34836-34845 (incorporated herein by reference in its entirety).


In some embodiments, the reverse transcriptase domain performs reverse transcription at least 5 or 10 times more efficiently (e.g., by cDNA production), e.g., when converting its RNA template to cDNA, for example, as compared to an RNA template lacking the protein binding motif (e.g., a 3′ UTR). In embodiments, efficiency of reverse transcription is measured as described in Yasukawa et al. (2017) Biochem Biophys Res Commun 492(2):147-153 (incorporated by reference herein in its entirety).


In some embodiments, the reverse transcriptase domain specifically binds a specific RNA template with higher frequency (e.g., about 5 or 10-fold higher frequency) than any endogenous cellular RNA, e.g., when expressed in cells (e.g., HEK293T cells). In embodiments, frequency of specific binding between the reverse transcriptase domain and the template RNA are measured by CLIP-seq, e.g., as described in Lin and Miles (2019) Nucleic Acids Res 47(11):5490-5501 (incorporated herein by reference in its entirety).


In some embodiments, a reverse transcriptase domain may comprise a mutation, e.g., as listed in Table 45. In embodiments, the mutation modifies, e.g., increases the stability and functionality of the RT domain. In some embodiments, the mutation modifies, e.g., increases processivity and template affinity of the RT domain. In some embodiments, the mutated RT domain may show at least 5 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 80 fold, at least 100 fold increase to processivity compared to an unmutated RT domain. In embodiments, a mutated RT domain may show at least at least 5 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 45 fold, at least 50 fold, at least 55 fold, at least 60 fold, at least 65 fold, at least 70 fold, at least 80 fold, at least 100 fold increase in template affinity compared to an unmutated RT domain. In some embodiments, a mutant RT domain may comprise one or more mutations selected from D200N/T330P/L603W, T306K, W313F, L139P, E607K.


Table 45 discloses mutations improve the properties of various reverse transcriptases. Core mutations expected to be the most impactful were applied across groups of retroviruses. Conservation of sequence across a group of viruses at one of these core mutations led to the installation of the mutation across that group (see Example 33, FIGS. 36 A and B). Sequence positions refer to the positions in MMLV RT. In some embodiments, a RT domain described herein comprises a mutation as described in Table 45.









TABLE 45







List of exemplary RT domain mutations














Group
L139
D200
T306
W313
T330
L603
E607





Gamma

D200N
T306K
W313F
T330P
L603W



Epsilon

D200N
T306K
W313F
T330P
L603W



Delta
L139P
D200N
X
X
T330P
L603W*
X


Beta
L139P
X
X
X
T330P
X
X


Spuma

D200N
T306K
X
T330P
L603W









Cas-RT Fusions


In some embodiments, a GeneWriter polypeptide comprises a RT domain fused to a Cas molecule. In some embodiments, the Cas molecule is the DBD and/or the endonuclease domain of the GeneWriter polypeptide. In some embodiments, the an RT domain comprises Cas9. In some embodiments, the Cas9 may comprise a mutation, e.g., a disclosed in Table 40A. Table 46 discloses a list of exemplary Cas-RT fusion proteins.


In some embodiments, a Cas molecule in a GeneWriter polypeptide has a similar activity to an otherwise similar Cas molecule that is not fused to a RT domain. In some embodiments, the activity is at least 40%, 50%, 60%, 70%, 80%, or 90% of that of the otherwise similar Cas molecule. In some embodiments, the Cas molecule in the GeneWriter polypeptide may have an indel formation activity at least 40%, 50%, 60%, 70%, 80%, or 90% of that of an otherwise similar Cas molecule that is not fused to a RT domain, e.g., in an assay according to Example 32.


In some embodiments, a GeneWriter polypeptide comprises an amino acid sequence according to Table 46 below, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto. In some embodiments, a nucleic acid encoding a GeneWriter polypeptide comprises a nucleic acid sequence according to Table 47, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.









TABLE 46







List of exemplary Gene Writer polypeptides comprising Cas-RT fusions















RT





Cas

retroviral
RT source



Name
domain
Linker
source
polypeptide
Gene Writer polypeptide sequence





Cas-
Cas9
SGGSS
Moloney
P03355
MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVG


RT(MMLV)
(N863A)
GGSSG
murine

WAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALL




SETPG
leukemia

FDSGETAEATRLKRTARRRYTRRKNRICYLQEIFS




TSESA
virus

NEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI




TPESS
(MMLV

VDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLA




GGSSG
or

LAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTY




GSS
MLVMS)

NQLFEENPINASGVDAKAILSARLSKSRRLENLIA




(SEQ


QLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDA




ID NO:


KLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLS




1589)


DAILLSDILRVNTEITKAPLSASMIKRYDEHHQDL







TLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGA







SQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQ







RTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNR







EKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEET







ITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKV







LPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSG







EQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS







VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEEN







EDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKV







MKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDF







LKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQG







DSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMG







RHKPENIVIEMARENQTTQKGQKNSRERMKRIEEG







IKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDM







YVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVL







TRSDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLI







TQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQI







TKHVAQILDSRMNTKYDENDKLIREVKVITLKSKL







VSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTAL







IKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKA







TAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNG







ETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQT







GGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSP







TVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERS







SFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELE







NGRKRMLASAGELQKGNELALPSKYVNFLYLASHY







EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFS







KRVILADANLDKVLSAYNKHRDKPIREQAENIIHL







FTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATL







IHQSITGLYETRIDLSQLGGDSGGSSGGSSGSETP







GTSESATPESSGGSSGGSSTLNIEDEYRLHETSKE







PDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLIIP







LKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGI







LVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKR







VEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFF







CLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQG







FKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDL







LLAATSELDCQQGTRALLQTLGNLGYRASAKKAQI







CQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKT







PRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPG







TLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPF







ELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDP







VAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILA







PHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQ







FGPVVALNPATLLPLPEEGLQHNCLDILAEAHGTR







PDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVT







TETEVIWAKALPAGTSAQRAELIALTQALKMAEGK







KLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEI







KNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEA







RGNRMADQAARKAAITETPDTSTLLIENSSPSGGS







KRTADGSEFEPKKKRKV (SEQ ID NO: 3560)





Cas-
Cas9
SGGSS
Porcine
Q4VFZ2
MAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVGWA


RT(PERV)
(N863A)
GGSSG
endogenous

VITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD




SETPG
retrovirus

SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNE




TSESA
(PERV)

MAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVD




TPESS


EVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALA




GGSSG


HMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQ




GSS


LFEENPINASGVDAKAILSARLSKSRRLENLIAQL




(SEQ


PGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKL




ID NO:


QLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDA




1589)


ILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTL







LKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQ







EEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT







FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREK







IEKILTFRIPYYVGPLARGNSRFAWMTRKSEETIT







PWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP







KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQ







KKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVE







ISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENED







ILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMK







QLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLK







SDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDS







LHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH







KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIK







ELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYV







DQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTR







SDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ







RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITK







HVAQILDSRMNTKYDENDKLIREVKVITLKSKLVS







DFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIK







KYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATA







KYFFYSNIMNFFKTEITLANGEIRKRPLIETNGET







GEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG







FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV







AYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF







EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENG







RKRMLASAGELQKGNELALPSKYVNFLYLASHYEK







LKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKR







VILADANLDKVLSAYNKHRDKPIREQAENIIHLFT







LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIH







QSITGLYETRIDLSQLGGDSGGSSGGSSGSETPGT







SESATPESSGGSSGGSSLDDEYRLYSPLVKPDQNI







QFWLEQFPQAWAETAGMGLAKQVPPQVIQLKASAT







PVSVRQYPLSKEAQEGIRPHVQRLIQQGILVPVQS







PWNTPLLPVRKPGTNDYRPVQDLREVNKRVQDIHP







TVPNPYNLLCALPPQRSWYTVLDLKDAFFCLRLHP







TSQPLFAFEWRDPGTGRTGQLTWTRLPQGFKNSPT







IFNEALHRDLANFRIQHPQVTLLQYVDDLLLAGAT







KQDCLEGTKALLLELSDLGYRASAKKAQICRREVT







YLGYSLRDGQRWLTEARKKTVVQIPAPTTAKQVRE







FLGKAGFCRLFIPGFATLAAPLYPLTKPKGEFSWA







PEHQKAFDAIKKALLSAPALALPDVTKPFTLYVDE







RKGVARGVLTQTLGPWRRPVAYLSKKLDPVASGWP







VCLKAIAAVAILVKDADKLTLGQNITVIAPHALEN







IVRQPPDRWMTNARMTHYQSLLLTERVTFAPPAAL







NPATLLPEETDEPVTHDCHQLLIEETGVRKDLTDI







PLTGEVLTWFTDGSSYVVEGKRMAGAAVVDGTRTI







WASSLPEGTSAQKAELMALTQALRLAEGKSINIYT







DSRYAFATAHVHGAIYKQRGWLTSAGREIKNKEEI







LSLLEALHLPKRLAIIHCPGHQKAKDPISRGNQMA







DRVAKQAAQGVNLLP (SEQ ID NO: 3561)





Cas-
Cas9
SGGSS
Murine
Q7SVK7
MAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVGWA


RT(MLVB
(N863A)
GGSSG
leukemia

VITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD


M)

SETPG
virus

SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNE




TSESA
(MLVBM)

MAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVD




TPESS


EVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALA




GGSSG


HMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQ




GSS


LFEENPINASGVDAKAILSARLSKSRRLENLIAQL




(SEQ


PGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKL




ID NO:


QLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDA




1589)


ILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTL







LKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQ







EEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT







FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREK







IEKILTFRIPYYVGPLARGNSRFAWMTRKSEETIT







PWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP







KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQ







KKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVE







ISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENED







ILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMK







QLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLK







SDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDS







LHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH







KPENIVIEMGRDMYVDQELDINRLSDYDVDHIVPQ







SFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKM







KNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKA







GFIKRQLVETRQITKHVAQILDSRMNTKYDENDKL







IREVKVITLKSKLVSDFRKDFQFYKVREINNYHHA







HDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVR







KMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLAN







GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSM







PQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKK







DWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKS







VKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDL







IIKLPKYSLFELENGRKRMLASAGELQKGNELALP







SKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKH







YLDEIIEQISEFSKRVILADANLDKVLSAYNKHRD







KPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRK







RYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDS







GGSSGGSSGSETPGTSESATPESSGGSSGGSSLGI







EDEYRLHETSTEPDVSLGSTWLSDFPQAWAETGGM







GLAVRQAPLIIPLKATSTPVSIQQYPMSHEARLGI







KPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDY







RPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQ







WYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPGMGI







SGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQH







PDLILLQYVDDILLAATSELDCQQGTRALLQTLGD







LGYRASAKKAQICQKQVKYLGYLLREGQRWLTEAR







KETVMGQPVPKTPRQLREFLGKAGFCRLFIPGFAE







MAAPLYPLTKPGTLFSWGPDQQKAYQEIKQALLTA







PALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWR







RPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAG







KLTMGQPLVILAPHAVEALVKQPPDRWLSNARMTH







YQAMLLDTDRVQFGPVVALNPATLLPLPEEGAPHD







CLEILAETHGTRPDLTDQPIPDADHTWYTDGSSFL







QEGQRKAGAAVTTETEVIWAGALPAGTSAQRAELI







ALTQALKMAEGKRLNVYTDSRYAFATAHIHGEIYR







RRGWLTSEGREIKNKSEILALLKALFLPKRLSIIH







CLGHQKGDSAEARGNRLADQAAREAAIKTPPDTST







LLI (SEQ ID NO:3562)





Cas-
Cas9
SGGSS
Mouse
P03365
MAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVGWA


RT
(N863A)
GGSSG
mammary

VITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD


(MMTVB)

SETPG
tumor

SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNE




TSESA
virus

MAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVD




TPESS
(MMTVB

EVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALA




GGSSG


HMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQ




GSS


LFEENPINASGVDAKAILSARLSKSRRLENLIAQL




(SEQ


PGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKL




ID NO:


QLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDA




1589)


ILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTL







LKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQ







EEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT







FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREK







IEKILTFRIPYYVGPLARGNSRFAWMTRKSEETIT







PWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP







KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQ







KKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVE







ISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENED







ILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMK







QLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLK







SDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDS







LHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH







KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIK







ELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYV







DQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTR







SDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ







RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITK







HVAQILDSRMNTKYDENDKLIREVKVITLKSKLVS







DFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIK







KYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATA







KYFFYSNIMNFFKTEITLANGEIRKRPLIETNGET







GEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG







FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV







AYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF







EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENG







RKRMLASAGELQKGNELALPSKYVNFLYLASHYEK







LKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKR







VILADANLDKVLSAYNKHRDKPIREQAENIIHLFT







LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIH







QSITGLYETRIDLSQLGGDSGGSSGGSSGSETPGT







SESATPESSGGSSGGSSVQEISDSRPMLHIYLNGR







RFLGLLDTGADKTCIAGRDWPANWPIHQTESSLQG







LGMACGVARSSQPLRWQHEDKSGIIHPFVIPTLPF







TLWGRDIMKDIKVRLMTDSPDDSQDLMIGAIESNL







FADQISWKSDQPVWLNQWPLKQEKLQALQQLVTEQ







LQLGHLEESNSPWNTPVFVIKKKSGKWRLLQDLRA







VNATMHDMGALQPGLPSPVAVPKGWEIIIIDLQDC







FFNIKLHPEDCKRFAFSVPSPNFKRPYQRFQWKVL







PQGMKNSPTLCQKFVDKAILTVRDKYQDSYIVHYM







DDILLAHPSRSIVDEILTSMIQALNKHGLVVSTEK







IQKYDNLKYLGTHIQGDSVSYQKLQIRTDKLRTLN







DFQKLLGNINWIRPFLKLTTGELKPLFEILNGDSN







PISTRKLTPEACKALQLMNERLSTARVKRLDLSQP







WSLCILKTEYTPTACLWQDGVVEWIHLPHISPKVI







TPYDIFCTQLIIKGRHRSKELFSKDPDYIVVPYTK







VQFDLLLQEKEDWPISLLGFLGEVHFHLPKDPLLT







FTLQTAIIFPHMTSTTPLEKGIVIFTDGSANGRSV







TYIQGREPIIKENTQNTAQQAEIVAVITAFEEVSQ







PFNLYTDSKYVTGLFPEIETATLSPRTKIYTELKH







LQRLIHKRQEKFYIGHIRGHTGLPGPLAQGNAYAD







SLTRILTA(SEQ ID NO: 3563)





Cas-
Cas9
SGGSS
Mason-
P07572
MAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVGWA


RT
(N863A)
GGSSG
Pfizer

VITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFD


(MPMV)

SETPG
monkey

SGETAEATRLKRTARRRYTRRKNRICYLQEIFSNE




TSESA
virus

MAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVD




TPESS
(MPMV)

EVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALA




GGSSG


HMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQ




GSS


LFEENPINASGVDAKAILSARLSKSRRLENLIAQL




(SEQ


PGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKL




ID NO:


QLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDA




1589)


ILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTL







LKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQ







EEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRT







FDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREK







IEKILTFRIPYYVGPLARGNSRFAWMTRKSEETIT







PWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLP







KHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQ







KKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVE







ISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENED







ILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMK







QLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLK







SDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDS







LHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH







KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIK







ELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYV







DQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTR







SDKARGKSDNVPSEEVVKKMKNYWRQLLNAKLITQ







RKFDNLTKAERGGLSELDKAGFIKRQLVETRQITK







HVAQILDSRMNTKYDENDKLIREVKVITLKSKLVS







DFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIK







KYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATA







KYFFYSNIMNFFKTEITLANGEIRKRPLIETNGET







GEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG







FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTV







AYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF







EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENG







RKRMLASAGELQKGNELALPSKYVNFLYLASHYEK







LKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKR







VILADANLDKVLSAYNKHRDKPIREQAENIIHLFT







LTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIH







QSITGLYETRIDLSQLGGDSGGSSGGSSGSETPGT







SESATPESSGGSSGGSSTAAIDILAPQQCAEPITW







KSDEPVWVDQWPLTNDKLAAAQQLVQEQLEAGHIT







ESSSPWNTPIFVIKKKSGKWRLLQDLRAVNATMVL







MGALQPGLPSPVAIPQGYLKIIIDLKDCFFSIPLH







PSDQKRFAFSLPSTNFKEPMQRFQWKVLPQGMANS







PTLCQKYVATAIHKVRHAWKQMYIIHYMDDILIAG







KDGQQVLQCFDQLKQELTAAGLHIAPEKVQLQDPY







TYLGFELNGPKITNQKAVIRKDKLQTLNDFQKLLG







DINWLRPYLKLTTGDLKPLFDTLKGDSDPNSHRSL







SKEALASLEKVETAIAEQFVTHINYSLPLIFLIFN







TALTPTGLFWQDNPIMWIHLPASPKKVLLPYYDAI







ADLIILGRDHSKKYFGIEPSTIIQPYSKSQIDWLM







QNTEMWPIACASFVGILDNHYPPNKLIQFCKLHTF







VFPQIISKTPLNNALLVFTDGSSTGMAAYTLTDTT







IKFQTNLNSAQLVELQALIAVLSAFPNQPLNIYTD







SAYLAHSIPLLETVAQIKHISETAKLFLQCQQLIY







NRSIPFYIGHVRAHSGLPGPIAQGNQRADLATKIV







ASNINTN (SEQ ID NO: 3564)
















TABLE 47







Exemplary Gene Writer polypeptide


coding mRNAs sequences











Name
mRNA (5′ to 3′)
Tail






Cas9-
AGGAAAUAAGAGAGAAAAGAAGAGU
(A)80



RT(MMLV)
AAGAAGAAAUAUAAGAGCCACCAUG
(SEQ




AAACGGACAGCCGACGGAAGCGAGU
ID




UCGAGUCACCAAAGAAGAAGCGGAA
NO:




AGUCGACAAGAAGUACAGCAUCGGC
3666)




CUGGACAUCGGCACCAACUCUGUGG





GCUGGGCCGUGAUCACCGACGAGUA





CAAGGUGCCCAGCAAGAAAUUCAAG





GUGCUGGGCAACACCGACCGGCACA





GCAUCAAGAAGAACCUGAUCGGAGC





CCUGCUGUUCGACAGCGGCGAAACA





GCCGAGGCCACCCGGCUGAAGAGAA





CCGCCAGAAGAAGAUACACCAGACG





GAAGAACCGGAUCUGCUAUCUGCAA





GAGAUCUUCAGCAACGAGAUGGCCA





AGGUGGACGACAGCUUCUUCCACAG





ACUGGAAGAGUCCUUCCUGGUGGAA





GAGGAUAAGAAGCACGAGCGGCACC





CCAUCUUCGGCAACAUCGUGGACGA





GGUGGCCUACCACGAGAAGUACCCC





ACCAUCUACCACCUGAGAAAGAAAC





UGGUGGACAGCACCGACAAGGCCGA





CCUGCGGCUGAUCUAUCUGGCCCUG





GCCCACAUGAUCAAGUUCCGGGGCC





ACUUCCUGAUCGAGGGCGACCUGAA





CCCCGACAACAGCGACGUGGACAAG





CUGUUCAUCCAGCUGGUGCAGACCU





ACAACCAGCUGUUCGAGGAAAACCC





CAUCAACGCCAGCGGCGUGGACGCC





AAGGCCAUCCUGUCUGCCAGACUGA





GCAAGAGCAGACGGCUGGAAAAUCU





GAUCGCCCAGCUGCCCGGCGAGAAG





AAGAAUGGCCUGUUCGGAAACCUGA





UUGCCCUGAGCCUGGGCCUGACCCC





CAACUUCAAGAGCAACUUCGACCUG





GCCGAGGAUGCCAAACUGCAGCUGA





GCAAGGACACCUACGACGACGACCU





GGACAACCUGCUGGCCCAGAUCGGC





GACCAGUACGCCGACCUGUUUCUGG





CCGCCAAGAACCUGUCCGACGCCAU





CCUGCUGAGCGACAUCCUGAGAGUG





AACACCGAGAUCACCAAGGCCCCCC





UGAGCGCCUCUAUGAUCAAGAGAUA





CGACGAGCACCACCAGGACCUGACC





CUGCUGAAAGCUCUCGUGCGGCAGC





AGCUGCCUGAGAAGUACAAAGAGAU





UUUCUUCGACCAGAGCAAGAACGGC





UACGCCGGCUACAUUGACGGCGGAG





CCAGCCAGGAAGAGUUCUACAAGUU





CAUCAAGCCCAUCCUGGAAAAGAUG





GACGGCACCGAGGAACUGCUCGUGA





AGCUGAACAGAGAGGACCUGCUGCG





GAAGCAGCGGACCUUCGACAACGGC





AGCAUCCCCCACCAGAUCCACCUGG





GAGAGCUGCACGCCAUUCUGCGGCG





GCAGGAAGAUUUUUACCCAUUCCUG





AAGGACAACCGGGAAAAGAUCGAGA





AGAUCCUGACCUUCCGCAUCCCCUA





CUACGUGGGCCCUCUGGCCAGGGGA





AACAGCAGAUUCGCCUGGAUGACCA





GAAAGAGCGAGGAAACCAUCACCCC





CUGGAACUUCGAGGAAGUGGUGGAC





AAGGGCGCUUCCGCCCAGAGCUUCA





UCGAGCGGAUGACCAACUUCGAUAA





GAACCUGCCCAACGAGAAGGUGCUG





CCCAAGCACAGCCUGCUGUACGAGU





ACUUCACCGUGUAUAACGAGCUGAC





CAAAGUGAAAUACGUGACCGAGGGA





AUGAGAAAGCCCGCCUUCCUGAGCG





GCGAGCAGAAAAAGGCCAUCGUGGA





CCUGCUGUUCAAGACCAACCGGAAA





GUGACCGUGAAGCAGCUGAAAGAGG





ACUACUUCAAGAAAAUCGAGUGCUU





CGACUCCGUGGAAAUCUCCGGCGUG





GAAGAUCGGUUCAACGCCUCCCUGG





GCACAUACCACGAUCUGCUGAAAAU





UAUCAAGGACAAGGACUUCCUGGAC





AAUGAGGAAAACGAGGACAUUCUGG





AAGAUAUCGUGCUGACCCUGACACU





GUUUGAGGACAGAGAGAUGAUCGAG





GAACGGCUGAAAACCUAUGCCCACC





UGUUCGACGACAAAGUGAUGAAGCA





GCUGAAGCGGCGGAGAUACACCGGC





UGGGGCAGGCUGAGCCGGAAGCUGA





UCAACGGCAUCCGGGACAAGCAGUC





CGGCAAGACAAUCCUGGAUUUCCUG





AAGUCCGACGGCUUCGCCAACAGAA





ACUUCAUGCAGCUGAUCCACGACGA





CAGCCUGACCUUUAAAGAGGACAUC





CAGAAAGCCCAGGUGUCCGGCCAGG





GCGAUAGCCUGCACGAGCACAUUGC





CAAUCUGGCCGGCAGCCCCGCCAUU





AAGAAGGGCAUCCUGCAGACAGUGA





AGGUGGUGGACGAGCUCGUGAAAGU





GAUGGGCCGGCACAAGCCCGAGAAC





AUCGUGAUCGAAAUGGCCAGAGAGA





ACCAGACCACCCAGAAGGGACAGAA





GAACAGCCGCGAGAGAAUGAAGCGG





AUCGAAGAGGGCAUCAAAGAGCUGG





GCAGCCAGAUCCUGAAAGAACACCC





CGUGGAAAACACCCAGCUGCAGAAC





GAGAAGCUGUACCUGUACUACCUGC





AGAAUGGGCGGGAUAUGUACGUGGA





CCAGGAACUGGACAUCAACCGGCUG





UCCGACUACGAUGUGGACCAUAUCG





UGCCUCAGAGCUUUCUGAAGGACGA





CUCCAUCGACAACAAGGUGCUGACC





AGAAGCGACAAGGCCCGGGGCAAGA





GCGACAACGUGCCCUCCGAAGAGGU





CGUGAAGAAGAUGAAGAACUACUGG





CGGCAGCUGCUGAACGCCAAGCUGA





UUACCCAGAGAAAGUUCGACAAUCU





GACCAAGGCCGAGAGAGGCGGCCUG





AGCGAACUGGAUAAGGCCGGCUUCA





UCAAGAGACAGCUGGUGGAAACCCG





GCAGAUCACAAAGCACGUGGCACAG





AUCCUGGACUCCCGGAUGAACACUA





AGUACGACGAGAAUGACAAGCUGAU





CCGGGAAGUGAAAGUGAUCACCCUG





AAGUCCAAGCUGGUGUCCGAUUUCC





GGAAGGAUUUCCAGUUUUACAAAGU





GCGCGAGAUCAACAACUACCACCAC





GCCCACGACGCCUACCUGAACGCCG





UCGUGGGAACCGCCCUGAUCAAAAA





GUACCCUAAGCUGGAAAGCGAGUUC





GUGUACGGCGACUACAAGGUGUACG





ACGUGCGGAAGAUGAUCGCCAAGAG





CGAGCAGGAAAUCGGCAAGGCUACC





GCCAAGUACUUCUUCUACAGCAACA





UCAUGAACUUUUUCAAGACCGAGAU





UACCCUGGCCAACGGCGAGAUCCGG





AAGCGGCCUCUGAUCGAGACAAACG





GCGAAACCGGGGAGAUCGUGUGGGA





UAAGGGCCGGGAUUUUGCCACCGUG





CGGAAAGUGCUGAGCAUGCCCCAAG





UGAAUAUCGUGAAAAAGACCGAGGU





GCAGACAGGCGGCUUCAGCAAAGAG





UCUAUCCUGCCCAAGAGGAACAGCG





AUAAGCUGAUCGCCAGAAAGAAGGA





CUGGGACCCUAAGAAGUACGGCGGC





UUCGACAGCCCCACCGUGGCCUAUU





CUGUGCUGGUGGUGGCCAAAGUGGA





AAAGGGCAAGUCCAAGAAACUGAAG





AGUGUGAAAGAGCUGCUGGGGAUCA





CCAUCAUGGAAAGAAGCAGCUUCGA





GAAGAAUCCCAUCGACUUUCUGGAA





GCCAAGGGCUACAAAGAAGUGAAAA





AGGACCUGAUCAUCAAGCUGCCUAA





GUACUCCCUGUUCGAGCUGGAAAAC





GGCCGGAAGAGAAUGCUGGCCUCUG





CCGGCGAACUGCAGAAGGGAAACGA





ACUGGCCCUGCCCUCCAAAUAUGUG





AACUUCCUGUACCUGGCCAGCCACU





AUGAGAAGCUGAAGGGCUCCCCCGA





GGAUAAUGAGCAGAAACAGCUGUUU





GUGGAACAGCACAAGCACUACCUGG





ACGAGAUCAUCGAGCAGAUCAGCGA





GUUCUCCAAGAGAGUGAUCCUGGCC





GACGCUAAUCUGGACAAAGUGCUGU





CCGCCUACAACAAGCACCGGGAUAA





GCCCAUCAGAGAGCAGGCCGAGAAU





AUCAUCCACCUGUUUACCCUGACCA





AUCUGGGAGCCCCUGCCGCCUUCAA





GUACUUUGACACCACCAUCGACCGG





AAGAGGUACACCAGCACCAAAGAGG





UGCUGGACGCCACCCUGAUCCACCA





GAGCAUCACCGGCCUGUACGAGACA





CGGAUCGACCUGUCUCAGCUGGGAG





GUGACUCUGGAGGAUCUAGCGGAGG





AUCCUCUGGCAGCGAGACACCAGGA





ACAAGCGAGUCAGCAACACCAGAGA





GCAGUGGCGGCAGCAGCGGCGGCAG





CAGCACCCUAAAUAUAGAAGAUGAG





UAUCGGCUACAUGAGACCUCAAAAG





AGCCAGAUGUUUCUCUAGGGUCCAC





AUGGCUGUCUGAUUUUCCUCAGGCC





UGGGCGGAAACCGGGGGCAUGGGAC





UGGCAGUUCGCCAAGCUCCUCUGAU





CAUACCUCUGAAAGCAACCUCUACC





CCCGUGUCCAUAAAACAAUACCCCA





UGUCACAAGAAGCCAGACUGGGGAU





CAAGCCCCACAUACAGAGACUGUUG





GACCAGGGAAUACUGGUACCCUGCC





AGUCCCCCUGGAACACGCCCCUGCU





ACCCGUUAAGAAACCAGGGACUAAU





GAUUAUAGGCCUGUCCAGGAUCUGA





GAGAAGUCAACAAGCGGGUGGAAGA





CAUCCACCCCACCGUGCCCAACCCU





UACAACCUCUUGAGCGGGCUCCCAC





CGUCCCACCAGUGGUACACUGUGCU





UGAUUUAAAGGAUGCCUUUUUCUGC





CUGAGACUCCACCCCACCAGUCAGC





CUCUCUUCGCCUUUGAGUGGAGAGA





UCCAGAGAUGGGAAUCUCAGGACAA





UUGACCUGGACCAGACUCCCACAGG





GUUUCAAAAACAGUCCCACCCUGUU





UAAUGAGGCACUGCACAGAGACCUA





GCAGACUUCCGGAUCCAGCACCCAG





ACUUGAUCCUGCUACAGUACGUGGA





UGACUUACUGCUGGCCGCCACUUCU





GAGCUAGACUGCCAACAAGGUACUC





GGGCCCUGUUACAAACCCUAGGGAA





CCUCGGGUAUCGGGCCUCGGCCAAG





AAAGCCCAAAUUUGCCAGAAACAGG





UCAAGUAUCUGGGGUAUCUUCUAAA





AGAGGGUCAGAGAUGGCUGACUGAG





GCCAGAAAAGAGACUGUGAUGGGGC





AGCCUACUCCGAAGACCCCUCGACA





ACUAAGGGAGUUCCUAGGGAAGGCA





GGCUUCUGUCGCCUCUUCAUCCCUG





GGUUUGCAGAAAUGGCAGCCCCCCU





GUACCCUCUCACCAAACCGGGGACU





CUGUUUAAUUGGGGCCCAGACCAAC





AAAAGGCCUAUCAAGAAAUCAAGCA





AGCUCUUCUAACUGCCCCAGCCCUG





GGGUUGCCAGAUUUGACUAAGCCCU





UUGAACUCUUUGUCGACGAGAAGCA





GGGCUACGCCAAAGGUGUCCUAACG





CAAAAACUGGGACCUUGGCGUCGGC





CGGUGGCCUACCUGUCCAAAAAGCU





AGACCCAGUAGCAGCUGGGUGGCCC





CCUUGCCUACGGAUGGUAGCAGCCA





UUGCCGUACUGACAAAGGAUGCAGG





CAAGCUAACCAUGGGACAGCCACUA





GUCAUUCUGGCCCCCCAUGCAGUAG





AGGCACUAGUCAAACAACCCCCCGA





CCGCUGGCUUUCCAACGCCCGGAUG





ACUCACUAUCAGGCCUUGCUUUUGG





ACACGGACCGGGUCCAGUUCGGACC





GGUGGUAGCCCUGAACCCGGCUACG





CUGCUCCCACUGCCUGAGGAAGGGC





UGCAACACAACUGCCUUGAUAUCCU





GGCCGAAGCCCACGGAACCCGACCC





GACCUAACGGACCAGCCGCUCCCAG





ACGCCGACCACACCUGGUACACGGA





UGGAAGCAGUCUCUUACAAGAGGGA





CAGCGUAAGGCGGGAGCUGCGGUGA





CCACCGAGACCGAGGUAAUCUGGGC





UAAAGCCCUGCCAGCCGGGACAUCC





GCUCAGCGGGCUGAACUGAUAGCAC





UCACCCAGGCCCUAAAGAUGGCAGA





AGGUAAGAAGCUAAAUGUUUAUACU





GAUAGCCGUUAUGCUUUUGCUACUG





CCCAUAUCCAUGGAGAAAUAUACAG





AAGGCGUGGGUGGCUCACAUCAGAA





GGCAAAGAGAUCAAAAAUAAAGACG





AGAUCUUGGCCCUACUAAAAGCCCU





CUUUCUGCCCAAAAGACUUAGCAUA





AUCCAUUGUCCAGGACAUCAAAAGG





GACACAGCGCCGAGGCUAGAGGCAA





CCGGAUGGCUGACCAAGCGGCCCGA





AAGGCAGCCAUCACAGAGACUCCAG





ACACCUCUACCCUCCUCAUAGAAAA





UUCAUCACCCUCUGGCGGCUCAAAA





AGAACCGCCGACGGCAGCGAAUUCG





AGCCCAAGAAGAAGAGGAAAGUCUG





AUUAAUUAAGCUGCCUUCUGCGGGG





CUUGCCUUCUGGCCAUGCCCUUCUU





CUCUCCCUUGCACCUGUACCUCUUG





GUCUUUGAAUAAAGCCUGAGUAGGA





AGUCUAG





(SEQ ID NO: 3565)







Cas9-
AGGAAAUAAGAGAGAAAAGAAGAGU
(A)80



RT(PERV)
AAGAAGAAAUAUAAGAGCCACCAUG
(SEQ




GCUCCCAAAAAGAAAAGGAAGGUGG
ID




GCAUUCACGGCGUGCCUGCGGCCGA
NO:




CAAAAAGUACAGCAUCGGCCUUGAU
3666)




AUCGGCACCAAUAGCGUGGGCUGGG





CCGUUAUCACAGACGAAUACAAGGU





ACCCAGCAAGAAGUUCAAGGUGCUG





GGGAAUACAGACAGGCACUCUAUCA





AGAAAAACCUUAUCGGGGCUCUGCU





GUUUGACUCAGGCGAGACCGCCGAG





GCCACCAGGUUGAAGAGGACCGCAA





GGCGAAGGUACACCCGGAGGAAGAA





CAGGAUCUGCUAUCUGCAGGAGAUC





UUCAGCAACGAGAUGGCCAAGGUGG





ACGACAGCUUCUUCCACAGGCUGGA





GGAGAGCUUCCUUGUCGAGGAGGAU





AAGAAGCACGAACGACACCCCAUCU





UCGGCAACAUAGUCGACGAGGUCGC





UUAUCACGAGAAGUACCCCACCAUC





UACCACCUGCGAAAGAAAUUGGUGG





AUAGCACCGAUAAAGCCGACUUGCG





ACUUAUCUACUUGGCUCUGGCGCAC





AUGAUUAAGUUCAGGGGCCACUUCC





UGAUCGAGGGCGACCUUAACCCCGA





CAACAGUGACGUAGACAAAUUGUUC





AUCCAGCUUGUACAGACCUAUAACC





AGCUGUUCGAGGAAAACCCUAUUAA





CGCCAGCGGGGUGGAUGCGAAGGCC





AUACUUAGCGCCAGGCUGAGCAAAA





GCAGGCGCUUGGAGAACCUGAUAGC





CCAGCUGCCCGGUGAAAAGAAGAAC





GGCCUCUUCGGUAAUCUGAUUGCCC





UGAGCCUGGGCCUGACCCCCAACUU





CAAGAGCAACUUCGACCUGGCAGAA





GAUGCCAAGCUGCAGUUGAGUAAGG





ACACCUAUGACGACGACUUGGACAA





UCUGCUCGCCCAAAUCGGCGACCAG





UACGCUGACCUGUUCCUCGCCGCCA





AGAACCUUUCUGACGCAAUCCUGCU





UAGCGAUAUCCUUAGGGUGAACACA





GAGAUCACCAAGGCCCCCCUGAGCG





CCAGCAUGAUCAAGAGGUACGACGA





GCACCAUCAGGACCUGACCCUUCUG





AAGGCCCUGGUGAGGCAGCAACUGC





CCGAGAAGUACAAGGAGAUCUUUUU





CGACCAGAGCAAGAACGGCUACGCC





GGCUACAUCGACGGCGGAGCCAGCC





AAGAGGAGUUCUACAAGUUCAUCAA





GCCCAUCCUGGAGAAGAUGGAUGGC





ACCGAGGAGCUGCUGGUGAAGCUGA





ACAGGGAAGAUUUGCUCCGGAAGCA





GAGGACCUUUGACAACGGUAGCAUC





CCCCACCAGAUCCACCUGGGCGAGC





UGCACGCAAUACUGAGGCGACAGGA





GGAUUUCUACCCCUUCCUCAAGGAC





AAUAGGGAGAAAAUCGAAAAGAUUC





UGACCUUCAGGAUCCCCUACUACGU





GGGCCCUCUUGCCAGGGGCAACAGC





CGAUUCGCUUGGAUGACAAGAAAGA





GCGAGGAGACCAUCACCCCCUGGAA





CUUCGAGGAAGUGGUGGACAAAGGA





GCAAGCGCGCAGUCUUUCAUCGAAC





GGAUGACCAAUUUCGACAAAAACCU





GCCUAACGAGAAGGUGCUGCCCAAG





CACAGCCUGCUUUACGAGUACUUCA





CCGUGUACAACGAGCUCACCAAGGU





GAAAUAUGUGACCGAGGGCAUGCGA





AAACCCGCUUUCCUGAGCGGCGAGC





AGAAGAAGGCCAUCGUGGACCUGCU





GUUCAAGACCAACAGGAAGGUGACC





GUGAAGCAGCUGAAGGAGGACUACU





UCAAGAAGAUCGAGUGCUUUGAUAG





CGUGGAAAUAAGCGGCGUGGAGGAC





AGGUUCAACGCCAGCCUGGGCACCU





ACCACGACUUGUUGAAGAUAAUCAA





AGACAAGGAUUUCCUGGAUAAUGAG





GAGAACGAGGAUAUACUCGAGGACA





UCGUGCUGACUUUGACCCUGUUUGA





GGACCGAGAGAUGAUUGAAGAAAGG





CUCAAAACCUACGCCCACCUGUUCG





ACGACAAAGUGAUGAAACAACUGAA





GAGACGAAGAUACACCGGCUGGGGC





AGACUGUCCAGGAAGCUCAUCAACG





GCAUUAGGGACAAGCAGAGCGGCAA





GACCAUCCUGGAUUUCCUGAAGUCC





GACGGCUUCGCCAACCGAAACUUCA





UGCAGCUGAUUCACGAUGACAGCUU





GACCUUCAAGGAGGACAUCCAGAAG





GCCCAGGUUAGCGGCCAGGGCGACU





CCCUGCACGAACAUAUUGCAAACCU





GGCAGGCUCCCCUGCGAUCAAGAAG





GGCAUACUGCAGACCGUUAAGGUUG





UGGACGAAUUGGUCAAGGUCAUGGG





CAGGCACAAGCCCGAAAACAUAGUU





AUAGAGAUGGCCAGAGAGAACCAGA





CCACCCAAAAGGGCCAGAAGAACAG





CCGGGAGCGCAUGAAAAGGAUCGAG





GAGGGUAUCAAGGAACUCGGAAGCC





AGAUCCUCAAAGAGCACCCCGUGGA





GAAUACCCAGCUCCAGAACGAGAAG





CUGUACCUGUACUACCUGCAGAACG





GCAGGGACAUGUACGUUGACCAGGA





GUUGGACAUCAACAGGCUUUCAGAC





UAUGACGUGGAUCACAUAGUGCCCC





AGAGCUUUCUUAAAGACGAUAGCAU





CGACAACAAGGUCCUGACCCGCUCC





GACAAAGCCAGGGGCAAAAGCGACA





ACGUGCCAAGCGAAGAGGUGGUUAA





AAAGAUGAAGAACUACUGGAGGCAA





CUGCUCAACGCGAAAUUGAUCACCC





AGAGAAAGUUCGAUAACCUGACCAA





GGCCGAGAGGGGCGGACUCUCCGAA





CUUGACAAAGCGGGCUUCAUAAAGA





GGCAGCUGGUCGAGACCCGACAGAU





CACGAAGCACGUGGCCCAAAUCCUC





GACAGCAGAAUGAAUACCAAGUACG





AUGAGAAUGACAAACUCAUCAGGGA





AGUGAAAGUGAUUACCCUGAAGAGC





AAGUUGGUGUCCGACUUUCGCAAAG





AUUUCCAGUUCUACAAGGUGAGGGA





GAUCAACAACUACCACCAUGCCCAC





GACGCAUACCUGAACGCCGUGGUCG





GCACCGCCCUGAUUAAGAAGUAUCC





AAAGCUGGAGUCCGAAUUUGUCUAC





GGCGACUACAAAGUUUACGAUGUGA





GGAAGAUGAUCGCUAAGAGCGAACA





GGAGAUCGGCAAGGCCACCGCUAAG





UAUUUCUUCUACAGCAACAUCAUGA





ACUUUUUCAAGACCGAGAUCACACU





UGCCAACGGCGAAAUCAGGAAGAGG





CCGCUUAUCGAGACCAACGGUGAGA





CCGGCGAGAUCGUGUGGGACAAGGG





CAGGGACUUCGCCACCGUGAGGAAA





GUCCUGAGCAUGCCCCAGGUGAAUA





UUGUGAAAAAAACUGAGGUGCAGAC





AGGCGGCUUUAGCAAGGAAUCCAUC





CUGCCCAAGAGGAACAGCGACAAGC





UGAUCGCCCGGAAGAAGGACUGGGA





CCCUAAGAAGUAUGGAGGCUUCGAC





AGCCCCACCGUAGCCUACAGCGUGC





UGGUGGUCGCGAAGGUAGAGAAGGG





GAAGAGCAAGAAACUGAAGAGCGUG





AAGGAGCUGCUCGGCAUAACCAUCA





UGGAGAGGUCCAGCUUUGAGAAGAA





CCCCAUUGACUUUUUGGAAGCCAAG





GGCUACAAAGAGGUCAAAAAGGACC





UGAUCAUCAAACUCCCCAAGUACUC





CCUGUUUGAAUUGGAGAACGGCAGA





AAGAGGAUGCUGGCGAGCGCUGGGG





AACUGCAAAAGGGCAACGAACUGGC





GCUGCCCAGCAAGUACGUGAAUUUU





CUGUACCUGGCGUCCCACUACGAAA





AGCUGAAAGGCAGCCCCGAGGACAA





CGAGCAGAAGCAGCUGUUCGUGGAG





CAGCACAAGCAUUACCUGGACGAGA





UAAUCGAGCAAAUCAGCGAGUUCAG





CAAGAGGGUGAUUCUGGCCGACGCG





AACCUGGAUAAGGUCCUCAGCGCCU





ACAACAAGCACCGAGACAAACCCAU





CAGGGAGCAGGCCGAGAAUAUCAUA





CACCUGUUCACCCUGACAAAUCUGG





GCGCACCUGCGGCAUUCAAAUACUU





CGAUACCACCAUCGACAGGAAAAGG





UACACUAGCACUAAGGAGGUGCUGG





AUGCCACCUUGAUCCACCAGUCCAU





UACCGGCCUGUAUGAGACCAGGAUC





GACCUGAGCCAGCUUGGAGGCGACU





CUGGAGGAUCUAGCGGAGGAUCCUC





UGGCAGCGAGACACCAGGAACAAGC





GAGUCAGCAACACCAGAGAGCAGUG





GCGGCAGCAGCGGCGGCAGCAGCCU





GGACGACGAGUACAGACUGUAUAGC





CCUCUGGUGAAGCCAGAUCAGAACA





UUCAGUUCUGGCUGGAACAGUUUCC





ACAGGCCUGGGCCGAAACAGCCGGA





AUGGGCCUGGCCAAGCAGGUGCCUC





CUCAGGUGAUCCAGCUGAAGGCCAG





CGCCACACCUGUGUCCGUGCGGCAG





UACCCUCUGUCCAAGGAGGCUCAGG





AGGGCAUCAGACCUCACGUCCAGCG





GCUGAUCCAGCAGGGGAUCCUGGUG





CCCGUGCAAAGCCCUUGGAACACCC





CUCUUCUGCCCGUGAGAAAACCCGG





CACAAACGACUACCGGCCUGUGCAG





GACCUGAGAGAAGUGAACAAGCGGG





UGCAGGACAUCCACCCCACAGUGCC





AAAUCCUUACAACCUGCUUUGUGCC





CUGCCCCCCCAGCGCAGCUGGUACA





CCGUUCUGGACCUGAAAGAUGCCUU





UUUCUGUCUGAGACUUCAUCCUACA





AGCCAGCCCCUGUUCGCCUUCGAGU





GGCGGGAUCCUGGCACCGGCCGGAC





AGGCCAGCUGACAUGGACCAGACUG





CCUCAGGGCUUCAAGAACAGCCCUA





CCAUCUUCAACGAGGCCCUGCACAG





AGACCUUGCCAACUUCAGAAUCCAA





CACCCACAGGUGACCCUGCUCCAGU





ACGUGGAUGACCUGCUGCUGGCCGG





CGCCACAAAACAAGAUUGCCUGGAA





GGCACCAAGGCCCUUCUGCUGGAGC





UGAGCGACCUGGGAUAUCGGGCCUC





UGCUAAGAAAGCUCAGAUCUGCAGG





AGAGAGGUGACCUACCUGGGCUACU





CUCUGAGAGAUGGCCAAAGAUGGCU





GACCGAGGCCAGAAAGAAAACCGUG





GUGCAAAUCCCCGCUCCUACAACAG





CCAAGCAGGUUAGAGAGUUCCUGGG





AAAGGCUGGAUUUUGCAGACUGUUC





AUCCCAGGCUUUGCCACCCUGGCCG





CCCCUCUGUACCCCCUGACCAAACC





UAAGGGCGAGUUCAGCUGGGCCCCA





GAGCACCAGAAGGCAUUCGACGCGA





UCAAGAAGGCUCUGCUGUCUGCCCC





UGCCCUGGCUCUGCCCGACGUGACA





AAGCCCUUCACCCUGUACGUGGACG





AACGGAAGGGCGUGGCUAGAGGCGU





UCUGACCCAGACCCUGGGUCCUUGG





AGAAGGCCUGUGGCCUACCUCAGUA





AGAAGCUGGAUCCUGUGGCCUCUGG





CUGGCCUGUGUGCCUGAAGGCCAUC





GCCGCCGUGGCCAUUCUGGUCAAGG





AUGCCGAUAAGCUGACCCUAGGCCA





GAAUAUCACCGUGAUCGCCCCUCAC





GCCCUCGAGAACAUCGUGCGGCAGC





CUCCCGACAGAUGGAUGACCAACGC





CAGAAUGACCCACUACCAGAGCCUG





UUGCUGACCGAGAGAGUGACCUUCG





CCCCUCCAGCUGCCCUGAAUCCCGC





CACUCUGCUGCCCGAGGAAACCGAC





GAGCCUGUGACCCACGACUGCCACC





AGCUGCUGAUCGAGGAAACCGGCGU





CAGAAAGGACCUGACAGAUAUCCCU





CUGACCGGAGAGGUGCUGACAUGGU





UCACCGACGGCAGCAGCUACGUCGU





GGAAGGCAAGCGGAUGGCCGGCGCC





GCUGUGGUCGACGGCACAAGAACCA





UCUGGGCUUCCAGCCUGCCUGAGGG





CACCAGCGCCCAGAAGGCCGAGCUG





AUGGCCCUCACACAGGCCCUGCGGC





UGGCUGAGGGCAAAAGCAUCAACAU





CUAUACAGACAGCCGUUACGCCUUC





GCCACAGCGCACGUGCACGGCGCCA





UCUACAAGCAGAGAGGAUGGCUGAC





CUCUGCCGGAAGAGAAAUCAAGAAC





AAGGAAGAAAUCCUGAGCCUGCUGG





AAGCCCUGCAUCUCCCAAAGAGACU





GGCCAUCAUCCACUGCCCCGGCCAC





CAGAAGGCCAAAGACCCUAUCAGCA





GAGGCAACCAGAUGGCCGACCGGGU





GGCCAAGCAAGCCGCCCAAGGCGUG





AAUCUGCUGCCUUAGUUAAUUAAGC





UGCCUUCUGCGGGGCUUGCCUUCUG





GCCAUGCCCUUCUUCUCUCCCUUGC





ACCUGUACCUCUUGGUCUUUGAAUA





AAGCCUGAGUAGGAAGUCUAG





(SEQ ID NO: 3566)









In some embodiments, a fusion protein may comprise a Cas molecule, e.g., a mutated Cas9, e.g., a Cas-nuclease containing a mutation inhibiting (e.g., inactivating) one endonuclease active site, e.g., the Cas9 nickase Cas9(N863A). In some embodiments, the fusion protein comprises a peptide linker, e.g., a glycine serine rich flexible peptide linker, e.g., a linker as disclosed in Tables 38 and/or 42, e.g., linker 10, in Table 42. In some embodiments, the fusion protein comprises a RT domain, e.g., a RT domain comprising a sequence from Table 1, Table 3, Table 30, Table 31, Table 41, Table 44, Table 50, or a fragment or variant thereof. In some embodiments, the Cas-RT fusion protein (or nucleic acid encoding the same) is formulated with a gRNA. In some embodiments, the linker length is between 2-40 amino acids, between 5-30 amino acids, between 5-20 amino acids, between 10-20 amino acids, or between 10-15 amino acids. In some embodiments, the Cas-RT fusion proteins has similar DNA binding activity to a Cas molecule that is not fused with a RT domain. In some embodiments, a Cas-RT may comprise a RT domain comprising a mutation. In embodiments, the mutant RT domain shows increased processability and template affinity compared to an unmutated RT domain. Target Site


In some embodiments, after Gene Writing, the target site surrounding the integrated sequence contains a limited number of insertions or deletions, for example, in less than about 50% or 10% of integration events, e.g., as determined by long-read amplicon sequencing of the target site, e.g., as described in Karst et al. (2020) bioRxiv doi.org/10.1101/645903 (incorporated by reference herein in its entirety). In some embodiments, the target site does not show multiple insertion events, e.g., head-to-tail or head-to-head duplications, e.g., as determined by long-read amplicon sequencing of the target site, e.g., as described in Karst et al. bioRxiv doi.org/10.1101/645903 (2020) (incorporated herein by reference in its entirety). In some embodiments, the target site contains an integrated sequence corresponding to the template RNA. In some embodiments, the target site does not contain insertions resulting from endogenous RNA in more than about 1% or 10% of events, e.g., as determined by long-read amplicon sequencing of the target site, e.g., as described in Karst et al. bioRxiv doi.org/10.1101/645903 (2020) (incorporated herein by reference in its entirety). In some embodiments, the target site contains the integrated sequence corresponding to the template RNA.


Second Strand Nicking

In some embodiments, a Gene Writer system described herein comprises nickase activity that nicks the first strand and the second strand of target DNA. As discussed herein, without wishing to be bound by theory, nicking of the first strand of the target site DNA is thought to provide a 3′ OH that can be used by an RT domain to reverse transcribe a sequence of a template RNA, e.g., a heterologous object sequence. Without wishing to be bound by theory, it is thought that introducing an additional nick to the second strand may bias the cellular DNA repair machinery to adopt the heterologous object sequence-based sequence more frequently than the original genomic sequence. In some embodiments, the additional nick to the second strand is made by the same endonuclease domain (e.g., nickase domain) as the nick to the first strand. In some embodiments, the same Gene Writer polypeptide performs both the nick to the first strand and the nick to the second strand. In some embodiments, the Gene Writer polypeptide comprises a CRISPR/Cas domain and the additional nick to the second strand is directed by an additional nucleic acid, e.g., comprising a second gRNA directing the CRISPR/Cas domain to nick the second strand. In other embodiments, the additional second strand nick is made by a different endonuclease domain (e.g., nickase domain) than the nick to the first strand. In some embodiments, that different endonuclease domain is situated in an additional polypeptide (e.g., a system of the invention further comprises the additional polypeptide), separate from the Gene Writer polypeptide. In some embodiments, the additional polypeptide comprises an endonuclease domain (e.g., nickase domain) described herein. In some embodiments, the additional polypeptide comprises a DNA binding domain, e.g., described herein.


It is contemplated herein that the position at which the second strand nick occurs relative to the first strand nick may influence the extent to which one or more of: desired Gene Writing DNA modifications are obtained, undesired double-strand breaks (DSBs) occur, undesired insertions occur, or undesired deletions occur. Without wishing to be bound by theory, second strand nicking may occur in two general orientations: inward nicks and outward nicks.


In some embodiments, in the inward nick orientation, the RT domain polymerizes (e.g., using the template RNA (e.g., the heterologous object sequence)) away the second strand nick. In some embodiments, in the inward nick orientation, the location of the nick to the first strand and the location of the nick to the second strand are positioned between the first PAM site and second PAM site (e.g., in a scenario wherein both nicks are made by a polypeptide (e.g., a Gene Writer polypeptide) comprising a CRISPR/Cas domain). In some embodiments, in the inward nick orientation, the location of the nick to the first strand and the location of the nick to the second strand are between the sites where the polypeptide and the additional polypeptide bind to the target DNA. In some embodiments, in the inward nick orientation, the location of the nick to the second strand is positioned on the same side of the binding sites of the polypeptide and additional polypeptide relative to the location of the nick to the first strand. In some embodiments, in the inward nick orientation, the location of the nick to the first strand and the location of the nick to the second strand are positioned between the PAM site and the site at a distance from the target site.


An example of a Gene Writer system that provides an inward nick orientation comprises a Gene Writer polypeptide comprising a CRISPR/Cas domain, a template RNA comprising a gRNA that directs nicking of the target site DNA on the first strand, and an additional nucleic acid comprising an additional gRNA that directs nicking at a site a distance from the location of the first nick, wherein the location of the first nick and the location of the second nick are between the PAM sites of the sites to which the two gRNAs direct the Gene Writer polypeptide. As a further example, another Gene Writer system that provides an inward nick orientation comprises a Gene Writer polypeptide comprising a zinc finger molecule and a first nickase domain wherein the zinc finger molecule binds to the target DNA in a manner that directs the first nickase domain to nick the first strand of the target site; an additional polypeptide comprising a CRISPR/Cas domain, and an additional nucleic acid comprising a gRNA that directs the additional polypeptide to nick a site a distance from the target site DNA on the second strand, wherein the location of the first nick and the location of the second nick are between the PAM site and the site to which the zinc finger molecule binds. As a further example, another Gene Writer system that provides an inward nick orientation comprises a Gene Writer polypeptide comprising a zinc finger molecule and a first nickase domain wherein the zinc finger molecule binds to the target DNA in a manner that directs the first nickase domain to nick the first strand of the target site; an additional polypeptide comprising a TAL effector molecule and a second nickase domain wherein the TAL effector molecule binds to a site a distance from the target site in a manner that directs the additional polypeptide to nick the second strand, wherein the location of the first nick and the location of the second nick are between the site to which the TAL effector molecule binds and the site to which the zinc finger molecule binds. In some embodiments, in the outward nick orientation, the RT domain polymerizes (e.g., using the template RNA (e.g., the heterologous object sequence)) toward the second strand nick.


In some embodiments, in the inward nick orientation when both the first and second nicks are made by a polypeptide comprising a CRISPR/Cas domain (e.g., a Gene Writer polypeptide), the first PAM site and second PAM site are positioned between the location of the nick to the first strand and the location of the nick to the second strand. In some embodiments, in the inward nick orientation, the polypeptide (e.g., the Gene Writer polypeptide) and the additional polypeptide bind to sites on the target DNA between the location of the nick to the first strand and the location of the nick to the second. In some embodiments, in the inward nick orientation, the location of the nick to the second strand is positioned on the opposite side of the binding sites of the polypeptide and additional polypeptide relative to the location of the nick to the first strand. In some embodiments, in the inward orientation, the PAM site and the site at a distance from the target site are positioned between the location of the nick to the first strand and the location of the nick to the second strand.


An example of a Gene Writer system that provides an outward nick orientation comprises a Gene Writer polypeptide comprising a CRISPR/Cas domain, a template RNA comprising a gRNA that directs nicking of the target site DNA on the first strand, and an additional nucleic acid comprising an additional gRNA that directs nicking at a site a distance from the location of the first nick, wherein the location of the first nick and the location of the second nick are outside of the PAM sites of the sites to which the two gRNAs direct the Gene Writer polypeptide (i.e., the PAM sites are between the the location of the first nick and the location of the second nick). As a further example, another Gene Writer system that provides an outward nick orientation comprises a Gene Writer polypeptide comprising a zinc finger molecule and a first nickase domain wherein the zinc finger molecule binds to the target DNA in a manner that directs the first nickase domain to nick the first strand of the target site; an additional polypeptide comprising a CRISPR/Cas domain, and an additional nucleic acid comprising a gRNA that directs the additional polypeptide to nick a site a distance from the target site DNA on the second strand, wherein the location of the first nick and the location of the second nick are outside the PAM site and the site to which the zinc finger molecule binds (i.e., the PAM site and the site to which the zinc finger molecule binds are between the the location of the first nick and the location of the second nick). As a further example, another Gene Writer system that provides an outward nick orientation comprises a Gene Writer polypeptide comprising a zinc finger molecule and a first nickase domain wherein the zinc finger molecule binds to the target DNA in a manner that directs the first nickase domain to nick the first strand of the target site; an additional polypeptide comprising a TAL effector molecule and a second nickase domain wherein the TAL effector molecule binds to a site a distance from the target site in a manner that directs the additional polypeptide to nick the second strand, wherein the location of the first nick and the location of the second nick are outside the site to which the TAL effector molecule binds and the site to which the zinc finger molecule binds (i.e., the site to which the TAL effector molecule binds and the site to which the zinc finger molecule binds are between the location of the first nick and the location of the second nick).


Without wishing to be bound by theory, it is thought that, for Gene Writer systems where a second strand nick is provided, an outward nick orientation is preferred in some embodiments. As is described herein, an inward nick may produce a higher number of double-strand breaks (DSBs) than an outward nick orientation. DSBs may be recognized by the DSB repair pathways in the nucleus of a cell, which can result in undesired insertions and deletions. An outward nick orientation may provide a decreased risk of DSB formation, and a corresponding lower amount of undesired insertions and deletions. In some embodiments, undesired insertions and deletions are insertions and deletions not encoded by the heterologous object sequence, e.g., an insertion or deletion produced by the double-strand break repair pathway unrelated to the modification encoded by the heterologous object sequence. In some embodiments, a desired Gene Writing modification comprises a change to the target DNA (e.g., a substitution, insertion, or deletion) encoded by the heterologous object sequence (e.g., and achieved by the Gene Writer writing the heterologous object sequence into the target site). In some embodiments, the first strand nick and the second strand nick are in an outward orientation.


In addition, the distance between the first strand nick and second strand nick may influence the extent to which one or more of: desired Gene Writing DNA modifications are obtained, undesired double-strand breaks (DSBs) occur, undesired insertions occur, or undesired deletions occur. Without wishing to be bound by theory, it is thought the second strand nick benefit, the biasing of DNA repair toward incorporation of the heterologous object sequence into the target DNA, increases as the distance between the first strand nick and second strand nick decreases. However, it is thought that the risk of DSB formation also increases as the distance between the first strand nick and second strand nick decreases. Correspondingly, it is thought that the number of undesired insertions and/or deletions may increase as the distance between the first strand nick and second strand nick decreases. In some embodiments, the distance between the first strand nick and second strand nick is chosen to balance the benefit of biasing DNA repair toward incorporation of the heterologous object sequence into the target DNA and the risk of DSB formation and of undesired deletions and/or insertions. In some embodiments, a system where the first strand nick and the second strand nick are at least a threshold distance apart has an increased level of desired Gene Writing modification outcomes, a decreased level of undesired deletions, and/or a decreased level of undesired insertions relative to an otherwise similar inward nick orientation system where the first nick and the second nick are less than the a threshold distance apart. In some embodiments the threshold distance(s) is given below.


In some embodiments, the first nick and the second nick are at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nucleotides apart. In some embodiments, the first nick and the second nick are no more than 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or 250 nucleotides apart. In some embodiments, the first nick and the second nick are 20-200, 30-200, 40-200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200, 110-200, 120-200, 130-200, 140-200, 150-200, 160-200, 170-200, 180-200, 190-200, 20-190, 30-190, 40-190, 50-190, 60-190, 70-190, 80-190, 90-190, 100-190, 110-190, 120-190, 130-190, 140-190, 150-190, 160-190, 170-190, 180-190, 20-180, 30-180, 40-180, 50-180, 60-180, 70-180, 80-180, 90-180, 100-180, 110-180, 120-180, 130-180, 140-180, 150-180, 160-180, 170-180, 20-170, 30-170, 40-170, 50-170, 60-170, 70-170, 80-170, 90-170, 100-170, 110-170, 120-170, 130-170, 140-170, 150-170, 160-170, 20-160, 30-160, 40-160, 50-160, 60-160, 70-160, 80-160, 90-160, 100-160, 110-160, 120-160, 130-160, 140-160, 150-160, 20-150, 30-150, 40-150, 50-150, 60-150, 70-150, 80-150, 90-150, 100-150, 110-150, 120-150, 130-150, 140-150, 20-140, 30-140, 40-140, 50-140, 60-140, 70-140, 80-140, 90-140, 100-140, 110-140, 120-140, 130-140, 20-130, 30-130, 40-130, 50-130, 60-130, 70-130, 80-130, 90-130, 100-130, 110-130, 120-130, 20-120, 30-120, 40-120, 50-120, 60-120, 70-120, 80-120, 90-120, 100-120, 110-120, 20-110, 30-110, 40-110, 50-110, 60-110, 70-110, 80-110, 90-110, 100-110, 20-100, 30-100, 40-100, 50-100, 60-100, 70-100, 80-100, 90-100, 20-90, 30-90, 40-90, 50-90, 60-90, 70-90, 80-90, 20-80, 30-80, 40-80, 50-80, 60-80, 70-80, 20-70, 30-70, 40-70, 50-70, 60-70, 20-60, 30-60, 40-60, 50-60, 20-50, 30-50, 40-50, 20-40, 30-40, or 20-30 nucleotides apart. In some embodiments, the first nick and the second nick are 40-100 nucleotides apart.


Without wishing to be bound by theory, it is thought that, for Gene Writer systems where a second strand nick is provided and an inward nick orientation is selected, increasing the distance between the first strand nick and second strand nick may be preferred. As is described herein, an inward nick orientation may produce a higher number of DSBs than an outward nick orientation, and may result in a higher amount of undesired insertions and deletions than an outward nick orientation, but increasing the distance between the nicks may mitigate that increase in DSBs, undesired deletions, and/or undesired insertions. In some embodiments, an inward nick orientation wherein the first nick and the second nick are at least a threshold distance apart has an increased level of desired Gene Writing modification outcomes, a decreased level of undesired deletions, and/or a decreased level of undesired insertions relative to an otherwise similar inward nick orientation system where the first nick and the second nick are less than the a threshold distance apart. In some embodiments the threshold distance is given below.


In some embodiments, the first strand nick and the second strand nick are in an inward orientation. In some embodiments, the first strand nick and the second strand nick are in an inward orientation and the first strand nick and second strand nick are at least 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 350, 400, 450, or 500 nucleotides apart, e.g., at least 100 nucleotides apart, (and optionally no more than 500, 400, 300, 200, 190, 180, 170, 160, 150, 140, 130, or 120 nucleotides apart). In some embodiments, the first strand nick and the second strand nick are in an inward orientation and the first strand nick and second strand nick are 100-200, 110-200, 120-200, 130-200, 140-200, 150-200, 160-200, 170-200, 180-200, 190-200, 100-190, 110-190, 120-190, 130-190, 140-190, 150-190, 160-190, 170-190, 180-190, 100-180, 110-180, 120-180, 130-180, 140-180, 150-180, 160-180, 170-180, 100-170, 110-170, 120-170, 130-170, 140-170, 150-170, 160-170, 100-160, 110-160, 120-160, 130-160, 140-160, 150-160, 100-150, 110-150, 120-150, 130-150, 140-150, 100-140, 110-140, 120-140, 130-140, 100-130, 110-130, 120-130, 100-120, 110-120, or 100-110 nucleotides apart.


Evolved Variants of Gene Writers

In some embodiments, the invention provides evolved variants of Gene Writers. Evolved variants can, in some embodiments, be produced by mutagenizing a reference Gene Writer, or one of the fragments or donains comprised therein. In some embodiments, one or more of the domains (e.g., the reverse transcriptase, DNA binding (including, for example, sequence-guided DNA binding elements), RNA-binding, or endonuclease domain) is evolved. One or more of such evolved variant domains can, in some embodiments, be evolved alone or together with other domains. An evolved variant domain or domains may, in some embodiments, be combined with unevolved cognate component(s) or evolved variants of the cognate component(s), e.g., which may have been evolved in either a parallel or serial manner.


In some embodiments, the process of mutagenizing a reference Gene Writer, or fragment or domain thereof comprises mutagenizing the reference Gene Writer or fragment or domain thereof. In embodiments, the mutagenesis comprises a continuous evolution method (e.g., PACE) or non-continuous evolution method (e.g., PANCE), e.g., as described herein. In some embodiments, the evolved Gene Writer, or a fragment or domain thereof, comprises one or more amino acid variations introduced into its amino acid sequence relative to the amino acid sequence of the reference Gene Writer, or fragrant or domain thereof. In embodiments, amino acid sequence variations may include one or more mutated residues (e.g., conservative substitutions, non-conservative substitutions, or a combination thereof) within the amino acid sequence of a reference Gene Writer, e.g., as a result of a change in the nucleotide sequence encoding the gene writer that results in, e g., a. change in the codon at any particular position in the coding sequence, the deletion of one or more amino acids (e.g., a truncated protein), the insertion of one or more amino acids, or any combination of the foregoing. The evolved variant (Gene Writer may include variants in one or more components or domains of the Gene Writer (e.g., variants introduced into a reverse transcriptase domain, endonuclease domain, DNA binding domain, RNA binding domain, or combinations thereof).


In some aspects, the invention provides Gene Writers, systems, kits, and methods using or comprising an evolved variant of a Gene Writer, e.g., employs an evolved variant of a Gene Writer or a Gene Writer produced or producible by PACE or PANCE. In embodiments, the unevolved reference Gene Writer is a Gene Writer as disclosed herein.


The term “phage-assisted continuous evolution (PAE),” as used herein, generally refers to continuous evolution that employs phage as viral vectors. Examples of PACE technology have been described, for example, in International PCT Application No. PCT/US 2009/056194, filed Sep. 8, 2009, published as WO 2010/028347 on Mar. 11, 2010; International PCT Application, PCT/US2011/066747, filed Dec. 22, 2011, published as WO 2012/088381 on Jun. 28, 2012; U.S. Pat. No. 9,023,594, issued May 5, 2015; U.S. Pat. No. 9,771,574, issued Sep. 26, 2017, U.S. Pat. No. 9,394,537, issued Jul. 19, 2016; International PCT Application, PCT/US2015/012022, filed Jan. 20, 2015, published as WO 2015/134121 on Sep. 11, 2015; U.S. Pat. No. 10,179,911, issued Jan. 15, 2019; and International PCT Application, PCT/US2016/027795, filed Apr. 15, 2016, published as WO 2016/168631 on Oct. 20, 2016, the entire contents of each of which are incorporated herein by reference.


The term “phage-assisted non-continuous evolution (PANCE),” as used herein, generally refers to non-continuous evolution that employs phage as viral vectors. Examples of PANCE technology have been described, for example, in Suzuki T. et al, Crystal structures reveal an elusive functional domain of pyrrolysyl-tRNA synthetase, Nat Chem Biol. 13(12): 1261-1266 (2017), incorporated herein by reference in its entirety. Briefly, PANCE is a technique for rapid in vivo directed evolution using serial flask transfers of evolving selection phage (SP), which contain a gene of interest to be evolved, across fresh host cells (e.g., E. coli cells). Genes inside the host cell may be held constant while genes contained in the SP continuously evolve. Following phage growth, an aliquot of infected cells may be used to transfect a subsequent flask containing host E. coli. This process can be repeated and/or continued until the desired phenotype is evolved, e.g., for as many transfers as desired.


Methods of applying PACE and PANCE to Gene Writers may be readily appreciated by the skilled artisan by reference to, inter ala, the foregoing references. Additional exemplary methods for directing continuous evolution of genome-modifying proteins or systems, e.g., in a population of host cells, e.g, using phage particles, can be applied to generate evolved variants of Gene Writers, or fragments or subdomains thereof. Non-limiting examples of such methods are described in International PCT Application, PCT/US2009/056194, filed Sep. 8, 2009, published as WO 2010/028347 on Mar. 11, 20101 International PCT Application, PCT/US2011/066747, filed Dec. 22, 2011, published as WO 2012/088381 on Jun. 28, 2012; U.S. Pat. No. 9,023,594, issued May 5, 2015; U.S. Pat. No. 9,771,574, issued Sep. 26, 2017; U.S. Pat. No. 9,394,537, issued Jul. 19, 2016; International PCT Application, PCT/US2015/012022, filed Jan. 20, 2015, published as WO 2015/134121 on Sep. 11, 2015; U.S. Pat. No. 10,179,911, issued Jan. 15, 2019; International Application No. PCT/US2019/37216, filed Jun. 14, 2019, International Patent Publication WO 2019/023680, published Jan. 31, 2019, International PCT Application, PCI/US2016/027795, filed Apr. 15, 2016, published as WO 2015/168631 on Oct. 20, 2016, and international Patent Publication No. PCT/US2019/47996, filed Aug. 23, 2019, each of which is incorporated herein by reference in its entirety.


In some non-limiting illustrative embodiments, a method of evolution of a evolved variant Gene Writer, of a fragment or domain thereof, comprises: (a) contacting a population of host cells with a population of viral vectors comprising the gene of interest (the starting Gene Writer or fragment or domain thereof), wherein: (1) the host cell is amenable to infection by the viral vector; (2) the host cell expresses viral genes required for the generation of viral particles; (3) the expression of at least one viral gene required for the production of an infectious viral particle is dependent on a function of the gene of interest; and/or (4) the viral vector allows for expression of the protein in the host cell, and can be replicated and packaged into a viral particle by the host cell. In some embodiments, the method comprises (b) contacting the host cells with a mutagen, using host cells with mutations that elevate mutation rate (e g., either by carrying a mutation plasmid or some genome modification—e.g., proofing-impaired DNA polymerase, SOS genes, such as UmuC, UmuD, and/or RecA, which mutations, if plasmid-bound, may be under control of an inducible promoter), or a combination thereof. In some embodiments, the method comprises (c) incubating the population of host cells under conditions allowing for viral replication and the production of viral particles, wherein host cells are removed from the host cell population, and fresh, uninfected host cells are introduced into the population of host cells, thus replenishing the population of host cells and creating a flow of host cells. In some embodiments, the cells are incubated under conditions allowing for the gene of interest to acquire a. mutation. In some embodiments. the method further comprises (d) isolating a. mutated version of the viral vector, encoding an evolved gene product (e.g., an evolved variant Gene Writer, or fragment or domain thereof), from the population of host cells.


The skilled artisan will appreciate a variety of features employable within the above-described framework. For example, in some embodiments, the viral vector or the phage is a filamentous phage, for example, an MI3 phage, e.g., an MI3 selection phage. In certain embodiments, the gene required for the production of infectious viral particles is the M13 gene III (gIII). In embodiments, the phage may lack a functional gill, but otherwise comprise gI, gII, giV, gV, gVI, gVill, gVIII, gX, and a gX. In some embodiments, the generation of infectious VSV particles involves the envelope protein VSV-G. Various embodiments can use different retroviral vectors, for example, Murine Leukemia Virus vectors, or Lentiviral vectors. in embodiments, the retroviral vectors can efficiently be packaged with VSV-G envelope protein, e.g., as a substitute for the native envelope protein of the virus.


In some embodiments, host cells are incubated according to a suitable number of viral life cycles, e.g., at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 200, at least 300, at least 400, at least, 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1250, at least 1500, at least 1750, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, at least 7500, at least 10000, or more consecutive viral life cycles, which in on illustrative and non-limiting examples of M13 phage is 10-20 minutes per virus life cycle. Similarly, conditions can be modulated to adjust the time a host cell remains in a population of host cells, e.g., about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 80, about 90, about 100, about 120, about 150, or about 180 minutes. Host cell populations can be controlled in part by density of the host cells, or, in some embodiments, the host cell density in an inflow, e.g., 102 cells/ml, about 107 cells/ml, about 105 cells/ml, about 5-10 cells/ml, about 106 cells/nl, about 5-10 cells/nl, about 107 cells/ml, about 5-107 cells/ml, about 108 cells/ml, about 5-108 cells/ml, about 109 cells/ml, about 5·109 cells/ml, about 1010 cells/ml, or about 5·1010 cells/ml.


Promoters

In some embodiments, one or more promoter or enhancer elements are operably linked to a nucleic acid encoding a Gene Writer protein or a template nucleic acid, e.g., that controls expression of the heterologous object sequence. In certain embodiments, the one or more promoter or enhancer elements comprise cell-type or tissue specific elements. In some embodiments, the promoter or enhancer is the same or derived from the promoter or enhancer that naturally controls expression of the heterologous object sequence. For example, the ornithine transcarbomylase promoter and enhancer may be used to control expression of the ornithine transcarbomylase gene in a system or method provided by the invention for correcting ornithine transcarbomylase deficiencies. In some embodiments, a promoter for use in the invention is for a gene described in any one of Tables 9-22, e.g., which may be used with an allele of the reference gene, or, in other embodiments, with a heterologous gene. In some embodiments, the promoter is a promoter of Table 33 or a functional fragment or variant thereof.


Exemplary tissue specific promoters that are commercially available can be found, for example, at a uniform resource locator (e.g., www.invivogen.com/tissue-specific-promoters). In some embodiments, a promoter is a native promoter or a minimal promoter, e.g., which consists of a single fragment from the 5′ region of a given gene. In some embodiments, a native promoter comprises a core promoter and its natural 5′ UTR. In some embodiments, the 5′ UTR comprises an intron, in other embodiments, these include composite promoters, which combine promoter elements of different origins or were generated by assembling a distal enhancer with a minimal promoter of the same origin.


Exemplary cell or tissue specific promoters are provided in the tables, below, and exemplary nucleic acid sequences encoding them are known in the art and can be readily accessed using a variety of resources, such as the NCBI database, including RefSeq, as well as the Eukaryotic Promoter Database (//epd.epfl.ch//index.php).









TABLE 33







Exemplary cell or tissue-specific promoters










Promoter
Target cells







B29 Promoter
B cells



CD14 Promoter
Monocytic Cells



CD43 Promoter
Leukocytes and platelets



CD45 Promoter
Hematopoeitic cells



CD68 promoter
macrophages



Desmin promoter
muscle cells



Elastase-1 promoter
pancreatic acinar cells



Endoglin promoter
endothelial cells



fibronectin promoter
differentiating cells, healing tissue



Flt-1 promoter
endothelial cells



GFAP promoter
Astrocytes



GPIIB promoter
megakaryocytes



ICAM-2 Promoter
Endothelial cells



INF-Beta promoter
Hematopoeitic cells



Mb promoter
muscle cells



Nphs1 promoter
podocytes



OG-2 promoter
Osteoblasts, Odonblasts



SP-B promoter
Lung



Syn1 promoter
Neurons



WASP promoter
Hematopoeitic cells



SV40/bAlb promoter
Liver



SV40/bAlb promoter
Liver



SV40/Cd3 promoter
Leukocytes and platelets



SV40/CD45 promoter
hematopoeitic cells



NSE/RU5′ promoter
Mature Neurons

















TABLE 34







Additional exemplary cell or tissue-specific promoters









Promoter
Gene Description
Gene Specificity





APOA2
Apolipoprotein A-II
Hepatocytes (from hepatocyte




progenitors)


SERPINA1
Serpin peptidase inhibitor,
Hepatocytes


(hAAT)
clade A (alpha-1
(from definitive endoderm



antiproteinase, antitrypsin),
stage)



member 1 (also named alpha




1 anti-tryps in)



CYP3A
Cytochrome P450, family 3,
Mature Hepatocytes



subfamily A, polypeptide



MIR122
MicroRNA 122
Hepatocytes




(from early stage embryonic




liver cells)




and endoderm







Pancreatic specific promoters









INS
Insulin
Pancreatic beta cells




(from definitive endoderm




stage)


IRS2
Insulin receptor substrate 2
Pancreatic beta cells


Pdx1
Pancreatic and duodenal
Pancreas



homeobox 1
(from definitive endoderm




stage)


Alx3
Aristaless-like homeobox 3
Pancreatic beta cells




(from definitive endoderm




stage)


Ppy
Pancreatic polypeptide
PP pancreatic cells




(gamma cells)







Cardiac specific promoters









Myh6
Myosin, heavy chain 6,
Late differentiation marker of


(aMHC)
cardiac muscle, alpha
cardiac muscle cells (atrial




specificity)


MYL2
Myosin, light chain 2,
Late differentiation marker


(MLC-2v)
regulatory, cardiac, slow
of cardiac muscle cells




(ventricular specificity)


ITNN13
Troponin I type 3 (cardiac)
Cardiomyocytes


(cTnl)

(from immature state)


ITNN13
Troponin I type 3 (cardiac)
Cardiomyocytes


(cTnl)

(from immature state)


NPPA
Natriuretic peptide precursor
Atrial specificity in adult cells


(ANF)
A (also named Atrial




Natriuretic Factor)



Slc8a1
Solute carrier family 8
Cardiomyocytes from early


(Ncx1)
(sodium/calcium exchanger),
developmental stages



member 1








CNS specific promoters









SYN1
Synapsin I
Neurons


(hSyn)




GFAP
Glial fibrillary acidic protein
Astrocytes


INA
lntemexin neuronal
Neuroprogenitors



intermediate filament




protein, alpha (a-internexin)



NES
Nestin
Neuroprogenitors and




ectoderm


MOBP
Myelin-associated
Oligodendrocytes



oligodendrocyte basic




protein



MBP
Myelin basic protein
Oligodendrocytes


TH
Tyrosine hydroxylase
Dopaminergic neurons


FOXA2
Forkhead box A2
Dopaminergic neurons (also


(HNF3

used as a marker of


beta)

endoderm)







Skin specific promoters









FLG
Filaggrin
Keratinocytes from granular




layer


K14
Keratin 14
Keratinocytes from granular




and basal layers


TGM3
Transglutaminase 3
Keratinocytes from granular




layer







Immune cell specific promoters









ITGAM
lntegrin, alpha M
Monocytes, macrophages,


(CD11B)
(complement component 3
granulocytes, natural killer



receptor 3 subunit)
cells







Urogential cell specific promoters









Pbsn
Probasin
Prostatic epithelium


Upk2
Uroplakin 2
Bladder


Sbp
Spermine binding protein
Prostate


Ferl14
Fer-1-like 4
Bladder







Endothelial cell specific promoters









ENG
Endoglin
Endothelial cells







Pluripotent and embryonic cell specific promoters









Oct4
POU class 5 homeobox 1
Pluripotent cells


(POU5F1)

(germ cells, ES cells, iPS




cells)


NANOG
Nanog homeobox
Pluripotent cells




(ES cells, iPS cells)


Synthetic
Synthetic promoter based on
Pluripotent cells (ES cells, iPS


Oct4
a Oct-4 core enhancer
cells)



element



T
Brachyury
Mesoderm


brachyury




NES
Nestin
Neuroprogenitors and




Ectoderm


SOX17
SRY (sex determining region
Endoderm



Y)-box 17



FOXA2
Forkhead box A2
Endoderm (also used as a


(HNFJ

marker of dopaminergic


beta)

neurons)


MIR122
MicroRNA 122
Endoderm and hepatocytes




(from early stage embryonic




liver cells~









Depending on the host/vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544; incorporated herein by reference in its entirety).


In some embodiments, a nucleic acid encoding a Gene Writer or template nucleic acid is operably linked to a control element, e.g, a transcriptional control element, such as a promoter. The transcriptional control element may, in some embodiment, be functional in either a eukaryotic cell, e.g., a mammalian cell; or a prokaryotic cell (e.g., bacterial or archaeal cell) In some embodiments, a nucleotide sequence encoding a polypeptide is operably linked to multiple control elements, e.g., that allow expression of the nucleotide sequence encoding the polypeptide in both prokaryotic and eukaryotic cells.


For illustration purposes, examples of spatially restricted promoters include, but are not limited to, neuron-specific promoters, adipocyte-specific promoters, cardiomyocyte-specific promoters, smooth muscle-specific promoters, photoreceptor-specific promoters, etc. Neuron-specific spatially restricted promoters include, but are not limited to, a neuron-specific enolase (NSE) promoter (see, e.g., EMBL HSENO2, X51956); an aromatic amino acid decarboxylase (AADC) promoter, a neurofilament promoter (see, e.g., GenBank H-UMNFL, L04147); a synapsin promoter (see, e.g., GenBank HUMSYNIB, M55301); a thy-I promoter (see, e.g., Chen et al. (1987) Cell 51:7-19; and Llewellyn, et al. (2010) Nat. Med. 16(10):1161-1166); a serotonin receptor promoter (see, e.g., (GenBank S62283); a tyrosine hydroxylase promoter (TH) (see, e.g., Oh et al. (2009) Gene Ther 16:437; Sasaoka et al. (1992) Mol. Brain Res. 16:274; Boundy et al. (1998) J. Neurosci 18:9989; and Kaneda et al. (1991) Neuron 6:583-594); a GnRH promoter (see, e.g., Radovick et al. (1991) Proc. Natl. Acad. Sci. USA 88:3402-3406); an L7 promoter (see, e.g., Oberdick et al (1990) Science 248:223-226); a DNMT promoter (see, e.g., Bartge et al. (1988) Proc. Natl. Acad. Sci. USA 85:3648-3652); an enkephalin promoter (see, e.g., Comb et al. (1988) EMBO J. 17:3793-3805); a myelin basic protein (MR1P) promoter; a Ca2+-calmodulin-dependent protein kinase III-alpha (CamKIIα) promoter (see, e.g., Mayford et al. (1996) Proc. Natl. Acad. Sci. USA 93:13250; and Casanova et al. (2001) Genesis 31:37); a CMV enhancer/platelet-derived growth factor-β promoter (see, e.g. Liu et al, (2004) Gene Therapy 11:52-60); and the like.


Adipocyte-specific spatially restricted promoters include, but are not limited to, the aP2 gene promoter/enhancer, e.g., a region from −5.4 kb to +21 bp of a human aP2 gene (see, e.g., Tozzo et al. (1997) Endocrinol. 138:1604: Ross et al. (1990) Proc. Natl. Acad. Sci. USA 87:9590; and Pavjani et al. (2005) Nat. Med, 11:797); a glucose transporter-4 (GLUT4) promoter (see, e.g., Knight et al. (2003) Proc. Natl. Acad. Sci. USA 100:14725); a fatty acid translocase (FAT/CD36) promoter (see, e g., Kuriki et al (2002) Biol Pharm. Bull. 25:1476; and Sato et al (2002) J. Biol. Chem. 277:15703); a stearoyl-CoA desaturase-1 (SCD1) promoter (Tabor et al. (1999) J. Biol. Chem. 274:20603); a leptin promoter (see, e.g., Mason et al (1998) Endocrinol. 139:1013; and Chen et al. (1999) Biochem. Biophys. Res. Comm. 262:187); an adiponectin promoter (see, e.g., Kita et al. (2005) Biochem Biophys Res. Comm. 331:484; and Chakrabarti (2010) Endocrinol. 151:2408); an adipsin promoter (see, e.g., Platt et al (1989) Proc. Natl. Acad. Sci. USA 86:7490); a resistin promoter (see, e.g., Seo et al. (2003) Molec. Endocrinol. 17:1522); and the like.


Cardiomyocyte-specific spatially restricted promoters include, but are not limited to, control sequences derived from the following genes: myosin light chain-2, α-myosin heavy chain, AE3, cardiac troponin C, cardiac actin, and the like. Franz et al. (1997) Cardiovasc. Res, 35:560-566; Robbins et al (1995) Ann N.Y. Acad. Sci. 752:492-505; Linn et al. (1995) Circ. Res. 76:584-591: Parmacek et al. (1994) Mol. Cell. Biol. 14:1870-1885; Hunter et al. (1993) Hypertension 22:608-617; and Sartorelli et al. (1992) Proc. Nat. Acad. Sci. USA. 89:4047-4051


Smooth muscle-specific spatially restricted promoters include, but are not limited to, an SM22α promoter (see, e.g., Akynrek et al. (2000) Mol. Med. 6:983; and U.S. Pat. No. 7,169,874); a smnoothelin promoter (see, e.g., WO 2001/018048); an c-smooth muscle actin promoter; and the like. For example, a 0.4 kb region of the SM22a promoter, within which lie two CArG elements, has been shown to mediate vascular smooth muscle cell-specific expression (see, e g., Kin, et al (1997) Mol. Cell. Biol. 17, 2266-2278; Li, et al., (1996) J. Cell Biol. 132, 849-859; and Moessler, et al. (1996) Development 122, 2415-2425).


Photoreceptor-specific spatially restricted promoters include, but are not limited to, a rhodopsin promoter; a rhodopsin kinase promoter (Young et al. (2003) Ophthalmol. Vis. Sci. 44:4076); a beta phosphodiesterase gene promoter (Nicoud et al (2007) J. Gene Med. 9.1015); a retinitis pigmentosa gene promoter (Nicoud et al. (2007) supra); an interphotoreceptor retinoid-binding protein (JRBP) gene enhancer (Nicoud et al (2007) supra): an IRBP gene promoter (Yokoyama et al. (1992) Exp Eye Res. 55:225); and the like.


Nonlimiting Exemplary Cell-Specific Promoters


Cell-specific promoters known in the art may be used to direct expression of a Gene Writer protein, e.g., as described herein Nonlimiting exemplary mammalian cell-specific promoters have been characterized and used in mice expressing Cre recombinase in a cell-specific manner. Certain nonlimiting exemplary mammalian cell-specific promoters are listed in Table 1 of U.S. Pat. No. 9,845,481, incorporated herein by reference.


In some embodiments, a cell-specific promoters is a promoter that is active in plants. Many exemplary cell-specific plant promoters are known in the art. See, e.g., U.S. Pat. Nos. 5,097,025; 5,783,393; 5,880,330; 5,981,727; 7,557,264; 6,291,666; 7,132,526; and 7,323,622; and U.S. Publication Nos. 2010/0269226; 2007/0180580; 2005/0034192; and 2005/0086712, which are incorporated by reference herein in their entireties for any purpose.


In some embodiments, a vector as described herein comprises an expression cassette. The term “expression cassette”, as used herein refers to a nucleic acid construct comprising nucleic acid elements sufficient for the expression of the nucleic acid molecule of the instant invention. Typically, an expression cassette comprises the nucleic acid molecule of the instant invention operatively linked to a promoter sequence. The term“operatively linked” refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operatively linked with a coding sequence when it is capable of affecting the expression of that coding sequence (eg. the coding sequence is under the transcriptional control of the promoter). Encoding sequences can be operatively linked to regulatory sequences in sense or antisense orientation. In certain embodiments, the promoter is a heterologous promoter. The term“heterologous promoter”, as used herein, refers to a promoter that is not found to be operatively linked to a given encoding sequence in nature in certain embodiments, an expression cassette may comprise additional elements, for example, an intron, an enhancer, a polyadenylation site, a woodchuck response element (WRE), and/or other elements known to affect expression levels of the encoding sequenceA“promoter” typically controls the expression of a coding sequence or functional RNA. In certain embodiments, a promoter sequence comprises proximal and more distal upstream elements and can further comprise an enhancer element. An “enhancer” can typically stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. In certain embodiments, the promoter is derived in its entirety from a native gene. In certain embodiments, the promoter is composed of different elements derived from different naturally occurring promoters. In certain embodiments, the promoter comprises a synthetic nucleotide sequence. It will be understood by those skilled in the art that different promoters will direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions or to the presence or the absence of a drug or transcriptional co-factor. Ubiquitous, cell-type-specific, tissue-specific, developmental stage-specific, and conditional promoters, for example, drug-responsive promoters (e.g., tetracycline-responsive promoters) are well known to those of skill in the art. Examples of promoter include, but are not limited to, the phosphoglycerate kinase (PKG) promoter, CAG (composite of the CMV enhancer the chicken beta actin promoter (CBIA) and the rabbit beta globin intron.), NSE (neuronal specific enolase), synapsin or NeuN promoters, the SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MILP), a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), SFFV promoter, rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like, Other promoters can be of human origin or from other species, including from mice. Common promoters include, e.g., the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, [beta]-actin, rat insulin promoter, the phosphoglycerate kinase promoter, the human alpha-1 antitrypsin (hAAT) promoter, the transthyretin promoter, the TBG promoter and other liver-specific promoters, the desmin promoter and similar muscle-specific promoters, the EF1-alpha promoter, the CAG promoter and other constitutive promoters, hybrid promoters with multi-tissue specificity, promoters specific for neurons like synapsin and glyceraldehyde-3-phosphate dehydrogenase promoter, all of which are promoters well known and readily available to those of skill in the art, can be used to obtain high-level expression of the coding sequence of interest. In addition, sequences derived from non-viral genes, such as the murine metallothionein gene, will also find use herein Such promoter sequences are commercially available from, e.g., Stratagene (San Diego, CA). Additional exemplary promoter sequences are described, for example, in WO2018213786A1 (incorporated by reference herein in its entirety).


In some embodiments, the apolipoprotein E enhancer (ApoE) or a functional fragment thereof is used, e.g., to drive expression in the liver. In some embodiments, two copies of the ApoE enhancer or a functional fragment thereof is used. In some embodiments, the ApoE enhancer or functional fragment thereof is used in combination with a promoter, e.g., the human alpha-1 antitrypsin (hAAT) promoter.


In some embodiments, the regulatory sequences impart tissue-specific gene expression capabilities. In some cases, the tissue-specific regulatory sequences bind tissue-specific transcription factors that induce transcription in a tissue specific manner. Various tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are known in the art. Exemplary tissue-specific regulatory sequences include, but are not limited to, the following tissue-specific promoters: a liver-specific thyroxin binding globulin (TUG) promoter, a insulin promoter, a glucagon promoter, a somatostatin promoter, a pancreatic polypeptide (PPY) promoter, a synapsin-1 (Syn) promoter, a creatine kinase (MCK) promoter, a mammalian desmin (DES) promoter, a α-myosin heavy chain (a-MHC) promoter, or a cardiac Troponin T (cTniT) promoter. Other exemplary promoters include Beta-actin promoter, hepatitis B virus core promoter, Sandig et al., Gene Ther., 3:1002-9 (1996); alpha-fetoprotein (AFP) promoter, Arbuthnot et al., Hum Gene Ther., 7:1503-14 (1996)) bone osteocalcin promoter (Stein et alt, Mol. Biol, Rep., 24.185-96 (1997)); bone sialoprotein promoter (When et al., J. Bone Miner. Res., 11:654-64 (1996)), CD2 promoter (Hansal et al, J Immunol, 161:1063-8 (1998); imnunoglobulin heavy chain promoter; T cell receptor α-chain promoter, neuronal such as neuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mo. Neurobiol., 13:503-15 (1993)), neurofilament light-chain gene promoter (Piccioli et al, Proc. Natl Acad. Sci. USA, 88:5611-5 (1991)), and the neuron-specific vgf gene promoter (Piccioli et al., Neuron, 15:373-84 (1995)) and others. Additional exemplary promoter sequences are described, for example, in U.S. patent Ser. No. 10/300,146 (incorporated herein by reference in its entirety). In some embodiments, a tissue-specific regulatory element, e g., a tissue-specific promoter, is selected from one known to be operably linked to a gene that is highly expressed in a given tissue, e.g., as measured by RNA-seq or protein expression data, or a combination thereof. Methods for analyzing tissue specificity by expression are taught in Fagerberg et al. Moll Cell Proteomics 13(2):397-406 (2014), which is incorporated herein by reference in its entirety.


In some embodiments, a vector described herein is a multicistronic expression construct Multicistronic expression constructs include, for example, constructs harboring a first expression cassette, e.g. comprising a first promoter and a first encoding nucleic acid sequence, and a second expression cassette, e.g. comprising a second promoter and a second encoding nucleic acid sequence. Such multicistronic expression constructs may, in some instances, be particularly useful in the delivery of non-translated gene products, such as hairpin RNAs, together with a polypeptide, for example, a gene writer and gene writer template. In some embodiments, multicistronic expression constructs may exhibit reduced expression levels of one or more of the included transgenes, for example, because of promoter interference or the presence of incompatible nucleic acid elements in close proximity. If a multicistronic expression construct is part of a viral vector, the presence of a self-complementary nucleic acid sequence may, in some instances, interfere with the formation of structures necessary for viral reproduction or packaging.


In some embodiments, the sequence encodes an RNA with a hairpin. In some embodiments, the hairpin RNA is an a guide RNA, a template RNA, shRNA, or a microRNA. In some embodiments, the first promoter is an RNA polymerase I promoter. In some embodiments, the first promoter is an RNA polymerase II promoter. In some embodiments, the second promoter is an RNA polymerase III promoter. In some embodiments, the second promoter is a U6 or H1I promoter. In some embodiments, the nucleic acid construct comprises the structure of AAV construct B1 or B2.


Without wishing to be bound by theory, multicistronic expression constructs may not achieve optimal expression levels as compared to expression systems containing only one cistron. One of the suggested causes of lower expression levels achieved with multicistronic expression constructs comprising two ore more promoter elements is the phenomenon of promoter interference (see, e.g., Curtin J A, Dane A P, Swanson A, Alexander I E, Ginn S L. Bidirectional promoter interference between two widely used internal heterologous promoters in a late-generation lentiviral construct. Gene Ther. 2008 March; 15(5):384-90; and Martin-Duque P, Jezzard S, Kaftansis L, Vassaux G. Direct comparison of the insulating properties of two genetic elements in an adenoviral vector containing two different expression cassettes. Hum Gene Ther. 2004 October: 15(10):995-1002; both references incorporated herein by reference for disclosure of promoter interference phenomenon). In some embodiments, the problem of promoter interference may be overcome, e.g., by producing multicistronic expression constructs comprising only one promoter driving transcription of multiple encoding nucleic acid sequences separated by internal ribosomal entry sites, or by separating cistrons comprising their own promoter with transcriptional insulator elements. In some embodiments, single-promoter driven expression of multiple cistrons may result in uneven expression levels of the cistrons. In some embodimems, a promoter cannot efficiently be isolated and isolation elements may not be compatible with some gene transfer vectors, for example, some retroviral vectors.


MicroRNAs


miRNAs and other small interfering nucleic acids generally regulate gene expression via target RNA transcript cleavage/degradation or translational repression of the target messenger RNA (mRNA). miRNAs may, in some instances, be natively expressed, typically as final 19-25 non-translated RNA products. miRNAs generally exhibit their activity through sequence-specific interactions with the 3′ untranslated regions (URI of target mRNAs. These endogenously expressed miRNAs may forms hairpin precursors that are subsequently processed into an miRNA duplex, and further into a mature single stranded miRNA molecule. This mature miRNA generally guides a multiprotein complex, miRISC, which identifies target 3′ UTR regions of target mRNAs based upon their complementarity to the mature miRNA. Useful transgene products may include, for example, miRNAs or miRNA binding sites that regulate the expression of a linked polypeptide A non-limiting list of miRNA genes; the products of these genes and their homologues are useful as transgenes or as targets for small interfering nucleic acids (e.g., miRNA sponges, antisense oligonucleotides), e.g., in methods such as those listed in U.S. Ser. No. 10/300,146, 22:25-25:48, incorporated by reference. In some embodiments, one or more binding sites for one or more of the foregoing miRNAs are incorporated in a transgene, e.g., a transgene delivered by a rAAV vector, e.g., to inhibit the expression of the transgene in one or more tissues of an animal harboring the transgene. In some embodiments, a binding site may be selected to control the expression of a trangenes in a tissue specific manner. For example, binding sites for the liver-specific miR-122 may be incorporated into a transgene to inhibit expression of that transgene in the liver. Additional exemplary miRNA sequences are described, for example, in U.S. patent Ser. No. 10/300,146 (incorporated herein by reference in its entirety). For liver-specific Gene Writing, however, overexpression of miR-122 may be utilized instead of using binding sites to effect miR-122-specific degradation. This miRNA is positively associated with hepatic differentiation and maturation, as well as enhanced expression of liver specific genes. Thus, in some embodiments, the coding sequence for miR-122 may be added to a component of a Gene Writing system to enhance a liver-directed therapy.


A miR inhibitor or miRNA inhibitor is generally an agent that blocks miRNA expression and/or processing. Examples of such agents include, but are not limited to, microRNA antagonists, microRNA specific antisense, microRNA sponges, and microRNA oligonucleotides (double-stranded, short oligonucleotides) that inhibit miRNA interaction with a Drosha complex. MicroRNA inhibitors, e.g., miRNA sponges, can be expressed in cells from transgenes (e.g., as described in Ebert, M. S. Nature Methods, Epub Aug. 12, 2007, incorporated by reference herein in its entirety). In some embodiments, microRNA sponges, or other miR inhibitors, are used with the AAVs. microRNA sponges generally specifically inhibit miRNAs through a complementary heptameric seed sequence. In some embodiments, an entire family of miRNAs can be silenced using a single sponge sequence. Other methods for silencing miRNA function (derepression of mRNA targets) in cells wil be apparent to one of ordinary skill in the art.


In some embodiments, a miRNA as described herein comprises a sequence listed in Table 4 of PCT Publication No. WO2020014209, incorporated herein by reference. Also incorporated herein by reference are the listing of exemplary miRNA sequences from WO2020014209.


In some embodiments, it is advantageous to silence one or more components of a Gene Writing system (e.g., mRNA encoding a Gene Writer polypeptide, a Gene Writer Template RNA, or a heterologous object sequence expressed from the genome after successful Gene Writing) in a portion of cells. In some embodiments, it is advantageous to restrict expression of a component of a Gene Writing system to select cell types within a tissue of interest.


For example, it is known that in a given tissue, e.g., liver, macrophages and immune cells, e.g., Kupffer cells in the liver, may engage in uptake of a delivery vehicle for one or more components of a Gene Writing system. In some embodiments, at least one binding site for at least one miRNA highly expressed in macrophages and immune cells, e.g., Kupffer cells, is included in at least one component of a Gene Writing system, e.g., nucleic acid encoding a Gene Writing polypeptide or a transgene. In some embodiments, a miRNA that targets the one or more binding sites is listed in a table referenced herein, e.g., miR-142, e.g., mature miRNA hsa-miR-142-5p or hsa-miR-142-3p.


In some embodiments, there may be a benefit to decreasing Gene Writer levels and/or Gene Writer activity in cells in which Gene Writer expression or overexpression of a transgene may have a toxic effect. For example, it has been shown that delivery of a transgene overexpression cassette to dorsal root ganglion neurons may result in toxicity of a gene therapy (see Hordeaux et al Sci Transl Med 12(569):eaba9188 (2020), incorporated herein by reference in its entirety). In some embodiments, at least one miRNA binding site may be incorporated into a nucleic acid component of a Gene Writing system to reduce expression of a system component in a neuron, e.g., a dorsal root ganglion neuron. In some embodiments, the at least one miRNA binding site incorporated into a nucleic acid component of a Gene Writing system to reduce expression of a system component in a neuron is a binding site of miR-182, e.g., mature miRNA hsa-miR-182-5p or hsa-miR-182-3p. In some embodiments, the at least one miRNA binding site incorporated into a nucleic acid component of a Gene Writing system to reduce expression of a system component in a neuron is a binding site of miR-183, e.g., mature miRNA hsa-miR-183-5p or hsa-miR-183-3p. In some embodiments, combinations of miRNA binding sites may be used to enhance the restriction of expression of one or more components of a Gene Writing system to a tissue or cell type of interest.


Table A5 below below provides exemplary miRNAs and corresponding expressing cells, e.g., a miRNA for which one can, in some embodiments, incorporate binding sites (complementary sequences) in the transgene or polypeptide nucleic acid, e.g., to decrease expression in that off-target cell.









TABLE A5







Exemplary miRNA from off-target


cells and tissues











Silenced



SEQ


cell
miRNA
Mature
miRNA
ID


type
name
miRNA
sequence
NO:





Kupffer
miR-142
hsa-miR-
cauaaaguaga
3567


cells

142-5p
aagcacuacu






Kupffer
miR-142
hsa-miR-
uguaguguuuc
1684


cells

142-3p
cuacuuuaugga






Dorsal
miR-182
hsa-miR-
uuuggcaauggu
3568


root

182-5p
agaacucacacu



ganglion






neurons









Dorsal
miR-182
hsa-miR-
ugguucuagacu
3569


root

182-3p
ugccaacua



ganglion






neurons









Dorsal
miR-183
hsa-miR-
uauggcacuggu
3570


root

183-5p
agaauucacu



ganglion






neurons









Dorsal
miR-183
hsa-miR-
gugaauuaccga
3571


root

183-3p
agggccauaa



ganglion






neurons









Hepatocytes
miR-122
hsa-miR-
uggagugugaca
3572




122-5p
augguguuug






Hepatocytes
miR-122
hsa-miR-
aacgccauuauc
3573




122-3p
acacuaaaua









Anticrispr Systems for Regulating GeneWriter Activity


Various approaches for modulating Cas molecule activity may be used in conjunction with the systems and methods described herein. For instance, in some embodiments, a polypeptide described herein (e.g., a Cas molecule or a GeneWriter comprising a Cas domain) can be regulated using an anticrispr agent (e.g., an anticrispr protein or anticrispr small molecule). In some embodiments, the Cas molecule or Cas domain comprises a responsive intein such as, for example, a 4-hydroxytamoxifen (4-HT)-responsive intein, an iCas molecule (e.g., iCas9); a 4-HT-responsive Cas (e.g., allosterically regulated Cas9 (arC9) or dead Cas9 (dC9)). The systems and methods described herein can also utilize a chemically-induced dimerization system of split protein fragments (e.g., rapamycin-mediated dimerization of FK506 binding protein 12 (FKBP) and FKBP rapamycin binding domain (FRB), an abscisic acid-inducible ABI-PYL1 and gibberellin-inducible GID1-GAI heterodimerization domains); a dimer of BCL-xL peptide and BH3 peptides, a A385358 (A3) small molecule, a degron system (e.g., a FKBP-Cas9 destabilized system, an auxin-inducible degron (AID) or an E. coli DHFR degron system), an aptamer or aptazyme fused with gRNA (e.g., tetracycline- and theophylline-responsive bioswitches), AcrIIA2 and AcrIIA4 proteins, and BRD0539.


In some embodiments, a small molecule-responsive intein (e.g., 4-hydroxytamoxifen (4-HT)-responsive intein) is inserted at specific sites within a Cas molecule (e.g., Cas9). In some embodiments, the insertion of a 4HT-responsive intein disrupts Cas9 enzymatic activity. In some embodiments, a Cas molecule (e.g., iCas9) is fused to the hormone binding domain of the estrogen receptor (ERT2). In some embodiments, the ligand binding domain of the human estrogen receptor-a can be inserted into a Cas molecule (e.g., Cas9 or dead Cas9 (dC9)), e.g., at position 231, yielding a 4HT-responsive anticrispr Cas9 (e.g., arC9 or dC9). In some embodiments, dCas9 can provide 4-HT dose-dependent repression of Cas9 function. In some embodiments, arC9 can provide 4-HT dose-dependent control of Cas9 function. In some embodiments, a Cas molecule (e.g., Cas9) is fused to split protein fragments. In some embodiments, chemically-induced dimerization of split protein fragments (e.g., rapamycin-mediated dimerization of FK506 binding protein 12 (FKBP) and FKBP rapamycin binding domain (FRB)) can induce low levels of Cas9 molecule activity. In some embodiments, a chemically-induced dimerization system (e.g., abscisic acid-inducible ABI-PYL1 and gibberellin-inducible GID1-GAI heterodimerization domains) can induce a dose-dependent and reversible transcriptional activation/repression of Cas9. In some embodiments, a Cas9 inducible system (ciCas9) comprises the replacement of a Cas molecule (e.g., Cas9) REC2 domain with a BCL-xl peptide and attachment of a BH3 peptide to the N- and C-termini of the modified Cas9.BCL. In some embodiments, the interaction between BCL-xL and BH3 peptides can keep Cas9 in an inactive state. In some embodiments, a small molecule (e.g., A-385358 (A3)) can disrupt the interaction between BLC-xl and BH3 peptides to activate Cas9. In some embodiments, a Cas9 inducible system can exhibit dose-dependent control of nuclease activity.


In some embodiments, a degron system can induce degradation of a Cas molecule (e.g., Cas9) upon activation or deactivation by an external factor (e.g., small-molecule ligand, light, temperature, or a protein). In some embodiments, a small molecule BRD0539 inhibits a Cas molecule (e.g., Cas9) reversibly. Additional information on anticrispr proteins or anticrispr small molecules can be found, for example, in Gangopadhyay, S. A. et al. Precision control of CRISPR-Cas9 using small molecules and light, Biochemistry, 2019, Maji, B. et al. A high-throughput platform to identify small molecule inhibitors of CRISPR-Cas9, and Pawluk Anti-CRISPR: discovery, mechanism and function Nature Reviews Microbiology volume 16, pages 12-17(2018), each of which is incorporated by reference in its entirety.


Self-Inactivating Modules for Regulating GeneWriter Activity


In some embodiments the Gene Writer systems described herein includes a self-inactivating module. The self-inactivating module leads to a decrease of expression of the Gene Writer polypeptide, the Gene Writer template, or both. Without wishing to be bound by the theory, the self-inactivating module provides for a temporary period of Gene Writer expression prior to inactivation. Without wishing to be bound by theory, the activity of the Gene Writer polypeptide at a target site introduces a mutation (e.g. a substitution, insertion, or deletion) into the DNA encoding the Gene Writer polypeptide or Gene Writer template which results in a decrease of Gene Writer polypeptide or template expression. In some embodiments of the self-inactivating module, a target site for the Gene Writer polypeptide is included in the DNA encoding the Gene Writer polypeptide or Gene Writer template. In some embodiments, one, two, three, four, five, or more copies of the target site are included in the DNA encoding the Gene Writer polypeptide or Gene Writer template. In some embodiments, the target site in the DNA encoding the Gene Writer polypeptide or Gene Writer template is the same target site as the target site on the genome. In some embodiments, the target site is a different target site than the target site on the genome. In some embodiments, the self-inactivation module target site uses the same or a different template RNA or guide RNA as the genome target site. In some embodiments, the target site is modified via target primed reverse transcription based upon a template RNA. In some embodiments the target side is nicked. The target site may be incorporated into an enhancer, a promoter, an untranslated region, an exon, an intron, an open reading frame, or a stuffer sequence.


In some embodiments, upon inactivation, the decrease of expression is 25%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or more lower than a Gene Writing system that does not contain the self-inactivating module. In some embodiments, a Gene Writer system that contains the self-inactivating module has a 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% 99%, or higher rate of integrations in target sites than off-target sites compared to a Gene Writing system that does not contain the self-inactivation module. a Gene Writer system that contains the self-inactivating module has a 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% 99%, or higher efficiency of target site modification compared to a Gene Writing system that does not contain the self-inactivation module. In some embodiments, the self-inactivating module is included when the Gene Writer polypeptide is delivered as DNA, e.g. via a viral vector.


Self-inactivating modules have been described for nucleases. See, e.g. in Li et al A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing, Mol Ther Methods Clin Dev. 2019 Mar. 15; 12: 111-122, P. Singhal, Self-Inactivating Cas9: a method for reducing exposure while maintaining efficacy in virally delivered Cas9 applications (available at www.editasmedicine.com/wp-content/uploads/2019/10/aef asgct_poster_2017_final_-_present_5-11-17_515pml_1494537387_1494558495_1497467403.pdf), and Epstein and Schaffer Engineering a Self-Inactivating CRISPR System for AAV Vectors Targeted Genome Editing IIVolume 24, SUPPLEMENT 1, S50, May 1, 2016, and WO2018106693A1.


Small Molecules


In some embodiments a polypeptide described herein (e.g., a Gene Writer polypeptide) is controllable via a small molecule. In some embodiments the polypeptide is dimerized via a small molecule.


In some embodiment, the polypeptide is controllable via Chemical Induction of Dimerization (CID) with small molecules. CID is generally used to generate switches of protein function to alter cell physiology. An exemplary high specificity, efficient dimerizer is rimiducid (AP1903), which has two identical, protein-binding surfaces arranged tail-to-tail, each with high affinity and specificity for a mutant of FKBP12: FKBP12(F36V) (FKBP12v36, FV36 or FV, Attachment of one or more FV domains onto one or more cell signaling molecules that normally rely on homodimerization can convert that protein to rimiducid control. Homodimerization with rimiducid is used in the context of an inducible caspase safety switch. This molecular switch that is controlled by a distinct dimerizer ligand, based on the heterodimerizing small molecule, rapamycin, or rapamycin analogs (“rapalogs”). Rapamycin binds to FKBP12, and its variants, and can induce heterodimerization of signaling domains that are fused to FKBP12 by binding to both FKBP12 and to polypeptides that contain the FKBP-rapamycin-binding (FRB) domain of mTOR. Provided in some embodiments of the present application are molecular switches that greatly augment the use of rapamycin, rapalogs and rimiducid as agents for therapeutic applications.


In some embodiments of the dual switch technology, a homodimerizer, such as AP1903 (rimiducid), directly induces dimerization or multimerization of polypeptides comprising an FKBP12 multimerizing region. In other embodiments, a polypeptide comprising an FKBP12 multimerization is multimerized, or aggregated by binding to a heterodimerizer, such as rapamycin or a rapalog, which also binds to an FRB or FRB variant multimerizing region on a chimeric poly peptide, also expressed in the modified cell, such as, for example, a chimeric antigen receptor. Rapamycin is a natural product macrolide that binds with high affinity (<1 nM) to FKBP12 and together initiates the high-affinity, inhibitory interaction with the FKBP-Rapamycin-Binding (FRB) domain of mTOR. FRB is small (89 amino acids) and can thereby be used as a protein “tag” or “handle” when appended to many proteins. Coexpression of a FRB-fused protein with a FKBP12-fused protein renders their approximation rapamycin-inducible (12-16). This can serve as the basis for a cell safety switch regulated by the orally available ligand, rapamycin, or derivatives of rapamycin (rapalogs) that do not inhibit mTOR at a low, therapeutic dose but instead bind with selected, Caspase-9-fused mutant FRB domains. (see Sabatini D M, et al., Cell 1994; 78(1):35-43; Brown E J, et alt, Nature. 1994, 369(6483):756-8; Chen J. et al., Proc Natl Acad Sci USA. 1995; 92(11):4947-51; and Choi J, Science. 1996: 273(5272)239-42).


In some embodiments, two levels of control are provided in the therapeutic cells. In embodiments, the first level of control may be tunable, i.e., the level of removal of the therapeutic cells may be controlled so that it results in partial removal of the therapeutic cells. In some embodiments, the chimeric antigen polypeptide comprises a binding site for rapamycin, or a rapamycin analog. In embodiments, also present in the therapeutic cell is a suicide gene, such as, for example, one encoding a caspase polypeptide. Using this controllable first level, the need for continued therapy may, in some embodiments, be balanced with the need to eliminate or reduce the level of negative side effects. In some embodiments, a rapamycin analog, a rapalog is administered to the patient, which then binds to both the caspase polypeptide and the chimeric antigen receptor, thus recruiting the caspase polypeptide to the location, and aggregating the caspase polypeptide. Upon aggregation, the caspase polypeptide induces apoptosis. The amount of rapamycin or rapamycin analog administered to the patient may vary; if the removal of a lower level of cells by apoptosis is desired, a lower level of rapamycin or rapamycin may be administered to the patient. In some embodiments, the second level of control may be designed to achieve the maximum level of cell elimination. This second level may be based, for example, on the use of rimiducid, or AP1903. If there is a need to rapidly eliminate up to 100% of the therapeutic cells, the AP1903 may be administered to the patient. The multimeric API 903 binds to the caspase polypeptide, leading to multimerization of the caspase polypeptide and apoptosis. In certain examples, second level may also be tunable, or controlled, by the level of AP 903 administered to the subject.


In certain embodiments, small molecules can be used to control genes, as described in for example, U.S. Ser. No. 10/584,351 at 47:53-56:47 (incorporated by reference herein in its entirety), together suitable ligands for the control features, e.g., in U.S. Ser. No. 10/584,351 at 56:48, et seq. as well as U10046049 at 43:27-52:20, incorporated by reference as well as the description of ligands for such control systems at 52:21, et seq.


Resolution of Gene Writing™ Events

After writing of the template nucleic acid into the target site, additional activities may be performed to increase the overall efficiency of incorporation. In some embodiments, a nick may be initiated in the genome on the non-written DNA strand to encourage copying of the newly written DNA onto the second strand. In some embodiments, the nick may be within at least 10, 20, 30, 40, 50, 60, 70 80, 90, or 100 bases of the target site. In some embodiments, this second nick is performed by the same polypeptide performing the writing. In other embodiments, the second nick may be performed by an additional polypeptide encoding nickase activity, e.g. a Cas9 nickase.


For some Gene Writer™ systems, the writing process may leave a 3′ flap containing the newly written DNA that must displace the flanking target sequence to anneal to the second genomic strand to complete the edit. In some embodiments, the 3′ flap is designed to have enhanced strand invasion capability. In some embodiments, 5′-3′ exonuclease activity is supplemented to chew back the exposed 5′ end of the displaced strand. In some embodiments, DNA ligase activity is supplemented to complete the reaction. In some embodiments, the exonuclease and/or ligase activities are optionally provided on the Gene Writer™ polypeptide. In some embodiments, the exonuclease and/or ligase activities are optionally provided separately from the Gene Writer™ polypeptide.


Based on the published mechanism of non-LTR retrotransposons, Gene Writing™ systems derived therefrom may not require supplementation of additional functions for resolution of the writing event. In some embodiments, the system may result in complete writing without requiring endogenous host factors. In some embodiments, the system may result in complete writing without the need for DNA repair. In some embodiments, the system may result in complete writing without eliciting a DNA damage response.


Chemically Modified Nucleic Acids and Nucleic Acid End Features

A nucleic acid described herein (e.g., a template nucleic acid, e.g., a template RNA; or a nucleic acid (e.g., mRNA) encoding a GeneWriter; or a gRNA) can comprise unmodified or modified nucleobases. Naturally occurring RNAs are synthesized from four basic ribonucleotides: ATP, CTP, UTP and GTP, but may contain post-transcriptionally modified nucleotides. Further, approximately one hundred different nucleoside modifications have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197). An RNA can also comprise wholly synthetic nucleotides that do not occur in nature.


In some embodiments, the chemically modification is one provided in PCT/US2016/032454, US Pat. Pub. No. 20090286852, of International Application No. WO/2012/019168, WO/2012/045075, WO/2012/135805, WO/2012/158736, WO/2013/039857, WO/2013/039861, WO/2013/052523, WO/2013/090648, WO/2013/096709, WO/2013/101690, WO/2013/106496, WO/2013/130161, WO/2013/151669, WO/2013/151736, WO/2013/151672, WO/2013/151664, WO/2013/151665, WO/2013/151668, WO/2013/151671, WO/2013/151667, WO/2013/151670, WO/2013/151666, WO/2013/151663, WO/2014/028429, WO/2014/081507, WO/2014/093924, WO/2014/093574, WO/2014/113089, WO/2014/144711, WO/2014/144767, WO/2014/144039, WO/2014/152540, WO/2014/152030, WO/2014/152031, WO/2014/152027, WO/2014/152211, WO/2014/158795, WO/2014/159813, WO/2014/164253, WO/2015/006747, WO/2015/034928, WO/2015/034925, WO/2015/038892, WO/2015/048744, WO/2015/051214, WO/2015/051173, WO/2015/051169, WO/2015/058069, WO/2015/085318, WO/2015/089511, WO/2015/105926, WO/2015/164674, WO/2015/196130, WO/2015/196128, WO/2015/196118, WO/2016/011226, WO/2016/011222, WO/2016/011306, WO/2016/014846, WO/2016/022914, WO/2016/036902, WO/2016/077125, or WO/2016/077123, each of which is herein incorporated by reference in its entirety. It is understood that incorporation of a chemically modified nucleotide into a polynucleotide can result in the modification being incorporated into a nucleobase, the backbone, or both, depending on the location of the modification in the nucleotide. In some embodiments, the backbone modification is one provided in EP 2813570, which is herein incorporated by reference in its entirety. In some embodiments, the modified cap is one provided in US Pat. Pub. No. 20050287539, which is herein incorporated by reference in its entirety.


In some embodiments, the chemically modified nucleic acid (e.g., RNA, e.g., mRNA) comprises one or more of ARCA: anti-reverse cap analog (m27.3′-OGP3G), GP3G (Unmethylated Cap Analog), m7GP3G (Monomethylated Cap Analog), m32.2.7GP3G (Trimethylated Cap Analog), m5CTP (5′-methyl-cytidine triphosphate), m6ATP (N6-methyl-adenosine-5′-triphosphate), s2UTP (2-thio-uridine triphosphate), and Ψ (pseudouridine triphosphate).


In some embodiments, the chemically modified nucleic acid comprises a 5′ cap, e.g.: a 7-methylguanosine cap (e.g., a 0-Me-m7G cap); a hypermethylated cap analog; an NAD+-derived cap analog (e.g., as described in Kiledjian, Trends in Cell Biology 28, 454-464 (2018)); or a modified, e.g., biotinylated, cap analog (e.g., as described in Bednarek et al., Phil Trans R Soc B 373, 20180167 (2018)).


In some embodiments, the chemically modified nucleic acid comprises a 3′ feature selected from one or more of: a polyA tail; a 16-nucleotide long stem-loop structure flanked by unpaired 5 nucleotides (e.g., as described by Mannironi et al., Nucleic Acid Research 17, 9113-9126 (1989)); a triple-helical structure (e.g., as described by Brown et al., PNAS 109, 19202-19207 (2012)); a tRNA, Y RNA, or vault RNA structure (e.g., as described by Labno et al., Biochemica et Biophysica Acta 1863, 3125-3147 (2016)); incorporation of one or more deoxyribonucleotide triphosphates (dNTPs), 2′O-Methylated NTPs, or phosphorothioate-NTPs; a single nucleotide chemical modification (e.g., oxidation of the 3′ terminal ribose to a reactive aldehyde followed by conjugation of the aldehyde-reactive modified nucleotide); or chemical ligation to another nucleic acid molecule.


In some embodiments, the the nucleic acid (e.g., template nucleic acid) comprises one or more modified nucleotides, e.g., selected from dihydrouridine, inosine, 7-methylguanosine, 5-methylcytidine (5mC), 5′ Phosphate ribothymidine, 2′-O-methyl ribothymidine, 2′-O-ethyl ribothymidine, 2′-fluoro ribothymidine, C-5 propynyl-deoxycytidine (pdC), C-5 propynyl-deoxyuridine (pdU), C-5 propynyl-cytidine (pC), C-5 propynyl-uridine (pU), 5-methyl cytidine, 5-methyl uridine, 5-methyl deoxycytidine, 5-methyl deoxyuridine methoxy, 2,6-diaminopurine, 5′-Dimethoxytrityl-N4-ethyl-2′-deoxycytidine, C-5 propynyl-f-cytidine (pfC), C-5 propynyl-f-uridine (pfU), 5-methyl f-cytidine, 5-methyl f-uridine, C-5 propynyl-m-cytidine (pmC), C-5 propynyl-f-uridine (pmU), 5-methyl m-cytidine, 5-methyl m-uridine, LNA (locked nucleic acid), MGB (minor groove binder) pseudouridine (Ψ), 1-N-methylpseudouridine (1-Me-Ψ), or 5-methoxyuridine (5-MO-U).


In some embodiments, the nucleic acid comprises a backbone modification, e.g., a modification to a sugar or phosphate group in the backbone. In some embodiments, the nucleic acid comprises a nucleobase modification.


In some embodiments, the nucleic acid comprises one or more chemically modified nucleotides of Table 6, one or more chemical backbone modifications of Table 7, one or more chemically modified caps of Table 7. For instance, in some embodiments, the nucleic acid comprises two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 or more) different types of chemical modifications. As an example, the nucleic acid may comprise two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 or more) different types of modified nucleobases, e.g., as described herein, e.g., in Table 6. Alternatively or in combination, the nucleic acid may comprise two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 or more) different types of backbone modifications, e.g., as described herein, e.g., in Table 7. Alternatively or in combination, the nucleic acid may comprise one or more modified cap, e.g., as described herein, e.g., in Table 8. For instance, in some embodiments, the nucleic acid comprises one or more type of modified nucleobase and one or more type of backbone modification; one or more type of modified nucleobase and one or more modified cap; one or more type of modified cap and one or more type of backbone modification; or one or more type of modified nucleobase, one or more type of backbone modification, and one or more type of modified cap.


In some embodiments, the nucleic acid comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or more) modified nucleobases. In some embodiments, all nucleobases of the nucleic acid are modified. In some embodiments, the nucleic acid is modified at one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or more) positions in the backbone. In some embodiments, all backbone positions of the nucleic acid are modified.









TABLE 6





Modified nucleotides
















5-aza-uridine
N2-methyl-6-thio-guanosine


2-thio-5-aza-midine
N2,N2-dimethyl-6-thio-guanosine


2-thiouridine
pyridin-4-one ribonucleoside


4-thio-pseudouridine
2-thio-5-aza-uridine


2-thio-pseudouridine
2-thiomidine


5-hydroxyuridine
4-thio-pseudomidine


3-methyluridine
2-thio-pseudowidine


5-carboxymethyl-uridine
3-methylmidine


1-carboxymethyl-pseudouridine
1-propynyl-pseudomidine


5-propynyl-uridine
1-methyl-1-deaza-pseudomidine


1-propynyl-pseudouridine
2-thio-1-methyl-1-deaza-


5-taurinomethyluridine
pseudouridine


1-taurinomethyl-pseudouridine
4-methoxy-pseudomidine


5-taurinomethyl-2-thio-uridine
5′-O-(1-Thiophosphate)-Adenosine


1-taurinomethyl-4-thio-uridine
5′-O-(1-Thiophosphate)-Cytidine


5-methyl-uridine
5′-O-(1-thiophosphate)-Guanosine


1-methyl-pseudouridine
5′-O-(1-Thiophophate)-Uridine


4-thio-1-methyl-pseudouridine
5′-O-(1-Thiophosphate)-


2-thio-1-methyl-pseudouridine
Pseudouridine


1-methyl-1-deaza-pseudouridine
2′-O-methyl-Adenosine


2-thio-1-methyl-1-deaza-
2′-O-methyl-Cytidine


pseudomidine
2′-O-methyl-Guanosine


dihydrouridine
2′-O-methyl-Uridine


dihydropseudouridine
2′-O-methyl-Pseudouridine


2-thio-dihydromidine
2′-O-methyl-Inosine


2-thio-dihydropseudouridine
2-methyladenosine


2-methoxyuridine
2-methylthio-N6-methyladenosine


2-methoxy-4-thio-uridine
2-methylthio-N6


4-methoxy-pseudouridine
isopentenyladenosine


4-methoxy-2-thio-pseudouridine
2-methylthio-N6-(cis-


5-aza-cytidine
hydroxyisopentenyl)adenosine


pseudoisocytidine
N6-methyl-N6-


3-methyl-cytidine
threonylcarbamoyladenosine


N4-acetylcytidine
N6-


5-formylcytidine
hydroxynorvalylcarbamoyladenosine


N4-methylcytidine
2-methylthio-N6-hydroxynorvalyl


5-hydroxymethylcytidine
carbamoyladenosine


1-methyl-pseudoisocytidine
2′-O-ribosyladenosine (phosphate)


pyrrolo-cytidine
1,2′-O-dimethylinosine


pyrrolo-pseudoisocytidine
5,2′-O-dimethylcytidine


2-thio-cytidine
N4-acetyl-2′-O-methylcytidine


2-thio-5-methyl-cytidine
Lysidine


4-thio-pseudoisocytidine
7-methylguanosine


4-thio-1-methyl-pseudoisocytidine
N2,2′-O-dimethylguanosine


4-thio-1-methyl-1-deaza-
N2,N2,2′-O-trimethylguanosine


pseudoisocytidine
2′-O-ribosylguanosine (phosphate)


1-methyl-1-deaza-
Wybutosine


pseudoisocytidine
Peroxywybutosine


zebularine
Hydroxywybutosine


5-aza-zebularine
undermodified hydroxywybutosine


5-methyl-zebularine
methylwyosine


5-aza-2-thio-zebularine
queuosine


2-thio-zebularine
epoxyqueuosine


2-methoxy-cytidine
galactosyl-queuosine


2-methoxy-5-methyl-cytidine
mannosyl-queuosine


4-methoxy-pseudoisocytidine
7-cyano-7-deazaguanosine


4-methoxy-l-methyl-
7-aminomethyl-7-deazaguanosine


pseudoisocytidine
archaeosine


2-aminopurine
5,2′-O-dimethyluridine


2,6-diaminopurine
4-thiouridine


7-deaza-adenine
5-methyl-2-thiouridine


7-deaza-8-aza-adenine
2-thio-2′-O-methyluridine


7-deaza-2-aminopurine
3-(3-amino-3-carboxypropyl)uridine


7-deaza-8-aza-2-aminopurine
5-methoxyuridine


7-deaza-2,6-diaminopurine
uridine 5-oxyacetic acid


7-deaza-8-aza-2,6-diarninopurine
uridine 5-oxyacetic acid methyl


1-methyladenosine
ester


N6-isopentenyladenosine
5-(carboxyhydroxymethyl)uridine)


N6-(cis-)adenosine
5-(carboxyhydroxymethyl)uridine


hydroxyisopentenyl
methyl ester


2-methylthio-N6-(cis-
5-methoxycarbonylmethyluridine


hydroxyisopentenyl)
5-methoxycarbonylmethyl-2′-O-


adenosine
methyluridine


N6-glycinylcarbamoyladenosine
5-methoxycarbonylmethyl-2-


N6-threonylcarbamoyladenosine
thiouridine


2-methylthio-N6-threonyl
5-aminomethyl-2-thiouridine


carbamoyladenosine
5-methylaminomethyluridine


N6,N6-dimethyladenosine
5-methylaminomethyl-2-thiouridine


7-methyladenine
5-methylaminomethyl-2-


2-methylthio-adenine
selenouridine


2-methoxy-adenine
5-carbamoylmethyluridine


inosine
5-carbamoylmethyl-2′-O-


1-methyl-inosine
methyluridine


wyosine
5-


wybutosine
carboxymethylaminomethyluridine


7-deaza-guanosine
5-carboxymethylaminomethyl-2′-O-


7-deaza-8-aza-guanosine
methyluridine


6-thio-guanosine
5-carboxymethylaminomethyl-2-


6-thio-7-deaza-guanosine
thiouridine


6-thio-7-deaza-8-aza-guanosine
N4,2′-O-dimethylcytidine


7-methyl-guanosine
5-carboxymethyluridine


6-thio-7-methyl-guanosine
N6,2′-O-dimethyladenosine


7-methylinosine
N,N6,O-2′-trimethyladenosine


6-methoxy-guanosine
N2,7-dimethylguanosine


1-methylguanosine
N2,N2,7-trimethylguanosine


N2-methylguanosine
3,2′-O-dimethyluridine


N2,N2-dimethylguanosine
5-methyldihydrouridine


8-oxo-guanosine
5-formyl-2′-O-methylcytidine


7-methyl-8-oxo-guanosine
1,2′-O-dimethylguanosine


1-methyl-6-thio-guanosine
4-demethylwyosine



Isowyosine



N6-acetyladenosine
















TABLE 7





Backbone modifications


















 
2′-O-Methyl backbone




Peptide Nucleic Acid (PNA) backbone




phosphorothioate backbone




morpholino backbone




carbamate backbone




siloxane backbone




sulfide backbone




sulfoxide backbone




sulfone backbone




formacetyl backbone




thioformacetyl backbone




methyleneformacetyl backbone




riboacetyl backbone




alkene containing backbone




sulfamate backbone




sulfonate backbone




sulfonamide backbone




methyleneimino backbone




methylenehydrazino backbone




amide backbone

















TABLE 8





Modified caps


















 
m7GpppA




m7GpppC




m2,7GpppG




m2,2,7GpppG




m7Gpppm7G




m7,2′OmeGpppG




m72′dGpppG




m7,3′OmeGpppG




m7,3′dGpppG




GppppG




m7GppppG




m7GppppA




m7GppppC




m2,7GppppG




m2,2,7GppppG




m7Gppppm7G




m7,2′OmeGppppG




m72′dGppppG




m7,3′OmeGppppG




m7,3′dGppppG










Production of Compositions and Systems

As will be appreciated by one of skill, methods of designing and constructing nucleic acid constructs and proteins or polypeptides (such as the systems, constructs and polypeptides described herein) are routine in the art. Generally, recombinant methods may be used. See, in general, Smales & James (Eds.), Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology), Humana Press (2005); and Crommelin, Sindelar & Meibohm (Eds.), Pharmaceutical Biotechnology: Fundamentals and Applications, Springer (2013). Methods of designing, preparing, evaluating, purifying and manipulating nucleic acid compositions are described in Green and Sambrook (Eds.), Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press (2012).


The disclosure provides, in part, a nucleic acid, e.g., vector, encoding a Gene Writer polypeptide described herein, a template nucleic acid described herein, or both. In some embodiments, a vector comprises a selective marker, e.g., an antibiotic resistance marker. In some embodiments, the antibiotic resistance marker is a kanamycin resistance marker. In some embodiments, the antibiotic resistance marker does not confer resistance to beta-lactam antibiotics. In some embodiments, the vector does not comprise an ampicillin resistance marker. In some embodiments, the vector comprises a kanamycin resistance marker and does not comprise an ampicillin resistance marker. In some embodiments, a vector encoding a Gene Writer polypeptide is integrated into a target cell genome (e.g., upon administration to a target cell, tissue, organ, or subject). In some embodiments, a vector encoding a Gene Writer polypeptide is not integrated into a target cell genome (e.g., upon administration to a target cell, tissue, organ, or subject). In some embodiments, a vector encoding a template nucleic acid (e.g., template RNA) is not integrated into a target cell genome (e.g., upon administration to a target cell, tissue, organ, or subject). In some embodiments, if a vector is integrated into a target site in a target cell genome, the selective marker is not integrated into the genome. In some embodiments, if a vector is integrated into a target site in a target cell genome, genes or sequences involved in vector maintenance (e.g., plasmid maintenance genes) are not integrated into the genome. In some embodiments, if a vector is integrated into a target site in a target cell genome, transfer regulating sequences (e.g., inverted terminal repeats, e.g., from an AAV) are not integrated into the genome. In some embodiments, administration of a vector (e.g., encoding a Gene Writer polypeptide described herein, a template nucleic acid described herein, or both) to a target cell, tissue, organ, or subject results in integration of a portion of the vector into one or more target sites in the genome(s) of said target cell, tissue, organ, or subject. In some embodiments, less than 99, 95, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1% of target sites (e.g., no target sites) comprising integrated material comprise a selective marker (e.g., an antibiotic resistance gene), a transfer regulating sequence (e.g., an inverted terminal repeat, e.g., from an AAV), or both from the vector.


Exemplary methods for producing a therapeutic pharmaceutical protein or polypeptide described herein involve expression in mammalian cells, although recombinant proteins can also be produced using insect cells, yeast, bacteria, or other cells under control of appropriate promoters. Mammalian expression vectors may comprise non-transcribed elements such as an origin of replication, a suitable promoter, and other 5′ or 3′ flanking non-transcribed sequences, and 5′ or 3′ non-translated sequences such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and termination sequences. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, splice, and polyadenylation sites may be used to provide other genetic elements required for expression of a heterologous DNA sequence. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described in Green & Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press (2012).


Various mammalian cell culture systems can be employed to express and manufacture recombinant protein. Examples of mammalian expression systems include CHO, COS, HEK293, HeLA, and BHK cell lines. Processes of host cell culture for production of protein therapeutics are described in Zhou and Kantardjieff (Eds.), Mammalian Cell Cultures for Biologics Manufacturing (Advances in Biochemical Engineering Biotechnology), Springer (2014). Compositions described herein may include a vector, such as a viral vector, e.g., a lentiviral vector, encoding a recombinant protein. In some embodiments, a vector, e.g., a viral vector, may comprise a nucleic acid encoding a recombinant protein.


Purification of protein therapeutics is described in Franks, Protein Biotechnology: Isolation, Characterization, and Stabilization, Humana Press (2013); and in Cutler, Protein Purification Protocols (Methods in Molecular Biology), Humana Press (2010).


In some embodiments, a Gene Writer™ system, polypeptide, and/or template nucleic acid (e.g., template RNA) conforms to certain quality standards. In some embodiments, a Gene Writer™ system, polypeptide, and/or template nucleic acid (e.g., template RNA) produced by a method described herein conforms to certain quality standards. Accordingly, the disclosure is directed in part to methods of manufacturing a Gene Writer™ system, polypeptide, and/or template nucleic acid (e.g., template RNA) that conforms to certain quality standards, e.g., in which said quality standards are assayed. The disclosure is further directed to methods of assaying said quality standards in a Gene Writer™ system, polypeptide, and/or template nucleic acid (e.g., template RNA). In some embodiments, quality standards include, but are not limited to:

    • (i) the length of the template RNA, e.g., whether the template RNA has a length that is above a reference length or within a reference length range, e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the template RNA present is greater than 100, 125, 150, 175, or 200 nucleotides long;
    • (ii) the presence, absence, and/or length of a polyA tail on the template RNA, e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the template RNA present contains a polyA tail (e.g., a polyA tail that is at least 5, 10, 20, 30, 50, 70, 100 nucleotides in length (SEQ ID NO: 3665));
    • (iii) the presence, absence, and/or type of a 5′ cap on the template RNA, e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the template RNA present contains a 5′ cap, e.g., whether that cap is a 7-methylguanosine cap, e.g., a O-Me-m7G cap;
    • (iv) the presence, absence, and/or type of one or more modified nucleotides (e.g., selected from pseudouridine, dihydrouridine, inosine, 7-methylguanosine, 1-N-methylpseudouridine (1-Me-Ψ), 5-methoxyuridine (5-MO-U), 5-methylcytidine (5mC), or a locked nucleotide) in the template RNA, e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the template RNA present contains one or more modified nucleotides;
    • (v) the stability of the template RNA (e.g., over time and/or under a pre-selected condition), e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the template RNA remains intact (e.g., greater than 100, 125, 150, 175, or 200 nucleotides long) after a stability test;
    • (vi) the potency of the template RNA in a system for modifying DNA, e.g., whether at least 1% of target sites are modified after a system comprising the template RNA is assayed for potency;
    • (vii) the length of the polypeptide, first polypeptide, or second polypeptide, e.g., whether the polypeptide, first polypeptide, or second polypeptide has a length that is above a reference length or within a reference length range, e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the polypeptide, first polypeptide, or second polypeptide present is greater than 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, or 2000 amino acids long (and optionally, no larger than 2500, 2000, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, or 600 amino acids long);
    • (viii) the presence, absence, and/or type of post-translational modification on the polypeptide, first polypeptide, or second polypeptide, e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the polypeptide, first polypeptide, or second polypeptide contains phosphorylation, methylation, acetylation, myristoylation, palmitoylation, isoprenylation, glipyatyon, or lipoylation, or any combination thereof;
    • (ix) the presence, absence, and/or type of one or more artificial, synthetic, or non-canonical amino acids (e.g., selected from ornithine, β-alanine, GABA, δ-Aminolevulinic acid, PABA, a D-amino acid (e.g., D-alanine or D-glutamate), aminoisobutyric acid, dehydroalanine, cystathionine, lanthionine, Djenkolic acid, Diaminopimelic acid, Homoalanine, Norvaline, Norleucine, Homonorleucine, homoserine, O-methyl-homoserine and O-ethyl-homoserine, ethionine, selenocysteine, selenohomocysteine, selenomethionine, selenoethionine, tellurocysteine, or telluromethionine) in the polypeptide, first polypeptide, or second polypeptide, e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the polypeptide, first polypeptide, or second polypeptide present contains one or more artificial, synthetic, or non-canonical amino acids;
    • (x) the stability of the polypeptide, first polypeptide, or second polypeptide (e.g., over time and/or under a pre-selected condition), e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the polypeptide, first polypeptide, or second polypeptide remains intact (e.g., greater than 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, or 2000 amino acids long (and optionally, no larger than 2500, 2000, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, or 600 amino acids long)) after a stability test;
    • (xi) the potency of the polypeptide, first polypeptide, or second polypeptide in a system for modifying DNA, e.g., whether at least 1% of target sites are modified after a system comprising the polypeptide, first polypeptide, or second polypeptide is assayed for potency; or
    • (xii) the presence, absence, and/or level of one or more of a pyrogen, virus, fungus, bacterial pathogen, or host cell protein, e.g., whether the system is free or substantially free of pyrogen, virus, fungus, bacterial pathogen, or host cell protein contamination.


In some embodiments, quality standards include, but are not limited to:

    • (i) the length of mRNA encoding the GeneWriter polypeptide, e.g., whether the mRNA has a length that is above a reference length or within a reference length range, e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the mRNA present is greater than 3000, 4000, or 5000 nucleotides long;
    • (ii) the presence, absence, and/or length of a polyA tail on the mRNA, e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the mRNA present contains a polyA tail (e.g., a polyA tail that is at least 5, 10, 20, 30, 50, 70, 100 nucleotides in length (SEQ ID NO: 3665));
    • (iii) the presence, absence, and/or type of a 5′ cap on the mRNA, e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the mRNA present contains a 5′ cap, e.g., whether that cap is a 7-methylguanosine cap, e.g., a O-Me-m7G cap;
    • (iv) the presence, absence, and/or type of one or more modified nucleotides (e.g., selected from pseudouridine, dihydrouridine, inosine, 7-methylguanosine, 1-N-methylpseudouridine (1-Me-Ψ), 5-methoxyuridine (5-MO-U), 5-methylcytidine (5mC), or a locked nucleotide) in the mRNA, e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the mRNA present contains one or more modified nucleotides;
    • (v) the stability of the mRNA (e.g., over time and/or under a pre-selected condition), e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the mRNA remains intact (e.g., greater than 100, 125, 150, 175, or 200 nucleotides long) after a stability test; or
    • (vi) the potency of the mRNA in a system for modifying DNA, e.g., whether at least 1% of target sites are modified after a system comprising the mRNA is assayed for potency.


Circular RNAs in Gene Writing System

Circular RNAs (circRNA) have been found to occur naturally in cells and have been found to have diverse functions, including both non-coding and protein coding roles in human cells. It has been shown that a circRNA can be engineered by incorporating a self-splicing intron into an RNA molecule (or DNA encoding the RNA molecule) that results in circularization of the RNA, and that an engineered circRNA can have enhanced protein production and stability (Wesselhoeft et al. Nature Communications 2018). It is contemplated that it may be useful to employ circular and/or linear RNA states during the formulation, delivery, or Gene Writing reaction within the target cell. Thus, in some embodiments of any of the aspects described herein, a Gene Writing system comprises one or more circular RNAs (circRNAs). In some embodiments of any of the aspects described herein, a Gene Writing system comprises one or more linear RNAs. In some embodiments, a nucleic acid as described herein (e.g., a nucleic acid molecule encoding a Gene Writer polypeptide, or both) is a circRNA. In some embodiments, a circular RNA molecule encodes the Gene Writer™ polypeptide. In some embodiments, the circRNA molecule encoding the Gene Writer™ polypeptide is delivered to a host cell. In some embodiments, a circular RNA molecule encodes a recombinase, e.g., as described herein. In some embodiments, the circRNA molecule encoding the recombinase is delivered to a host cell. In some embodiments, the circRNA molecule encoding the Gene Writer polypeptide is linearized (e.g., in the host cell) prior to translation. Circular RNAs (circRNA) have been found to occur naturally in cells and have been found to have diverse functions, including both non-coding and protein coding roles in human cells. It has been shown that a circRNA can be engineered by incorporating a self-splicing intron into an RNA molecule (or DNA encoding the RNA molecule) that results in circularization of the RNA, and that an engineered circRNA can have enhanced protein production and stability (Wesselhoeft et al. Nature Communications 2018). In some embodiments, the Gene Writer™ polypeptide is encoded as circRNA.


In some embodiments, the Gene Writer™ polypeptide is encoded as circRNA. While in certain embodiments the template nucleic acid is a DNA, such as a ssDNA, in some embodiments it can be provided as an RNA, e.g., with a reverse transcriptase.


In some embodiments, the circRNA comprises one or more ribozyme sequences. In some embodiments, the ribozyme sequence is activated for autocleavage, e.g., in a host cell, e.g., thereby resulting in linearization of the circRNA. In some embodiments, the ribozyme is activated when the concentration of magnesium reaches a sufficient level for cleavage, e.g., in a host cell. In some embodiments the circRNA is maintained in a low magnesium environment prior to delivery to the host cell. In some embodiments, the ribozyme is a protein-responsive ribozyme. In some embodiments, the ribozyme is a nucleic acid-responsive ribozyme.


In some embodiments, the circRNA is linearized in the nucleus of a target cell. In some embodiments, linearization of a circRNA in the nucleus of a cell involves components present in the nucleus of the cell, e.g., to activate a cleavage event. For example, the B2 and ALU retrotransposons contain self-cleaving ribozymes whose activity is enhanced by interaction with the Polycomb protein, EZH2 (Hernandez et al. PNAS 117(1):415-425 (2020)). Thus, in some embodiments, a ribozyme, e.g., a ribozyme from a B2 or ALU element, that is responsive to a nuclear element, e.g., a nuclear protein, e.g., a genome-interacting protein, e.g., an epigenetic modifier, e.g., EZH2, is incorporated into a circRNA, e.g., of a Gene Writing system. In some embodiments, nuclear localization of the circRNA results in an increase in autocatalytic activity of the ribozyme and linearization of the circRNA.


In some embodiments, an inducible ribozyme (e.g., in a circRNA as described herein) is created synthetically, for example, by utilizing a protein ligand-responsive aptamer design. A system for utilizing the satellite RNA of tobacco ringspot virus hammerhead ribozyme with an MS2 coat protein aptamer has been described (Kennedy et al. Nucleic Acids Res 42(19):12306-12321 (2014), incorporated herein by reference in its entirety) that results in activation of the ribozyme activity in the presence of the MS2 coat protein. In embodiments, such a system responds to protein ligand localized to the cytoplasm or the nucleus. In some embodiments the protein ligand is not MS2. Methods for generating RNA aptamers to target ligands have been described, for example, based on the systematic evolution of ligands by exponential enrichment (SELEX) (Tuerk and Gold, Science 249(4968):505-510 (1990); Ellington and Szostak, Nature 346(6287):818-822 (1990); the methods of each of which are incorporated herein by reference) and have, in some instances, been aided by in silico design (Bell et al. PNAS 117(15):8486-8493, the methods of which are incorporated herein by reference). Thus, in some embodiments, an aptamer for a target ligand is generated and incorporated into a synthetic ribozyme system, e.g., to trigger ribozyme-mediated cleavage and circRNA linearization, e.g., in the presence of the protein ligand. In some embodiments, circRNA linearization is triggered in the cytoplasm, e.g., using an aptamer that associates with a ligand in the cytoplasm. In some embodiments, circRNA linearization is triggered in the nucleus, e.g., using an aptamer that associates with a ligand in the nucleus. In embodiments, the ligand in to the nucleus comprises an epigenetic modifier or a transcription factor. In some embodiments the ligand that triggers linearization is present at higher levels in on-target cells than off-target cells.


It is further contemplated that a nucleic acid-responsive ribozyme system can be employed for circRNA linearization. For example, biosensors that sense defined target nucleic acid molecules to trigger ribozyme activation are described, e.g., in Penchovsky (Biotechnology Advances 32(5):1015-1027 (2014), incorporated herein by reference). By these methods, a ribozyme naturally folds into an inactive state and is only activated in the presence of a defined target nucleic acid molecule (e.g., an RNA molecule). In some embodiments, a circRNA of a Gene Writing system comprises a nucleic acid-responsive ribozyme that is activated in the presence of a defined target nucleic acid, e.g., an RNA, e.g., an mRNA, miRNA, guide RNA, gRNA, sgRNA, ncRNA, lncRNA, tRNA, snRNA, or mtRNA. In some embodiments the nucleic acid that triggers linearization is present at higher levels in on-target cells than off-target cells.


In some embodiments of any of the aspects herein, a Gene Writing system incorporates one or more ribozymes with inducible specificity to a target tissue or target cell of interest, e.g., a ribozyme that is activated by a ligand or nucleic acid present at higher levels in a target tissue or target cell of interest. In some embodiments, the Gene Writing system incorporates a ribozyme with inducible specificity to a subcellular compartment, e.g., the nucleus, nucleolus, cytoplasm, or mitochondria. In some embodiments, the ribozyme that is activated by a ligand or nucleic acid present at higher levels in the target subcellular compartment. In some embodiments, an RNA component of a Gene Writing system is provided as circRNA, e.g., that is activated by linearization. In some embodiments, linearization of a circRNA encoding a Gene Writing polypeptide activates the molecule for translation. In some embodiments, a signal that activates a circRNA component of a Gene Writing system is present at higher levels in on-target cells or tissues, e.g., such that the system is specifically activated in these cells.


In some embodiments, an RNA component of a Gene Writing system is provided as a circRNA that is inactivated by linearization. In some embodiments, a circRNA encoding the Gene Writer polypeptide is inactivated by cleavage and degradation. In some embodiments, a circRNA encoding the Gene Writing polypeptide is inactivated by cleavage that separates a translation signal from the coding sequence of the polypeptide. In some embodiments, a signal that inactivates a circRNA component of a Gene Writing system is present at higher levels in off-target cells or tissues, such that the system is specifically inactivated in these cells.


In some embodiments, nucleic acid (e.g., encoding a polypeptide, or a template DNA, or both) delivered to cells is covalently closed linear DNA, or so-called “doggybone” DNA. During its lifecycle, the bacteriophage N15 employs protelomerase to convert its genome from circular plasmid DNA to a linear plasmid DNA (Ravin et al. J Mol Biol 2001). This process has been adapted for the production of covalently closed linear DNA in vitro (see, for example, WO2010086626A1). In some embodiments, a protelomerase is contacted with a DNA containing one or more protelomerase recognition sites, wherein protelomerase results in a cut at the one or more sites and subsequent ligation of the complementary strands of DNA, resulting in the covalent linkage between the complementary strands. In some embodiments, nucleic acid (e.g., encoding a transposase, or a template DNA, or both) is first generated as circular plasmid DNA containing a single protelomerase recognition site that is then contacted with protelomerase to yield a covalently closed linear DNA. In some embodiments, nucleic acid (e.g., encoding a transposase, or a template DNA, or both) flanked by protelomerase recognition sites on plasmid or linear DNA is contacted with protelomerase to generate a covalently closed linear DNA containing only the DNA contained between the protelomerase recognition sites. In some embodiments, the approach of flanking the desired nucleic acid sequence by protelomerase recognition sites results in covalently closed circular DNA lacking plasmid elements used for bacterial cloning and maintenance. In some embodiments, the plasmid or linear DNA containing the nucleic acid and one or more protelomerase recognition sites is optionally amplified prior to the protelomerase reaction, e.g., by rolling circle amplification or PCR.


In some embodiments, nucleic acid (e.g., encoding a polypeptide, or a template nucleic acid, or both) delivered to cells is designed as minicircles, where plasmid backbone sequences not pertaining to Gene Writing™ are removed before administration to cells. For example, a minicircle may lack a bacterial origin of replication and a selectable marker. In some embodiments, the mnicircle does not comprise any bacterial sequence. Minicircles have been shown to result in higher transfection efficiencies and gene expression as compared to plasmids with backbones containing bacterial parts (e.g., bacterial origin of replication, antibiotic selection cassette) and have been used to improve the efficiency of transposition (Sharma et al Mol Ther Nucleic Acids 2013). In some embodiments, the DNA vector encoding the Gene Writer™ polypeptide is delivered as a minicircle. In some embodiments, the DNA vector containing the Gene Writer™ template nucleic acid (e.g., template RNA) is delivered as a minicircle. In some embodiments, the bacterial parts are flanked by recombination sites, e.g., attP/attB, loxP, FRT sites. In some embodiments, the addition of a cognate recombinase results in intramolecular recombination and excision of the bacterial parts. In some embodiments, the recombinase sites are recognized by phiC31 recombinase. In some embodiments, the recombinase sites are recognized by Cre recombinase. In some embodiments, the recombinase sites are recognized by FLP recombinase. In addition to plasmid DNA, minicircles can be generated by excising the desired construct, e.g., transposase expression cassettes or therapeutic expression cassette, from a viral backbone. Previously, it has been shown that excision and circularization of the donor sequence from a viral backbone may be important for transposase-mediated integration efficiency (Yant et al Nat Biotechnol 2002). In some embodiments, minicircles are first formulated and then delivered to target cells. In other embodiments, minicircles are formed from a DNA vector (e.g., plasmid DNA, rAAV, scAAV, ceDNA, doggybone DNA) intracellularly by co-delivery of a recombinase, resulting in excision and circularization of the recombinase recognition site-flanked nucleic acid, e.g., a nucleic acid encoding the Gene Writer™ polypeptide, template nucleic acid (e.g., template RNA) or nucleic acid encoding same, or both.


For optimizing protein expression, it can be helpful to provide tunable controls that can be used to modulate protein activity. In some embodiments, a tunable system may comprise at least one effector module that is responsive to at least one stimulus. The system may be, but is not limited to, a destabilizing domain (DD) system. This system is further taught in PCT/US2018/020704, as well as U.S. Provisional Patent Application No. 62/320,864 filed Apr. 11, 2016, 62/466,596 filed Mar. 3, 2017 and the International Publication WO2017/180587 (the contents each of which are herein incorporated by reference in their entirety). In some embodiments, the tunable system may comprise a first effector module. In some embodiments, the effector module may comprise a first stimulus response element (SRE) operably linked to at least one payload. In one aspect, the payload may be an immunotherapeutic agent. In one aspect, the first SRE of the composition may be responsive to or interact with at least one stimulus. In some embodiments, the first SRE may comprise a destabilizing domain (DD). The DD may be derived from a parent protein or from a mutant protein having one, two, three, or more amino acid mutations compared to the parent protein. In some embodiments, the parent protein may be selected from, but is not limited to, human protein FKBP, comprising the amino acid sequence of PCT/US201i/020704 SEQ. ID NO. 3; human DIFR (hDHFR), comprising the amino acid sequence of PCT/US2018/020704 SEQ. ID NO. 2; E. coli DHFR, comprising the amino acid sequence of PCT/US2018/020704 SEQ. ID NO. 1; PDE5, comprising the amino acid sequence of PCT/US2018/020704 SEQ. ID NO. 4; PPAR, gamma comprising the amino acid sequence of PCT/US2018/020704 SEQ. ID NO. 5; CA2, comprising the amino acid sequence of PCT/US201S/020704 SEQ. ID NO. 6; or NQ02, comprising the amino acid sequence of PCT/US2018/020704 SEQ. ID NO. 7. In some embodiments, the tunable controls are applied to the Gene Writer polypeptide, such that, e.g., a DD and stimulus can be used to modulate template integration efficiency. In some embodiments, the tunable controls are applied to one or more peptides encoded within the heterologous object sequence of the template, such that, e.g., a DD and stimulus can be used to modulate activity of a genomically integrated payload. In certain embodiments, the payload comprising the DD may be a therapeutic protein, e.g., a functional copy of an endogenously mutated gene. In certain embodiments, the payload comprising the DD may be a heterologous protein, e.g., a CAR.


Kits, Articles of Manufacture, and Pharmaceutical Compositions

In an aspect the disclosure provides a kit comprising a Gene Writer or a Gene Writing system, e.g., as described herein. In some embodiments, the kit comprises a Gene Writer polypeptide (or a nucleic acid encoding the polypeptide) and a template RNA (or DNA encoding the template RNA). In some embodiments, the kit further comprises a reagent for introducing the system into a cell, e.g., transfection reagent, LNP, and the like. In some embodiments, the kit is suitable for any of the methods described herein. In some embodiments, the kit comprises one or more elements, compositions (e.g., pharmaceutical compositions), Gene Writers, and/or Gene Writer systems, or a functional fragment or component thereof, e.g., disposed in an article of manufacture. In some embodiments, the kit comprises instructions for use thereof.


In an aspect, the disclosure provides an article of manufacture, e.g., in which a kit as described herein, or a component thereof, is disposed.


In an aspect, the disclosure provides a pharmaceutical composition comprising a Gene Writer or a Gene Writing system, e.g., as described herein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.


In some embodiments, the pharmaceutical composition comprises a template RNA and/or an RNA encoding the polypeptide. In embodiments, the pharmaceutical composition has one or more (e.g., 1, 2, 3, or 4) of the following characteristics:

    • (a) less than 1% (e.g., less than 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) DNA template relative to the template RNA and/or the RNA encoding the polypeptide, e.g., on a molar basis;
    • (b) less than 1% (e.g., less than 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) uncapped RNA relative to the template RNA and/or the RNA encoding the polypeptide, e.g., on a molar basis;
    • (c) less than 1% (e.g., less than 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) partial length RNAs relative to the template RNA and/or the RNA encoding the polypeptide, e.g., on a molar basis;
    • (d) substantially lacks unreacted cap dinucleotides.


Chemistry, Manufacturing, and Controls (CMC)

Purification of protein therapeutics is described, for example, in Franks, Protein Biotechnology: Isolation, Characterization, and Stabilization, Humana Press (2013); and in Cutler, Protein Purification Protocols (Methods in Molecular Biology), Humana Press (2010).


In some embodiments, a Gene Writer™ system, polypeptide, and/or template nucleic acid (e.g., template RNA) conforms to certain quality standards. In some embodiments, a Gene Writer™ system, polypeptide, and/or template nucleic acid (e.g., template RNA) produced by a method described herein conforms to certain quality standards. Accordingly, the disclosure is directed, in some aspects, to methods of manufacturing a Gene Writer™ system, polypeptide, and/or template nucleic acid (e.g., template RNA) that conforms to certain quality standards, e.g., in which said quality standards are assayed. The disclosure is also directed, in some aspects, to methods of assaying said quality standards in a Gene Writer™ system, polypeptide, and/or template nucleic acid (e.g., template RNA). In some embodiments, quality standards include, but are not limited to, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) of the following:

    • (i) the length of the template RNA, e.g., whether the template RNA has a length that is above a reference length or within a reference length range, e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the template RNA present is greater than 100, 125, 150, 175, or 200 nucleotides long;
    • (ii) the presence, absence, and/or length of a polyA tail on the template RNA, e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the template RNA present contains a polyA tail (e.g., a polyA tail that is at least 5, 10, 20, 30, 50, 70, 100 nucleotides in length (SEQ ID NO: 3665));
    • (iii) the presence, absence, and/or type of a 5′ cap on the template RNA, e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the template RNA present contains a 5′ cap, e.g., whether that cap is a 7-methylguanosine cap, e.g., a O-Me-m7G cap;
    • (iv) the presence, absence, and/or type of one or more modified nucleotides (e.g., selected from pseudouridine, dihydrouridine, inosine, 7-methylguanosine, 1-N-methylpseudouridine (1-Me-P), 5-methoxyuridine (5-MO-U), 5-methylcytidine (5mC), or a locked nucleotide) in the template RNA, e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the template RNA present contains one or more modified nucleotides;
    • (v) the stability of the template RNA (e.g., over time and/or under a pre-selected condition), e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the template RNA remains intact (e.g., greater than 100, 125, 150, 175, or 200 nucleotides long) after a stability test;
    • (vi) the potency of the template RNA in a system for modifying DNA, e.g., whether at least 1% of target sites are modified after a system comprising the template RNA is assayed for potency;
    • (vii) the length of the polypeptide, first polypeptide, or second polypeptide, e.g., whether the polypeptide, first polypeptide, or second polypeptide has a length that is above a reference length or within a reference length range, e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the polypeptide, first polypeptide, or second polypeptide present is greater than 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, or 2000 amino acids long (and optionally, no larger than 2500, 2000, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, or 600 amino acids long);
    • (viii) the presence, absence, and/or type of post-translational modification on the polypeptide, first polypeptide, or second polypeptide, e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the polypeptide, first polypeptide, or second polypeptide contains phosphorylation, methylation, acetylation, myristoylation, palmitoylation, isoprenylation, glipyatyon, or lipoylation, or any combination thereof,
    • (ix) the presence, absence, and/or type of one or more artificial, synthetic, or non-canonical amino acids (e.g., selected from ornithine, P-alanine, GABA, 6-Aminolevulinic acid, PABA, a D-amino acid (e.g., D-alanine or D-glutamate), aminoisobutyric acid, dehydroalanine, cystathionine, lanthionine, Djenkolic acid, Diaminopimelic acid, Homoalanine, Norvaline, Norleucine, Homonorleucine, homoserine, O-methyl-homoserine and O-ethyl-homoserine, ethionine, selenocysteine, selenohomocysteine, selenomethionine, selenoethionine, tellurocysteine, or telluromethionine) in the polypeptide, first polypeptide, or second polypeptide, e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the polypeptide, first polypeptide, or second polypeptide present contains one or more artificial, synthetic, or non-canonical amino acids;
    • (x) the stability of the polypeptide, first polypeptide, or second polypeptide (e.g., over time and/or under a pre-selected condition), e.g., whether at least 80, 85, 90, 95, 96, 97, 98, or 99% of the polypeptide, first polypeptide, or second polypeptide remains intact (e.g., greater than 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, or 2000 amino acids long (and optionally, no larger than 2500, 2000, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, or 600 amino acids long)) after a stability test;
    • (xi) the potency of the polypeptide, first polypeptide, or second polypeptide in a system for modifying DNA, e.g., whether at least 1% of target sites are modified after a system comprising the polypeptide, first polypeptide, or second polypeptide is assayed for potency; or
    • (xii) the presence, absence, and/or level of one or more of a pyrogen, virus, fungus, bacterial pathogen, or host cell protein, e.g., whether the system is free or substantially free of pyrogen, virus, fungus, bacterial pathogen, or host cell protein contamination.


In some embodiments, a system or pharmaceutical composition described herein is endotoxin free.


In some embodiments, the presence, absence, and/or level of one or more of a pyrogen, virus, fungus, bacterial pathogen, and/or host cell protein is determined. In embodiments, whether the system is free or substantially free of pyrogen, virus, fungus, bacterial pathogen, and/or host cell protein contamination is determined.


In some embodiments, a pharmaceutical composition or system as described herein has one or more (e.g., 1, 2, 3, or 4) of the following characteristics:

    • (a) less than 1% (e.g., less than 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) DNA template relative to the template RNA and/or the RNA encoding the polypeptide, e.g., on a molar basis;
    • (b) less than 1% (e.g., less than 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) uncapped RNA relative to the template RNA and/or the RNA encoding the polypeptide, e.g., on a molar basis;
    • (c) less than 1% (e.g., less than 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) partial length RNAs relative to the template RNA and/or the RNA encoding the polypeptide, e.g., on a molar basis;
    • (d) substantially lacks unreacted cap dinucleotides.


Applications

By integrating coding genes into a RNA sequence template, the Gene Writer™ system can address therapeutic needs, for example, by providing expression of a therapeutic transgene in individuals with loss-of-function mutations, by replacing gain-of-function mutations with normal transgenes, by providing regulatory sequences to eliminate gain-of-function mutation expression, and/or by controlling the expression of operably linked genes, transgenes and systems thereof. In certain embodiments, the RNA sequence template encodes a promotor region specific to the therapeutic needs of the host cell, for example a tissue specific promotor or enhancer. In still other embodiments, a promotor can be operably linked to a coding sequence. In embodiments, the Gene Writer™ gene editor system can provide therapeutic transgenes expressing, e.g., replacement blood factors or replacement enzymes, e.g., lysosomal enzymes. For example, the compositions, systems and methods described herein are useful to express, in a target human genome, agalsidase alpha or beta for treatment of Fabry Disease; imiglucerase, taliglucerase alfa, velaglucerase alfa, or alglucerase for Gaucher Disease; sebelipase alpha for lysosomal acid lipase deficiency (Wolman disease/CESD); laronidase, idursulfase, elosulfase alpha, or galsulfase for mucopolysaccharidoses; alglucosidase alpha for Pompe disease. For example, the compositions, systems and methods described herein are useful to express, in a target human genome factor I, II, V, VII, X, XI, XII or XIII for blood factor deficiencies.


In some embodiments, the heterologous object sequence encodes an intracellular protein (e.g., a cytoplasmic protein, a nuclear protein, an organellar protein such as a mitochondrial protein or lysosomal protein, or a membrane protein). In some embodiments, the heterologous object sequence encodes a membrane protein, e.g., a membrane protein other than a CAR, and/or an endogenous human membrane protein. In some embodiments, the heterologous object sequence encodes an extracellular protein. In some embodiments, the heterologous object sequence encodes an enzyme, a structural protein, a signaling protein, a regulatory protein, a transport protein, a sensory protein, a motor protein, a defense protein, or a storage protein. Other proteins include an immune receptor protein, e.g. a synthetic immune receptor protein such as a chimeric antigen receptor protein (CAR), a T cell receptor, a B cell receptor, or an antibody.


A Gene Writing™ system may be used to modify immune cells. In some embodiments, a Gene Writing™ system may be used to modify T cells. In some embodiments, T-cells may include any subpopulation of T-cells, e.g., CD4+, CD8+, gamma-delta, naïve T cells, stem cell memory T cells, central memory T cells, or a mixture of subpopulations. In some embodiments, a Gene Writing™ system may be used to deliver or modify a T-cell receptor (TCR) in a T cell. In some embodiments, a Gene Writing™ system may be used to deliver at least one chimeric antigen receptor (CAR) to T-cells. In some embodiments, a Gene Writing™ system may be used to deliver at least one CAR to natural killer (NK) cells. In some embodiments, a Gene Writing™ system may be used to deliver at least one CAR to natural killer T (NKT) cells. In some embodiments, a Gene Writing™ system may be used to deliver at least one CAR to a progenitor cell, e.g., a progenitor cell of T, NK, or NKT cells. In some embodiments, cells modified with at least one CAR (e.g., CAR-T cells, CAR-NK cells, CAR-NKT cells), or a combination of cells modified with at least one CAR (e.g., a mixture of CAR-NK/T cells) are used to treat a condition as identified in the targetable landscape of CAR therapies in MacKay, et al. Nat Biotechnol 38, 233-244 (2020), incorporated by reference herein in its entirety. In some embodiments, the immune cells comprise a CAR specific to a tumor or a pathogen antigen selected from a group consisting of AChR (fetal acetylcholine receptor), ADGRE2, AFP (alpha fetoprotein), BAFF-R, BCMA, CAIX (carbonic anhydrase IX), CCR1, CCR4, CEA (carcinoembryonic antigen), CD3, CD5, CD8, CD7, CD10, CD13, CD14, CD15, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49f, CD56, CD61, CD64, CD68, CD70, CD74, CD99, CD117, CD123, CD133, CD138, CD44v6, CD267, CD269, CDS, CLEC12A, CS1, EGP-2 (epithelial glycoprotein-2), EGP-40 (epithelial glycoprotein-40), EGFR(HER1), EGFR-VIII, EpCAM (epithelial cell adhesion molecule), EphA2, ERBB2 (HER2, human epidermal growth factor receptor 2), ERBB3, ERBB4, FBP (folate-binding protein), Flt3 receptor, folate receptor-a, GD2 (ganglioside G2), GD3 (ganglioside G3), GPC3 (glypican-3), GPI00, hTERT (human telomerase reverse transcriptase), ICAM-1, integrin B7, interleukin 6 receptor, IL13Ra2 (interleukin-13 receptor 30 subunit alpha-2), kappa-light chain, KDR (kinase insert domain receptor), LeY (Lewis Y), L1CAM (L1 cell adhesion molecule), LILRB2 (leukocyte immunoglobulin like receptor B2), MART1, MAGE-A1 (melanoma associated antigen A1), MAGE-A3, MSLN (mesothelin), MUC16 (mucin 16), MUCI (mucin I), KG2D ligands, NY-ESO-1 (cancer-testis antigen), PRI (proteinase 3), TRBC1, TRBC2, TFM-3, TACI, tyrosinase, survivin, hTERT, oncofetal antigen (h5T4), p53, PSCA (prostate stem cell antigen), PSMA (prostate-specific membrane antigen), hROR1, TAG-72 (tumor-associated glycoprotein 72), VEGF-R2 (vascular endothelial growth factor R2), WT-1 (Wilms tumor protein), and antigens of HIV (human immunodeficiency virus), hepatitis B, hepatitis C, CMV (cytomegalovirus), EBV (Epstein-Barr virus), HPV (human papilloma virus).


In some embodiments, immune cells, e.g., T-cells, NK cells, NKT cells, or progenitor cells are modified ex vivo and then delivered to a patient. In some embodiments, a Gene Writer™ system is delivered by one of the methods mentioned herein, and immune cells, e.g., T-cells, NK cells, NKT cells, or progenitor cells are modified in vivo in the patient.


In some embodiments, a Gene Writer™ system described herein is delivered to a tissue or cell from the cerebrum, cerebellum, adrenal gland, ovary, pancreas, parathyroid gland, hypophysis, testis, thyroid gland, breast, spleen, tonsil, thymus, lymph node, bone marrow, lung, cardiac muscle, esophagus, stomach, small intestine, colon, liver, salivary gland, kidney, prostate, blood, or other cell or tissue type. In some embodiments, a Gene Writer™ system described herein is used to treat a disease, such as a cancer, inflammatory disease, infectious disease, genetic defect, or other disease. A cancer can be cancer of the cerebrum, cerebellum, adrenal gland, ovary, pancreas, parathyroid gland, hypophysis, testis, thyroid gland, breast, spleen, tonsil, thymus, lymph node, bone marrow, lung, cardiac muscle, esophagus, stomach, small intestine, colon, liver, salivary gland, kidney, prostate, blood, or other cell or tissue type, and can include multiple cancers.


In some embodiments, a Gene Writer™ system described herein described herein is administered by enteral administration (e.g. oral, rectal, gastrointestinal, sublingual, sublabial, or buccal administration). In some embodiments, a Gene Writer™ system described herein is administered by parenteral administration (e.g., intravenous, intramuscular, subcutaneous, intradermal, epidural, intracerebral, intracerebroventricular, epicutaneous, nasal, intra-arterial, intra-articular, intracavernous, intraocular, intraosseous infusion, intraperitoneal, intrathecal, intrauterine, intravaginal, intravesical, perivascular, or transmucosal administration). In some embodiments, a Gene Writer™ system described herein is administered by topical administration (e.g., transdermal administration).


In some embodiments, a Gene Writing system can be used to make an insertion, deletion, substitution, or combination thereof in a cell, tissue, or subject. In some embodiments, an insertion, deletion, substitution, or combination thereof, increases or decreases expression (e.g. transcription or translation) of a gene. In some embodiments, an insertion, deletion, substitution, or combination thereof, increases or decreases expression (e.g. transcription or translation) of a gene by altering, adding, or deleting sequences in a promoter or enhancer, e.g. sequences that bind transcription factors. In some embodiments, an insertion, deletion, substitution, or combination thereof alters translation of a gene (e.g. alters an amino acid sequence), inserts or deletes a start or stop codon, alters or fixes the translation frame of a gene. In some embodiments, an insertion, deletion, substitution, or combination thereof alters splicing of a gene, e.g. by inserting, deleting, or altering a splice acceptor or donor site. In some embodiments, an insertion, deletion, substitution, or combination thereof alters transcript or protein half-life. In some embodiments, an insertion, deletion, substitution, or combination thereof alters protein localization in the cell (e.g. from the cytoplasm to a mitochondria, from the cytoplasm into the extracellular space (e.g. adds a secretion tag)). In some embodiments, an insertion, deletion, substitution, or combination thereof alters (e.g. improves) protein folding (e.g. to prevent accumulation of misfolded proteins). In some embodiments, an insertion, deletion, substitution, or combination thereof, alters, increases, decreases the activity of a gene, e.g. a protein encoded by the gene.


In some embodiments, a Gene Writing system can be used to make multiple modifications (e.g., multiple insertions, deletions, or substitutions, and all combinations thereof) to a target cell, either simultaneously or sequentially. In some embodiments, a Gene Writing system can be used to further modify an already modified cell. In some embodiments, a Gene Writing system can be use to modify a cell edited by a complementary technology, e.g., a gene edited cell, e.g., a cell with one or more CRISPR knockouts. In some embodiments, the previously edited cell is a T-cell. In some embodiments, the previous modifications comprise gene knockouts in a T-cell, e.g., endogenous TCR (e.g., TRAC, TRBC), HLA Class I (B2M), PD1, CD52, CTLA-4, TIM-3, LAG-3, DGK. In some embodiments, a Gene Writing system is used to insert a TCR or CAR into a T-cell that has been previously modified.


In some embodiments, a Gene Writer™ system as described herein can be used to modify an animal cell, plant cell, or fungal cell. In some embodiments, a Gene Writer™ system as described herein can be used to modify a mammalian cell (e.g., a human cell). In some embodiments, a Gene Writer™ system as described herein can be used to modify a cell from a livestock animal (e.g., a cow, horse, sheep, goat, pig, llama, alpaca, camel, yak, chicken, duck, goose, or ostrich). In some embodiments, a Gene Writer™ system as described herein can be used as a laboratory tool or a research tool, or used in a laboratory method or research method, e.g., to modify an animal cell, e.g., a mammalian cell (e.g., a human cell), a plant cell, or a fungal cell.


In some embodiments, a Gene Writer™ system as described herein can be used to express a protein, template, or heterologous object sequence (e.g., in an animal cell, e.g., a mammalian cell (e.g., a human cell), a plant cell, or a fungal cell). In some embodiments, a Gene Writer™ system as described herein can be used to express a protein, template, or heterologous object sequence under the control of an inducible promoter (e.g., a small molecule inducible promoter). In some embodiments, a Gene Writing system or payload thereof is designed for tunable control, e.g., by the use of an inducible promoter. For example, a promoter, e.g., Tet, driving a gene of interest may be silent at integration, but may, in some instances, activated upon exposure to a small molecule inducer, e.g., doxycycline. In some embodiments, the tunable expression allows post-treatment control of a gene (e.g., a therapeutic gene), e.g., permitting a small molecule-dependent dosing effect. In embodiments, the small molecule-dependent dosing effect comprises altering levels of the gene product temporally and/or spatially, e.g., by local administration. In some embodiments, a promoter used in a system described herein may be inducible, e.g., responsive to an endogenous molecule of the host and/or an exogenous small molecule administered thereto.


In some embodiments, a Gene Writing system is used to make changes to non-coding and/or regulatory control regions, e.g., to tune the expression of endogenous genes. In some embodiments, a Gene Writing system is used to induce upregulation or downregulation of gene expression. In some embodiments, a regulatory control region comprises one or more of a promoter, enhancer, UTR, CTCF site, and/or a gene expression control region.


In some embodiments, a Gene Writing system may be used to treat or prevent a repeat expansion disease (e.g., a disease of Table 26), or to reduce the severity or a symptom thereof. In some embodiments, the repeat expansion disease comprises expansion of a trinucleotide repeat. In some embodiments, the subject has at least 10, 20, 30, 40, or 50 copies of the repeat. In embodiments, the repeat expansion disease is an inherited disease. Non-limiting examples of repeat expansion diseases include Huntington's disease (HD) and myotonic dystrophy. For example, healthy individuals may possess between 10 and 35 tandem copies of the CAG trinucleotide repeat, while Huntington's patients frequently possess >40 copies, which can result, e.g., in an elongated and dysfunctional Huntingtin protein. In some embodiments, a Gene Writer corrects a repeat expansion, e.g., by recognizing DNA at the terminus of the repeat region and nicking one strand (FIG. 30). In some embodiments, the template RNA component of the Gene Writer comprises a region with a number of repeats characteristic of a healthy subject, e.g., about 20 repeats (e.g., between 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, or 35-40 repeats). In some embodiments, the template RNA component of the Gene Writer is copied by TPRT into the target site. In some embodiments, a second strand nick and second strand synthesis then results in the integration of the newly copied DNA comprising a correct number of repeats (e.g., as described herein). In some embodiments, the system recognizes DNA at the terminus of the repeat region and the template carries the information for the new number of repeats. In embodiments, a Gene Writer can be used in this way regardless of the number of repeats present in an individual and/or in an individual cell. Owing to the presence of multiple repeats, an alternative non-GeneWriter therapeutic (e.g., a CRISPR-based homologous recombination therapeutic) might, in some embodiments, result in unpredictable repair behavior. Further non-limiting examples of repeat expansion diseases and the causative repeats can be found, for example, in La Spada and Taylor Nat Rev Genet 11(4):247-258 (2010), which is incorporated herein by reference in its entirety.


In some embodiments, a Gene Writing system may be used to treat a healthy individual, e.g., as a preventative therapy. Gene Writing systems can, in some embodiments, be targeted to generate mutations, e.g., that have been shown to be protective towards a disease of interest. An exemplary list of such diseases and protective mutation targets can be found in Table 22.


In some embodiments, a Gene Writer system described herein is used to treat an indication of any of Tables 9-12. For instance, in some embodiments the GeneWriter system modifies a target site in genomic DNA in a cell, wherein the target site is in a gene of any of Tables 9-12, e.g., in a subject having the corresponding indication listed in any of Tables 9-12. In some embodiments, the cell is a liver cell and the target site is in a gene of Table 9, e.g., in a subject having the corresponding indication listed in Table 9. In some embodiments, the cell is an HSC and the target site is in a gene of Table 10, e.g., in a subject having the corresponding indication listed in Table 10. In some embodiments, the cell is a CNS cell and the target site is in a gene of Table 11, e.g., in a subject having the corresponding indication listed in Table 11. In some embodiments, the cell is a cell of the eye and the target site is in a gene of Table 12, e.g., in a subject having the corresponding indication listed in Table 12. In some embodiments, the target site is in a coding region in the gene. In some embodiments, the target site is in a promoter. In some embodiments, the target site is in a 5′ UTR or a 3′ UTR of the gene of any of Tables 9-12. In some embodiments, the target site is in an intron or exon of the gene. In some embodiments, the GeneWriter corrects a mutation in the gene. In some embodiments, the GeneWriter inserts a sequence that had been deleted from the gene (e.g., through a disease-causing mutation). In some embodiments, the GeneWriter deletes a sequence that had been duplicated in the gene (e.g., through a disease-causing mutation). In some embodiments, the GeneWriter replaces a mutation (e.g., a disease-causing mutation) with the corresponding wild-type sequence. In some embodiments, the mutation is a substitution, insertion, deletion, or inversion.









TABLE 9







Indications and genetic targets, e.g., in the liver








Disease
Gene Affected





Acute intermittent porphyria
HMBS


Alpha-1-antitrypsin deficiency (AAT)
SERPINA1


Arginase deficiency
ARG1


Argininosuccinate lyase deficiency
ASL


Carbamoyl phosphate synthetase I deficiency
CPS1


Citrin deficiency
SLC25A13


Citrullinemia type I
ASS1


Crigler-Najjar syndrome (Hyperbilirubinemia)
UGT1A1


Fabry disease
GLA


Familial hypercholesterolemia 4 (homozygous
LDLRAP1


familial cholesterolemia)



Glutaric aciduria I
GCDH


Glutaric aciduria II (multiple acyl-CoA
GA IIA: ETFA


dehydrogenase deficiency)
GA IIB: ETFB



GA IIC: ETFDH


Glycogen storage disease type IV
GBE1


Hemophilia A
F8


Hemophilia B
F9


Hereditary hemochromatosis
HFE


Homocystinuria
CBS


Maple syrup urine disease (MSUD)
Type Ia: BCKDHA



Type Ib: BCKDHB



Type II: DBT


Methylmalonic acidemia (methylmalonyl-CoA
MMUT


mutase deficiency)



MPS 1S (Scheie syndrome)
IDUA


MPS2
IDS


MPS3 (San Filippo Syndrome)
Type IIIa: SGSH



Type IIIb: NAGLU



Type IIIc: HGSNAT



Type IIId: GNS


MPS4
Type IVA: GALNS



Type IVB: GLB1


MPS6
ARSB


MPS7
GUSB


Ornithine transcarbamylase deficiency
OTC


Phenylketonuria (phenylalanine hydroxylase
PAH


deficiency)



Polycystic Liver Disease
PCLD1: PRKCSH



PCLD2: SEC63



PLCD3: ALG8



PCLD4: LRP5


Pompe disease
GAA


Primary Hyperoxaluria 1 (oxalosis)
AGXT


Progressive familial intrahepatic cholestasis type 1
ATP8B1


Progressive familial intrahepatic cholestasis type 2
ABCB11


Progressive familial intrahepatic cholestasis type 3
ABCB4


Propionic acidemia
PCCB; PCCA


Pyruvate carboxylase deficiency
PC


Tyrosinemia type I
FAH


Wilson's disease
ATP7B
















TABLE 10







Indications and genetic targets for HSCs








Disease
Gene Affected





Adrenoleukodystrophy (CALD)
ABCD1


Alpha-mannosidosis
MAN2B1


Blackfan-Diamond Anemia



Congenital amegakaryocytic thrombocytopenia
MPL


Dyskeratosis Congenita
TERC


Fanconi anemia
FANC


Gaucher disease
GBA


Globoid cell leukodystrophy (Krabbe disease)
GALC


Hemophagocytic lymphohistiocytosis
PRF1; STX11;



STXBP2; UNC13D


Malignant infantile osteopetrosis-autosomal
Many genes


recessive osteopetrosis
implicated


Metachromatic leukodystrophy
PSAP


MPS 1S (Scheie syndrome)
IDUA


MPS2
IDS


MPS7
GUSB


Mucolipidosis II
GNPTAB


Niemann-Pick disease A and B
SMPD1


Niemann-Pick disease C
NPC1


Pompe disease
GAA


Pyruvate kinase deficiency (PKD)
PKLR


Sickle cell disease (SCD)
HBB


Tay Sachs
HEXA


Thalassemia
HBB
















TABLE 11







Indications and genetic targets for the CNS








Disease
Gene Affected





Alpha-mannosidosis
MAN2B1


Ataxia-telangiectasia
ATM


CADASIL
NOTCH3


Canavan disease
ASPA


Carbamoyl-phosphate synthetase 1 deficiency
CPS1


CLN1 disease
PPT1


CLN2 Disease
TPP1


CLN3 Disease (Juvenile neuronal ceroid
CLN3


lipofuscinosis, Batten Disease)



Coffin-Lowry syndrome
RPS6KA3


Congenital myasthenic syndrome 5
COLQ


Cornelia de Lange syndrome (NIPBL)
NIPBL


Cornelia de Lange syndrome (SMC1A)
SMC1A


Dravet syndrome (SCN1A)
SCN1A


Glycine encephalopathy (GLDC)
GLDC


GM1 gangliosidosis
GLB1


Huntington's Disease
HTT


Hydrocephalus with stenosis of the aqueduct
L1CAM


of Sylvius



Leigh Syndrome
SURF1


Metachromatic leukodystrophy (ARSA)
ARSA


MPS type 2
IDS


MPS type 3
Type 3a: SGSH



Type 3b: NAGLU


Mucolipidosis IV
MCOLN1


Neurofibromatosis Type 1
NF1


Neurofibromatosis type 2
NF2


Pantothenate kinase-associated neurodegeneration
PANK2


Pyridoxine-dependent epilepsy
ALDH7A1


Rett syndrome (MECP2)
MECP2


Sandhoff disease
HEXB


Semantic dementia (Frontotemporal dementia)
MAPT


Spinocerebellar ataxia with axonal neuropathy
SETX


(Ataxia with Oculomotor Apraxia)



Tay-Sachs disease
HEXA


X-linked Adrenoleukodystrophy
ABCD1
















TABLE 12







Indications and genetic targets for the eye










Disease
Gene Affected







Achromatopsia
CNGB3



Amaurosis Congenita (LCA1)
GUCY2D



Amaurosis Congenita (LCA10)
CEP290



Amaurosis Congenita (LCA2)
RPE65



Amaurosis Congenita (LCA8)
CRB1



Choroideremia
CHM



Cone Rod Dystrophy (ABCA4)
ABCA4



Cone Rod Dystrophy (GUCY2D)
GUCY2D



Cystinosis, Ocular Nonnephropathic
CTNS



Doyne Honeycomb Retinal Dystrophy (DHRD)
EFEMP1



Familial Oculoleptomeningeal Amyloidosis
TTR



Keratitis-ichthyosis-deafness (KID)
GJB2



Lattice corneal dystrophy type I
TGFBI



Macular Corneal Dystrophy (MCD)
CHST6



Meesmann Corneal Dystrophy
KRT12; KRT3



Optic Atrophy
OPA1



Retinitis Pigmentosa (AR)
USH2A



Retinitis Rigmentosa (AD)
RHO



Sorsby Fundus Dystrophy
TIMP3



Stargardt Disease
ABCA4










Additional Suitable Indications

Exemplary suitable diseases and disorders that can be treated by the systems or methods provided herein, for example, those comprising Gene Writers, include, without limitation: Baraitser-Winter syndromes 1 and 2; Diabetes mellitus and insipidus with optic atrophy and deafness; Alpha-I-antitrypsin deficiency; Heparin cofactor II deficiency; Adrenoleukodystrophy Keppen-Lubinsky syndrome; Treacher collins syndrome 1; Mitochondrial complex I, II, III, III (nuclear type 2, 4, or 8) deficiency; Hypermanganesemia with dystonia, polycythemia and cirrhosis; Carcinoid tumor of intestine; Rhabdoid tumor predisposition syndrome 2; Wilson disease; Hyperphenylalaninemia, bh4-deficient, a, due to partial pts deficiency, BH4-deficient, D, and non-pku; Hyperinsulinemic hypoglycemia familial 3, 4, and 5; Keratosis follicularis: Oral-facial-digital syndrome; SeSAME syndrome; Deafness, nonsyndromic sensorineural, mitochondrial: Proteinuria: Insulin-dependent diabetes mellitus secretory diarrhea syndrome; Moyamoya disease 5: Diamond-Blackfan anemia 1, 5, 8, and 10: Pseudoachondroplastic spondyloepiphysial dysplasia syndrome; Brittle cornea syndrome 2; Methylmalonic acidemia with homocystinuria, Adams-Oliver syndrome 5 and 6; autosomal recessive Agammaglobulinemia 2; Cortical malformations, occipital; Febrile seizures, familial, 1I; Mucopolysaccharidosis type VI, type VI (severe), and type VII; Marden Walker like syndrome; Pseudoneonatal adrenoleukodystrophy; Spheroid body myopathy; Cleidocranial dysostosis; Multiple Cutaneous and Mucosal Venous Malformations: Liver failure acute infantile: Neonatal intrahepatic cholestasis caused by citrin deficiency; Ventricular septal defect 1; Oculodentodigital dysplasia; Wilms tumor 1; Weill-Marchesani-like syndrome; Renal adysplasia; Cataract 1, 4, autosomal dominant, autosomal dominant, multiple types, with microcornea, coppock-like, juvenile, with microcornea and glucosuria, and nuclear diffuse nonprogressive; Odontohypophosphatasia; Cerebro-oculo-facio-skeletal syndrome; Schizophrenia 15; Cerebral amyloid angiopathy, APP-related; Hemophagocytic lymphohistiocytosis, familial, 3; Porphobilinogen synthase deficiency; Episodic ataxia type 2; Trichorhinophalangeal syndrome type 3; Progressive familial heart block type 1; Glioma susceptibility 1; Lichtenstein-Knorr Syndrome; Hypohidrotic X-linked ectodermal dysplasia; Banter syndrome types 3, 3 with hypocalciuria, and 4; Carbonic anhydrase VA deficiency, hyperammonemia due to: Cardiomyopathy; Poikiloderma, hereditary fibrosing, with tendon contractures, myopathy, and pulmonary fibrosis; Combined d-2- and 1-2-hydroxyglutaric aciduria: Arginase deficiency; Cone-rod dystrophy 2 and 6; Smith-Lemli-Opitz syndrome; Mucolipidosis III Gamma; Blau syndrome; Wemer syndrome; Meningioma; Iodotyrosyl coupling defect, Dubin-Johnson syndrome; 3-Oxo-5 alpha-steroid delta 4-dehydrogenase deficiency; Boucher Neuhauser syndrome; Iron accumulation in brain: Mental Retardation, X-Linked 102 and syndromic 13; familial, Pituitary adenoma predisposition; Hypoplasia of the corpus callosum: Hyperalphalipoproteinemia 2; Deficiency of ferroxidase; Growth hormone insensitivity with immunodeficiency; Marinesco-Sj\xc3\xb6gren syndrome; Martsolf syndrome; Gaze palsy, familial horizontal, with progressive scoliosis; Mitchell-Riley syndrome; Hypocalciuric hypercalciuria, familial, types 1 and 3; Rubinstein-Taybi syndrome; Epstein syndrome; Juvenile retinoschisis; Becker muscular dystrophy; Loeys-Dietz syndrome 1, 2, 3; Congenital muscular hypertrophy-cerebral syndrome; Familial juvenile gout; Spermatogenic failure 11, 3, and 8; Orofacial cleft 11 and 7, Cleft lip/palate-ectodermal dysplasia syndrome; Mental retardation, X-linked, nonspecific, syndromic, Hedera type, and syndromic, wu type; Combined oxidative phosphorylation deficiencies 1, 3, 4, 12, 15, and 25; Frontotemporal dementia; Kniest dysplasia; Familial cardiomyopathy; Benign familial hematuria; Pheochromocytoma; Aminoglycoside-induced deafness; Gamma-aminobutyric acid transaminase deficiency: Oculocutaneous albinism type IB, type 3, and type 4; Renal coloboma syndrome; CNS hypomyelination; Hennekam lymphangiectasia-lymphedema syndrome 2; Migraine, familial basilar; Distal spinal muscular atrophy, X-linked 3; X-linked periventricular heterotopia; Microcephaly; Mucopolysaccharidosis, MPS-1-H/S, MPS-II, MPS-IIA, MPS-II-B, MPS-II-C, MPS-IV-A, MPS-IV-B; Infantile Parkinsonism-dystonia; Frontotemporal dementia with TDP43 inclusions, TARIDBP-related; Hereditary diffuse gastric cancer; Sialidosis type I and II; Microcephaly-capilliary malformation syndrome; Hereditary breast and ovarian cancer syndrome; Brain small vessel disease with hemorrhage; Non-ketotic hyperglycinemia; Navajo neurohepatopathy: Auriculocondylar syndrome 2; Spastic paraplegia 15, 2, 3, 35, 39, 4, autosomal dominant, 55, autosomal recessive, and 5A; Autosomal recessive cuts laxa type IA and IB: Hemolytic anemia, nonspherocytic, due to glucose phosphate isomerase deficiency; Hutchinson-Gilford syndrome; Familial amyloid nephropathy with urticaria and deafness; Supravalvar aortic stenosis; Diffuse palmoplantar keratoderma, Bothnian type; Holt-Oram syndrome; Coffin Siris/Intellectual Disability; Left-rigirt axis malformations; Rapadilino syndrome; Nanophthalmos 2; Craniosynostosis and dental anomalies; Paragangliomas 1; Snyder Robinson syndrome; Ventricular fibrillation: Activated PI3K-delta syndrome; Howel-Evans syndrome; Larsen syndrome, dominant type; Van Maldergem syndrome 2; MYH-associated polyposis; 6-pymvoyl-tetrahydropterin synthase deficiency; Alagille syndromes 1 and 2; Lymphangiomyomatosis; Muscle eye brain disease; WFSl-Related Disorders; Primary hypertrophic osteoarthropathy, autosomal recessive 2; Infertility: Nestor-Guillermo progeria syndrome; Mitochondrial trifunctional protein deficiency; Hypoplastic left heart syndrome 2; Primary dilated cardiomyopathy; Retinitis pigmentosa; Hirschsprung disease 3; Upshaw-Schulman syndrome; Desbuquois dysplasia 2; Diarrhea 3 (secretory sodium, congenital, syndromic) and 5 (with tufting enteropathy, congenital); Pachyonychia congenita 4 and type 2; Cerebral autosomal dominant and recessive arteriopathy with subcortical infarcts and leukoencephalopathy; Vi tel li form dystrophy; type II, type IV, IV (combined hepatic and myopathic), type V, and type VI; Atypical Rett syndrome; Atrioventricular septal defect 4: Papillon-Lef\xc3\xa8vre syndrome; -Leber amaurosis; X-linked hereditary motor and sensory neuropathy; Progressive sclerosing poliodystrophy; Goldmann-Favre syndrome; Renal-hepatic-pancreatic dysplasia; Pallister-Hall syndrome; Amyloidogenic transthyretin amyloidosis, Melnick-Needles syndrome: Hyperimmunoglobulin E syndrome; Posterior column ataxia with retinitis pigmentosa; Chondrodysplasia punctata 1, X-linked recessive and 2 X-linked dominant; Ectopia lentis, isolated autosomal recessive and dominant: Familial cold urticarial: Familial adenomatous polyposis 1 and 3; Porokeratosis 8, disseminated superficial actinic type; PIK3CA Related Overgrowth Spectrum; Cerebral cavernous malformations 2; Exudative vitreoretinopathy 6: Megalencephaly cutis marmorata telangiectatica congenital; TARP syndrome; Diabetes mellitus, permanent neonatal, with neurologic features; Short-rib thoracic dysplasia 11 or 3 with or without polydactyly; Hypertrichosis osteochondrodysplasia; beta Thalassemia; Niemann-Pick disease type C1, C2, type A, and type C1, adult form; Charcot-Marie-Tooth disease types 1B, 2B32, 2C, 2F, 2l, 2U (axonal), IC (demyelinating), dominant intermediate C, recessive intermediate A, 2A2, 4C, 4D, 4H, IF, IVF, and X: Tyrosinemia type I; Paroxysral atrial fibrillation; UV-sensitive syndrome; Tooth agenesis, selective, 3 and 4; Merosin deficient congenital muscular dystrophy; Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency; Congenital aninidia; Left ventricular noncompaction 5; Deficiency of aromatic-L-amino-acid decarboxylase; Coronary heart disease; Leukonychia totalis; Distal arthrogryposis type 2B; Retinitis pigmentosa 10, 11, 12, 14, 15, 17, and 19; Robinow Sorauf syndrome; Tenorio Syndrome; Prolactinoma; Neurofibromatosis, type 1 and type 2; Congenital muscular dystrophy-dystroglycanopathy with brain and eye anomalies, types A2, A7, A8, A11, and A14; Heterotaxy, visceral, 2, 4, and 6, autosomal; Jankovic Rivera syndrome, Lipodystrophy, familial partial, type 2 and 3: Hemoglobin H disease, nondeletional; Multicentric osteolysis, nodulosis and arthropathy; Thyroid agenesis; deficiency of Acyl-CoA dehydrogenase family, member 9; Alexander disease; Phytanic acid storage disease; Breast-ovarian cancer, familial 1, 2, and 4; Proline dehydrogenase deficiency; Childhood hypophosphatasia; Pancreatic agenesis and congenital heart disease; Vitamin 1)-dependent rickets, types 1 and 2; Iridogoniodysgenesis dominant type and type 1; Autosomal recessive hypohidrotic ectodermal dysplasia syndrome; Mental retardation, X-linked, 3, 21, 30, and 72; Hereditary hemorrhagic telangiectasia type 2; Blepharophimosis, ptosis, and epicanthus inversus; Adenine phosphoribosyltransferase deficiency; Seizures, benign familial infantile, 2; Acrodysostosis 2, with or without hormone resistance; Tetralogy of Fallot; Retinitis pigmentosa 2, 20, 25, 35, 36, 38, 39, 4, 40, 43, 45, 48, 66, 7, 70, 72; Lysosonmal acid lipase deficiency; Eichsfeld type congenital muscular dystrophy: Walker-Warburg congenital muscular dystrophy; TNF receptor-associated periodic fever syndrome (TRAPS); Progressive myoclonus epilepsy with ataxia; Epilepsy, childhood absence 2, 12 (idiopathic generalized, susceptibility to) 5 (nocturnal frontal lobe), nocturnal frontal lobe type 1, partial, with variable foci, progressive myoclonic 3, and X-linked, with variable learning disabilities and behavior disorders; Long QT syndrome; Dicarboxylic aminoaciduria; Brachydactyly types A1 and A2; Pseudoxanthona elasticum-like disorder with multiple coagulation factor deficiency; Multisystemic smooth muscle dysfunction syndrome; Syndactyly Cenani Lenz type; Joubert syndrome 1, 6, 7, 9/15 (digenic), 14, 16, and 17, and Orofaciodigital syndrome xiv; Digitorenocerebral syndrome; Retinoblastoma; Dyskinesia, familial, writh facial myokymia; Hereditary sensory and autonomic neuropathy type IIE and IIA; familial hyperinsulinism; Megalencephalic leukoencephalopathy with subcortical cysts 1 and 2a; Aase syndrome; Wiedemann-Steiner syndrome; Ichthyosis exfoliative; Myotonia congenital; Granulomatous disease, chronic, X-linked, variant; Deficiency of 2-methylbutyryl-CoA dehydrogenase; Sarcoidosis, early-onset; Glaucoma, congenital and Glaucoma, congenital, Coloboma; Breast cancer, susceptibility to; Ceroid lipofuscinosis neuronal 2, 6, 7, and 10; Congenital generalized lipodystrophy type 2; Fructose-bisphosphatase deficiency; Congenital contractual arachnodactyly; Lynch syndrome I and II; Phosphoglycerate dehydrogenase deficiency; Burn-Mckeown syndrome; Myocardial infarction 1: Achromatopsia 2 and 7; Retinitis Pigmentosa 73; Protan defect, Polymicrogyria, asymmetric, bilateral frontoparietal; Spinal muscular atrophy, distal, autosomal recessive, 5; Methylmalonic aciduria due to methylmalonyl-CoA mutase deficiency; Familial porencephaly; Hurler syndrome; Oto-palato-digital syndrome, types I and II; Sotos syndrome 1 or 2; Cardioencephalomyopathy, fatal infantile, due to cytochrome c oxidase deficiency; Parastremmatic dwarfism; Thyrotropin releasing hormone resistance, generalized; Diabetes mellitus, type 2, and insulin-dependent, 20; Thoracic aortic aneurysms and aortic dissections; Estrogen resistance; Maple syrup urine disease type 1A and type 3; Hypospadias 1 and 2, X-linked; Metachromatic leukodystrophy juvenile, late infantile, and adult types: Early T cell progenitor acute lymphoblastic leukemia; Neuropathy, Hereditary Sensory, Type IC; Mental retardation, autosomal dominant 31; Retinitis pigmentosa 39; Breast cancer, early-onset; May-Hegglin anomaly; Gaucher disease type I and Subacute neuronopathic; Terntamy syndrome; Spinal muscular atrophy, lower extremity predominant 2, autosomal dominant; Fanconi anemia, complementation group E, I, N, and O; Alkaptonuria; Hirschsprung disease; Combined malonic and methylmalonic aciduria; Arrhythmogenic right ventricular cardiomyopathy types 5, 8, and 10; Congenital lipomatous overgrowth, vascular malformations, and epidermal nevi; Timothy syndrome; Deficiency of guanidinoacetate methyltransferase; Myoclonic dystonia; Kanzaki disease; Neutral 1 amino acid transport defect: Neurohypophyseal diabetes insipidus; Thyroid hormone metabolism, abnormal; Benign scapuloperoncal muscular dystrophy with cardiomyopathy; Hypoglycemia with deficiency of glycogen synthetase in the liver; Hypertrophic cardiomyopathy; Myasthenic Syndrome, Congenital, 11, associated with acetylcholine receptor deficiency; Mental retardation X-linked syndromic 5; Stormorken syndrome; Aplastic anemia; Intellectual disability; Normokalemic periodic paralysis, potassiuni-sensitive; Danon disease; Nephronophthisis 13, 15 and 4; Thyrotoxic periodic paralysis and Thyrotoxic periodic paralysis 2; Infertility associated with multi-tailed spermatozoa and excessive DNA; Glaucoma, primary open angle, juvenile-onset; Afibrinogenemia and congenital Afibrinogenemia; Polycystic kidney disease 2, adult type, and infantile type; Familial porphyria cutanea tarda; Cerebello-oculo-renal syndrome (nephronophthisis, oculomotor apraxia and cerebellar abnormalities); Frontotemporal Dementia Chromosome 3-Linked and Frontotemporal dementia ubiquitin-positive; Metatrophic dysplasia; Immuonodeficiency-centromnerc instability-facial anomalies syndrome 2: Anemia, nonspherocytic hemolytic, due to G6PD deficiency; Bronchiectasis with or without elevated sweat chloride 3; Congenital myopathy with fiber type disproportion; Carney complex, type 1; Cryptorchidism, unilateral or bilateral; ichtyliosis bullosa of Simiens; Isolated uttropin deficiency; DFNA 2 Nonsyndromic Hearing Loss; Klein-Waardenberg syndrome; Gray platelet syndrome; Bile acid synthesis defect, congenital, 2; 46, XY sex reversal, type 1, 3, and 5; Acute intermittent porphyria; Cornelia de Fange syndromes 1 and 5; Hyperglycinuria; Cone-rod dystrophy 3; Dysfibrinogenemia; Karak syndrome; Congenital muscular dystrophy-dystroglycanopathy without mental retardation, type B5; Infantile nystagmus, X-linked; Dyskeratosis congenita, autosomal recessive, 1, 3, 4, and 5; Microcephaly with or without chorioretinopathy, lymphedema, or mental retardation; Hyperlysinemia; Bardet-Biedl syndromes 1, 11, 16, and 19; Autosomal recessive centronuclear myopathy; Frasier syndrome; Caudal regression syndrome; Fibrosis of extraocular muscles, congenital, 1, 2, 3a (with or without extraocular involvement), 3b; Prader-Willi-like syndrome; Malignant melanoma; Bloom syndrome; Darier disease, segmental; Multicentric osteolysis nephropathy; Hemochromatosis type 1, 2B, and 3; Cerebellar ataxia infantile with progressive external ophthalmoplegi and Cerebellar ataxia, mental retardation, and dysequilibrium syndrome 2; Hypoplastic left heart syndrome: Epilepsy, Hearing Loss, And Mental Retardation Syndrome; Transferrin serum level quantitative trait locus 2; Ocular albinism, type I; Marfan syndrome; Congenital muscular dystrophy-ystroglycanopatly with brain and eye anomalies, type A14 and B14; Hyperammonemia, type III; Cryptophthalmos syndrome; Alopecia universalis congenital; Adulk hypophosphatasia; Mannose-binding protein deficiency; Bull eye macular dystrophy; Autosomal dominant torsion dystonia 4; Nephrotic syndrome, type 3, type 5, with or without ocular abnormalities, type 7, and type 9; Seizures, Early infantile epileptic encephalopathy 7: Persistent hyperinsulinemic hypoglycemia of infancy; Thrombocytopenia, X-linked; Neonatal hypotonia; Orstavik Lindemann Solberg syndrome; Pulmonary hypertension, primary, 1, with hereditary hemorrhagic telangiectasia; Pituitary dependent hypercortisolism; Epidermodysplasia verruciformis; Epidermolysis bullosa, junctional, localisata variant; Cytochrome c oxidase i deficiency; Kindler syndrome; Myosclerosis, autosomal recessive; Truncus arteriosus; Duane syndrome type 2; ADULT syndrome; Zellweger syndrome spectrum; Leukoencephalopathy with ataxia, with Brainstem and Spinal Cord Involvement and Lactate Elevation, with vanishing white matter, and progressive, with ovarian failure; Antithrombin III deficiency; Holoprosencephaly 7; Roberts-SC phocomelia syndrome; Mitochondrial DNA-depletion syndrome 3 and 7, hepatocerebral types, and 13 (encephalomyopathic type); Porencephaly 2; Microcephaly, normal intelligence and immunodeficiency; Giant axonal neuropathy; Sturge-Weber syndrome, Capillary malformations, congenital, 1; Fabry disease and Fabry disease, cardiac variant: Glutamate formiminotransferase deficiency; Fanconi-Bickel syndrome; Acromicric dysplasia; Epilepsy, idiopathic generalized, susceptibility to, 12; Basal ganglia calcification, idiopathic, 4; Polyglucosan body myopathy 1 with or without immunodeficiency; Malignant tumor of prostate; Congenital ectodermal dysplasia of face; Congenital heart disease; Age-related macular degeneration 3, 6, 11, and 12; Congenital myotonia, autosomal dominant and recessive forms; Hypomagnesemia 1, intestinal; Sulfite oxidase deficiency, isolated; Pick disease; Plasminogen deficiency, type 1; Syndactyly type 3; Cone-rod dystrophy amylogenesis imperfecta; Pseudoprimary hyperaldosteronisin; Terminal osseous dysplasia; Bartter syndrome antenatal type 2; Congenital muscular dystrophy-dystroglycanopathy with mental retardation, types B2, B3, B5, and B15; Familial infantile myasthenia; Lymphoproliferative syndrome 1, 1 (X-linked), and 2; Hypercholesterolaemia and Hypercholesterolemia, autosomal recessive; Neoplasm of ovary; Infantile GM1 gangliosidosis; Syndromic X-linked mental retardation 16; Deficiency of ribose-5-phosphate isomerase; Alzheimer disease, types, 1, 3, and 4; Andersen Tawil syndrome; Multiple synostoses syndrome 3; Chilbain lupus 1; Hemophagocytic lymphohistiocytosis, familial, 2 Axenfeld-Rieger syndrome type 3; Myopathy, congenital with cores; Osteoarthritis with mild chondrodysplasia; Peroxisome biogenesis disorders: Severe congenital neutropenia; Hereditary neuralgic amyotrophy; Palmoplantar keratoderma, nonepidermolytic, focal or diffuse: Dysplasminogenemia; Familial colorectal cancer; Spastic ataxia 5, autosomal recessive, Charlevoix-Saguenay type, 1, 10, or 11, autosomal recessive; Frontometaphyseal dysplasia 1 and 3; Hereditary factors II, IX, VIII deficiency disease: Spondylocheirodysplasia, Ehlers-Danlos syndrome-like, with immune dysregulation, Aggrecan type, with congenital joint dislocations, short limb-hand type, Sedaghatian type, with cone-rod dystrophy, and Kozlowski type: Ichthyosis prematurity syndrome; Stickler syndrome type 1; Focal segmental glomerulosclerosis 5; 5-Oxoprolinase deficiency; Microphthalmia syndromic 5, 7, and 9; Juvenile polyposis/hereditary hemorrhagic telangiectasia syndrome; Deficiency of butyryl-CoA dehydrogenase; Maturity-onset diabetes of the young, type 2; Mental retardation, syndromic, Claes-Jensen type, X-linked; Deafness, cochlear, with myopia and intellectual impairment, without vestibular involvement, autosomal dominant, X-linked 2; Spondylocarpotarsal synostosis syndrome; Sting-associated vasculopathy, infantile-onset; Neutral lipid storage disease with myopathy; immune dysfunction with T-cell inactivation due to calcium entry defect 2; Cardiofaciocutaneous syndrome; Corticosterone methyloxidase type 2 deficiency; Hereditary myopathy with early respiratory failure; Interstitial nephritis, karyomegalic: Trimethylaminuria; Hyperimmunoglobulin D) with periodic fever; Malignant hyperthermia susceptibility type 1; Trichomegaly with mental retardation, dwarfism and pigmentary degeneration of retina: Breast adenocarcinoma; Complement factor B deficiency; Ullrich congenital muscular dystrophy; Left ventricular noncompaction cardiomyopathy; Fish-eye disease: Finnish congenital nephrotic syndrome; Limb-girdle muscular dystrophy, type IB, 2A, 2B, 2D, C1, C5, C9, C14; Idiopathic fibrosing alveolitis, chronic form; Primary familial hypertrophic cardiomyopathy; Angiotensin i-converting enzyme, benign serum increase: Cd8 deficiency, familial; Proteus syndrome; Glucose-6-phosphate transport defect; Boreson-Forssnan-Lehmann syndrome; Zellweger syndrome; Spinal muscular atrophy, type II; Prostate cancer, hereditary. 2; Thrombocytopenia, platelet dysfunction, hemolysis, and imbalanced globin synthesis; Congenital disorder of glycosylation types IB, ID, IC, IH, IJ, IJK, IN, IP, 2C, 2J, 2K, IIm; Junctional epidermolysis bullosa gravis of Herlitz; Generalized epilepsy with febrile seizures plus 3, type 1, type 2; Schizophrenia 4; Coronary artery disease, autosomal dominant 2; Dyslkeratosis congenita, autosomal dominant, 2 and 5: Subcortical laminar heterotopia, X-linked; Adenylate kinase deficiency; X-linked severe combined immunodeficiency; Coproporphyria; Amyloid Cardiomyopathy, Transthyretin-related: Hypocalcemia, autosomal dominant 1; Brugada syndrome; Congenital myasthenic syndrome, acetazolamide-responsive; Primary hypomagnesemia; Sclerosteosis; Frontotemporal dementia and/or amyotrophic lateral sclerosis 3 and 4: Mevalonic aciduria; Schwannomatosis 2; Hereditary motor and sensory neuropathy with optic atrophy; Porphyria cutanea tarda; Osteochondritis dissecans; Seizures, benign familial neonatal, 1, and/or myokymia; Long QT syndrome, LQT1 subtype; Mental retardation, anterior maxillary protrusion, and strabismus; Idiopathic hypercalcemia of infancy; Hypogonadotropic hypogonadism 11 with or without anosmia; Polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy; Primary autosomal recessive microcephaly 10, 2, 3, and 5; Interrupted aortic arch; Congenital amnegakaryocytic thrombocytopenia; Hermansky-Pudlak syndrome 1, 3, 4, and 6; Long QT syndrome 1, 2, 2/9, 2/5, (digenic), 3, 5 and 5, acquired, susceptibility to; Andermann syndrome; Retinal cone dystrophy 3B; Erythropoietic protoporphyria; Sepiapterin reductase deficiency; Very long chain acyl-CoA dehydrogenase deficiency; Hyperferritinenmia cataract syndrome; Silver spastic paraplegia syndrome; Charcot-Marie-Tooth disease; Atrial septal defect 2; Carnevale syndrome: Hereditary insensitivity to pain with anhidrosis; Catecholaminergic polymorphic ventricular tachycardia; hypokalemic periodic paralysis 1 and 2: Sudden infant death syndrome; Hypochloric microcytic anemia with iron overload; GLUT1 deficiency syndrome 2; Leukodystrophy, Hypomyelinating, 11 and 6; Cone monochromatism; Osteopetrosis autosomal dominant type 1 and 2, recessive 4, recessive I, recessive 6; Severe congenital neutropenia 3, autosomal recessive or dominant; Methionine adenosyltransferase deficiency, autosomal dominant; Paroxysmal familial ventricular fibrillation: Pyruvate kinase deficiency of red cells; Schneckenbecken dysplasia; Torsades de pointes; Distal myopathy Markesbery-Griggs type; Deficiency of UDP glucose-hexose-1-phosphate uridylyltransferase; Sudden cardiac death; Neu-Laxova syndrome 1; Atransferrinemia; Hyperparathyroidism 1 and 2; Cutaneous malignant melanoma L; Symphalangism, proximal, lb; Progressive pseudorheumatoid dysplasia; Werdnig-Hoffmann disease; Achondrogenesis type 2; Holoprosencephaly 2, 3, 7, and 9; Schindler disease, type 1, Cerebroretinal microangiopathy with calcifications and cysts; leterotaxy, visceral, X-linked; Tuberous sclerosis syndrome; Kartagener syndrome; Thyroid hormone resistance, generalized, autosomal dominant; Bestrophinopathy, autosomal recessive; Nail disorder, nonsyndromic congenital, 8; Mohr-Tranebjaerg syndrome; Cone-rod dystrophy 12; Hearing impairment; Ovarioleukodystrophy; Renal tubular acidosis, proximal, with ocular abnormalities and mental retardation; Dihydropteridine reductase deficiency; Focal epilepsy with speech disorder with or without mental retardation; Ataxia-telangiectasia syndrome; Brown-Vialetto-Van laere syndrome and Brown-Vialetto-Van Laere syndrome 2; Cardiomyopathy; Peripheral demyelinating neuropathy, central dysmyelination; Comeal dystrophy, Fuchs endothelial, 4; Cowden syndrome 3; Dystonia 2 (torsion, autosomal recessive), 3 (torsion, X-linked), 5 (Dopa-responsive type), 10, 12, 16, 25, 26 (Myoclonic); Epiphyseal dysplasia, multiple, with myopia and conductive deafness; Cardiac conduction defect, nonspecific; Branchiootic syndromes 2 and 3; Peroxisome biogenesis disorder 14B, 2A, 4A, 5B, 6A, 7A, and 7B: Familial renal glucosuria; Candidiasis, familial, 2, 5, 6, and 8; Autoimmune disease, multisystem, infantile-onset, Early infantile epileptic encephalopathy 2, 4, 7, 9, 10, 11, 13, and 14; Segawa syndrome, autosomal recessive; Deafness, autosomal dominant 3a, 4, 12, 13, 15, autosomal dominant nonsyndromic sensorineural 17, 20, and 65; Congenital dyserythropoietic anemia, type I and II, Enhanced s-cone syndrome; Adult neuronal ceroid lipofuscinosis; Atrial fibrillation, familial, 11, 12, 13, and 16; Norum disease; Osteosarcoma; Partial albinism; Biotinidase deficiency; Combined cellular and humoral immune defects with granulomas; Alpers encephalopathy; Holocarboxylase synthetase deficiency; Maturity-onset diabetes of the young, type 1, type 2, type 11, type 3, and type 9; Variegate porphyria; Infantile cortical hyperostosis; Testosterone 17-beta-dehydrogenase deficiency; L-2-hydroxyglutaric aciduria; Tyrosinase-negative oculocutaneous albinism, Primary ciliary dyskinesia 24; Pontocerebellar hypoplasia type 4; Ciliary dyskinesia, primary, 7, I1, 15, 20 and 22; Idiopathic basal ganglia calcification 5, Brain atrophy; Craniosynostosis 1 and 4; Keratoconus 1; Rasopathy; Congenital adrenal hyperplasia and Congenital adrenal hypoplasia, X-linked; Mitochondrial DNA depletion syndrome 11, 12 (cardiomyopathic type), 2, 4B (MNGIE type), 8B (MNGIE type); Brachydactyly with hypertension, Cornea plana 2; Aarskog syndrome; Multiple epiphyseal dysplasia 5 or Dominant; Comeal endothelial dystrophy type 2; Aminoacylase I deficiency; Delayed speech and language development; Nicolaides-Baraitser syndrome, Enterokinase deficiency; Ectrodactyly, ectodermal dysplasia, and cleft lip/palate syndrome 3; Arthrogryposis multiplex congenita, distal, X-linked; Perrault syndrome 4; Jervell and Lange-Nielsen syndrome 2; Hereditary Nonpolyposis Colorectal Neoplasms; Robinow syndrome, autosomal recessive. autosomal recessive, with brachy-syn-polydactyly; Neurofibrosarcoma, Cytochrome-c oxidase deficiency; Vesicoureteral reflux 8; Dopamine beta hydroxylase deficiency; Carbohydrate-deficient glycoprotein syndrome type I and 11; Progressive familial intrahepatic cholestasis 3; Benign familial neonatal-infantile seizures; Pancreatitis, chronic, susceptibility to; Rhizomelic chondrodysplasia punctata type 2 and type 3; Disordered steroidogenesis due to cytochrome p450 oxidoreductase deficiency; Deafness with labyrinthine aplasia microtia and microdontia (FAMM); Rothmund-Thomson syndrome, Cortical dysplasia, complex. with other brain malformations 5 and 6; Myasthenia, familial infantile, 1; Trichorhinophalangeal dysplasia type I; Worth disease; Splenic hypoplasia; Molybdenum cofactor deficiency, complementation group A; Sebastian syndrome; Progressive familial intrahepatic cholestasis 2 and 3; Weill-Marchesani syndrome 1 and 3, Microcephalic osteodysplastic primordial dwarfism type 2; Surfactant metabolism dysfunction, pulmonary, 2 and 3; Severe X-linked myotubular myopathy; Pancreatic cancer 3; Platelet-type bleeding disorder 15 and 8; Tyrosinase-positive oculocutaneous albinism; Borrone Di Rocco Crovato syndrome; ATR-X syndrome; Sucrase-isomaltase deficiency; Complement component 4, partial deficiency of, due to dysfunctional c1 inhibitor; Congenital central hypoventilation; Infantile hypophosphatasia; Plasminogen activator inhibitor type I deficiency; Malignant lymphoma, non-Hodgkin; Hyperornithinemia-hyperammonemia-homocitrullinuria syndrome; Schwartz Jampel syndrome type 1; Fetal hemoglobin quantitative trait locus 1; Myopathy, distal, with anterior tibial onset, Noonan syndrome I and 4, LEOPARD syndrome 1, Glaucoma 1, open angle, e, F, and G; Kenny-Caffey syndrome type 2; PTEN hamartoma tumor syndrome; Duchenne muscular dystrophy, Insulin-resistant diabetes mellitus and acanthosis nigricans, Microphthalmia, isolated 3, 5, 6, 8, and with coloboma 6, Raine syndrome, Premature ovarian failure 4, 5, 7, and 9; Allan-Hemdon-Dudley syndrome; Citrullinemia type I; Alzheimer disease, familial, 3, with spastic paraparesis and apraxia; Familial hemiplegic migraine types 1 and 2; Ventriculomegaly with cystic kidney disease; Pseudoxanthoma elasticum; Homocysteinemia due to MTHFR deficiency, CBS deficiency, and Homocystinuria, pyridoxine-responsive; Dilated cardiomyopathy 1A, 1AA, 1C, 1G, 1BB, 1DD, 1FF, 1HH, II, IK K, IN, IS, 1Y, and 3B; Muscle AM P guanine oxidase deficiency; Familial cancer of breast; Hereditary sideroblastic anemia; Myoglobinuria, acute recurrent, autosomal recessive; Neuroferritinopathy, Cardiac arrhythmia; Glucose transporter type I deficiency syndrome; Holoprosencephaly sequence; Angiopathy, hereditary, with nephropathy, aneurysms, and muscle cramps; Isovaleryl-CoA dehydrogenase deficiency; Kallmann syndrome 1, 2, and 6; Permanent neonatal diabetes mellitus; Acrocallosal syndrome, Schinzel type; Gordon syndrome; MYI-H9 related disorders; Donnai Barrow syndrome; Severe congenital neutropenia and 6, autosomal recessive; Charcot-Marie-Tooth disease, types ID and IVF; Coffin-Lowry syndrome; mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase deficiency; Flypomagnesemia, seizures, and mental retardation; Ischiopatellar dysplasia; Multiple congenital anomalies-hypotonia-seizures syndrome 3; Spastic paraplegia 50, autosomal recessive, Short stature with nonspecific skeletal abnormalities; Severe myoclonic epilepsy in infancy; Propionic academia; Adolescent nephronophthisis; Macrocephaly, macrosomia, facial dysmorphism syndrome; Stargardt disease 4; Ehlers-Danlos syndrome type 7 (autosomal recessive), classic type, type 2 (progeroid), hydroxylysine-deficient, type 4, type 4 variant, and due to tenascin-X deficiency; Myopia 6, Coxa plana; Familial cold autoinflammatory syndrome 2; Malformation of the heart and great vessels; von Willebrand disease type 2M and type 3; Deficiency of galactokinase; Brugada syndrome 1; X-linked ichthyosis with steryl-sulfatase deficiency; Congenital ocular coloboma; Flistiocytosis-lymphadenopathy plus syndrome; Aniridia, cerebellar ataxia, and mental retardation; Left ventricular noncompaction 3; Amyotrophic lateral sclerosis types 1, 6, 15 (with or without frontotemporal dementia). 22 (with or without frontotemporal dementia), and 10; Osteogenesis imperfecta type 12, type 5, type 7, type 8, type 1, type III, with normal sclerae, dominant form, recessive perinatal lethal; Hematologic neoplasm; Favism, susceptibility to; Pulmonary Fibrosis And/Or Bone Marrow Failure, Telomere-Related, 1 and 3; Dominant hereditary optic atrophy; Dominant dystrophic epidermolysis bullosa with absence of skin; Muscular dystrophy, congenital, megaconial type; Multiple gastrointestinal atresias; McCune-Albright syndrome; Nail-patella syndrome; McLeod neuroacanthocytosis syndrome; Common variable immunodeficiency 9; Partial hypoxanthine-guanine phosphoribosyltransferase deficiency; Pseudohypoaldosteronism type I autosomal dominant and recessive and type 2; Urocanate hydratase deficiency, Heterotopia; Meckel syndrome type 7, Ch\xc3\xa9diak-Higashi syndrome, Chediak-Higashi syndrome, adult type; Severe combined immunodeficiency due to ADA deficiency, with microcephaly, growth retardation, and sensitivity to ionizing radiation, atypical, autosomal recessive, T cell-negative. B cell-positive, NK cell-negative of NK-positive; Insulin resistance, Deficiency of steroid II-beta-monooxygenase; Popliteal pterygium syndrome; Pulmonary arterial hypertension related to hereditary hemorrhagic telangiectasia; Deafness, autosomal recessive IA, 2, 3, 6, 8, 9, 12, 15, 16, 18b, 22, 28, 31, 44, 49, 63, 77, 86, and 89; Primary hyperoxaluria, type 1, type, and type II I; Paramyotonia congenita of von Eulenburg; Desbuquois syndrome; Camitine palmitoyltransferase I, I, II (late onset), and II (infantile) deficiency; Secondary hypothyroidism; Mandibulofacial dysostosis, Treacher Collins type, autosomal recessive; Cowden syndrome 1; Li-Fraumeni syndrome 1; Asparagine synthetase deficiency, Malattia leventinese; Optic atrophy 9; Infantile convulsions and paroxysmal choreoathetosis, familial; Ataxia with vitamin E deficiency; Islet cell hyperplasia; Miyoshi muscular dystrophy 1; Thrombophilia, hereditary, due to protein C deficiency, autosomal dominant and recessive; Fechtner syndrome; Properdin deficiency, X-linked; Mental retardation, stereotypic movements, epilepsy, and/or cerebral malformations; Creatine deficiency, X-linked, Pilomatrixoma, Cyanosis, transient neonatal and atypical nephropathic; Adult onset ataxia with oculomotor apraxia; Hemangioma, capillary infantile; PC-K6a; Generalized dominant dystrophic epidermolysis bullosa; Pelizaeus-Merzbacher disease; Myopathy, centronuclear, 1, congenital, with excess of muscle spindles, distal, 1, lactic acidosis, and sideroblastic anemia 1, mitochondrial progressive with congenital cataract, hearing loss, and developmental delay, and tubular aggregate, 2; Benign familial neonatal seizures 1 and 2; Primary pulmonary hypertension; Lymphedema, primary, with myelodysplasia, Congenital long QT syndrome; Familial exudative vitreoretinopathy, X-linked; Autosomal dominant hypohidrotic ectodermal dysplasia; Primordial dwarfism; Familial pulmonary capillary hemangiomatosis, Carnitine acylcamitine translocase deficiency; Visceral myopathy, Familial Mediterranean fever and Familial mediterranean fever, autosomal dominant; Combined partial and complete 17-alpha-hydroxylase/17, 20-lyase deficiency, Oto-palato-digital syndrome, type 1; Nephrolithiasis/osteoporosis, hypophosphatemic, 2; Familial type 1 and 3 hyperlipoproteinemia; 5 Phenotypes; CHARGE association; Fuhrmann syndrome; Hypotrichosis-lymphedema-telangiectasia syndrome; Chondrodysplasia Blomstrand type, Acroerythrokeratoderma; Slowed nerve conduction velocity, autosomal dominant; Hereditary cancer-predisposing syndrome; Craniodiaphyseal dysplasia, autosomal dominant; Spinocerebellar ataxia autosomal recessive 1 and 16; Proprotein convertase 1/3 deficiency; D-2-hydroxyglutaric aciduria 2; Hyperekplexia 2 and Hyperekplexia hereditary; Central core disease; Opitz G/BBB syndrome, Cystic fibrosis; Thiel-Behnke comeal dystrophy; Deficiency of bisphosphoglycerate mutase; Mitochondrial short-chain Enoyl-CoA Hydratase I deficiency; Ectodermal dysplasia skin fragility syndrome; Wolfram-like syndrome, autosomal dominant; Microcytic anemia; Pyruvate carboxylase deficiency; Leukocyte adhesion deficiency type I and III; Multiple endocrine neoplasia, types 1 and 4, Transient bullous dermolysis of the newborn; Primrose syndrome; Non-small cell lung cancer; Congenital muscular dystrophy; Lipase deficiency combined; COLE-CARPENTER SYNDROME 2; Atrioventricular septal defect and common atrioventricular junction; Deficiency of xanthine oxidase; Waardenburg syndrome type 1, 4C, and 2E (with neurologic involvement); Stickler syndrome, types l(nonsyndromic ocular) and 4, Comeal fragility keratoglobus, blue sclerae and joint hypermobility; Microspherophakia; Chudley-McCullough syndrome; Epidermolysa bullosa simplex and limb girdle muscular dystrophy, simplex with mottled pigmentation, simplex with pyloric atresia, simplex, autosomal recessive, and with pyloric atresia; Rett disorder; Abnormality of neuronal migration; Growth hormone deficiency with pituitary anomalies; Leigh disease, Keratosis palmoplantaris striata 1; Weissenbacher-Zweymuller syndrome; Medium-chain acyl-coenzyme A dehydrogenase deficiency; UDPglucose-4-epimerase deficiency; susceptibility to Autism, X-linked 3; Rhegmatogenous retinal detachment, autosomal dominant; Familial febrile seizures 8; Ulna and fibula absence of with severe limb deficiency; Left ventricular noncompaction 6; Centromeric instability of chromosomes 1.9 and 16 and immunodeficiency; Hereditary diffuse leukoencephalopathy with spheroids, Cushing syndrome; Dopamine receptor d2, reduced brain density of; C-like syndrome, Renal dysplasia, retinal pigmentary dystrophy, cerebellar ataxia and skeletal dysplasia; Ovarian dysgenesis 1; Pierson syndrome; Polyneuropathy, hearing loss, ataxia, retinitis pigmentosa, and cataract; Progressive intrahepatic cholestasis; autosomal dominant, autosomal recessive, and X-linked recessive Alport syndromes; Angelman syndrome; Amish infantile epilepsy syndrome; Autoimmune lymphoproliferative syndrome, type la, Hydrocephalus; Marfanoid habitus; Bare lymphocyte syndrome type 2, complementation group E; Recessive dystrophic epidermolysis bullosa; Factor H, VII, X, v and factor viii, combined deficiency of 2, xiii, a subunit, deficiency, Zonular pulverulent cataract 3; Warts, hypogammaglobulinemia, infections, and myelokathexis; Benign hereditary chorea; Deficiency of hyaluronoglucosaminidase; Microcephaly, hiatal hernia and nephrotic syndrome; Growth and mental retardation, mandibulofacial dysostosis, microcephaly, and cleft palate; Lymphedema, hereditary, id: Delayed puberty, Apparent mineralocorticoid excess; Generalized arterial calcification of infancy 2; METHYLMALONIC ACIDURIA, mut(0) TYPE; Congenital heart disease, multiple types, 2; Familial hypoplastic, glomerulocystic kidney; Cerebrooculofacioskeletal syndrome 2, Stargardt disease 1; Mental retardation, autosomal recessive 15, 44, 46, and 5; Prolidase deficiency; Methylmalonic aciduria cblB type, Oguchi disease, Endocrine-cerebroosteodysplasia; Lissencephaly 1, 2 (X-linked), 3, 6 (with microcephaly), X-linked; Somatotroph adenoma; Gamstorp-Wohlfart syndrome; Lipid proteinosis; Inclusion body myopathy 2 and 3, Enlarged vestibular aqueduct syndrome; Osteoporosis with pseudoglioma; Acquired long QT syandrome; Phenylketonuria; CHOPS syndrome; Global developmental delay: Bietti crystalline corneoretinal dystrophy; Noonan syndrome-like disorder with or without juvenile myelomonocytic leukemia; Congenital erythropoietic porphyria; Atrophia bulborum hereditaria; Paragangliomas 3; Van der Woude syndrome, Aromatase deficiency, Birk Barel mental retardation dysmorphism syndrome; Amyotrophic lateral sclerosis type 5; Methemoglobinemia types 1 and 2; Congenital stationary night blindness, type IA, IB, 1C, IE, IF, and 2A, Seizures; Thyroid cancer, follicular; Lethal congenital contracture syndrome 6; Distal hereditary motor neuropathy type 2B; Sex cord-stromal tumor; Epileptic encephalopathy, childhood-onset, early infantile, 1, 19, 23, 25, 30, and 32; Myofibrillar myopathy 1 and ZASP-related; Cerebellar ataxia infantile with progressive external ophthalmoplegia; Purine-nucleoside phosphorylase deficiency: Forebrain defects; Epileptic encephalopathy Lennox-Gastaut type, Obesity: 4, Left ventricular noncompaction 10; Verheij syndrome; Mowat-Wilson syndrome; Odontotrichomelic syndrome; Patterned dystrophy of retinal pigment epithelium, Lig4 syndrome: Barakat syndrome: IRAK4 deficiency; Somatotroph adenoma; Branched-chain ketoacid dehydrogenase kinase deficiency; Cystinuria; Familial aplasia of the vermis; Succinyl-CoA acetoacetate transferase deficiency, Scapuloperoneal spinal muscular atrophy: Pigmentary retinal dystrophy, Glanzmann thrombasthenia; Primary open angle glaucoma juvenile onset 1; Aicardi Goutieres syndromes 1, 4, and 5, Renal dysplasia; Intrauterine growth retardation, metaphyseal dysplasia, adrenal hypoplasia congenita, and genital anomalies; Beaded hair: Short stature. onychodysplasia, facial dysmorphism, and hypotrichosis; Metachromatic leukodystrophy; Cholestanol storage disease; Three M syndrome 2; Leber congenital amaurosis 11, 12, 13, 16, 4, 7, and 9; Mandibuloacral dysplasia with type A or B lipodystrophy, atypical, Meier-Gorlin syndromes 1 and 4; Hypotrichosis 8 and 12; Short QT syndrome 3; Ectodermal dysplasia 1 ib; Anonychia; Pseudohypoparathyroidism type 1A, Pseudopseudohypoparathyroidism; Leber optic atrophy: Bainbridge-Ropers syndrome; Weaver syndrome; Short stature, auditory canal atresia, mandibular hypoplasia, skeletal abnormalities; Deficiency of alpha-mannosidase; Macular dystrophy, vitelliform, adult-onset; Glutaric aciduria, type 1; Gangliosidosis GM 1 type 1 (with cardiac involvement) 3; Mandibuloacral dysostosis: Hereditary lymphedema type 1; Atrial standstill 2; Kabuki make-up syndrome, Bethlem myopathy and Bethlem myopathy 2; Myeloperoxidase deficiency; Fleck comeal dystrophy: Hereditary acrodermatitis enteropathica; Hypobetalipoproteinemia, familial, associated with apob32; Cockayne syndrome type A; Hyperparathyroidism, neonatal severe; Ataxia-telangiectasia-like disorder; Pendred syndrome; I blood group system; Familial benign pemphigus; Visceral heterotaxy 5, autosomal; Nephrogenic diabetes insipidus, Nephrogenic diabetes insipidus, X-linked, Minicore myopathy with external ophthalmoplegia; Perry syndrome; hypohidrotic/hair/tooth type, autosomal recessive; Hereditary pancreatitis; Mental retardation and microcephaly with pontine and cerebellar hypoplasia, Glycogen storage disease 0 (muscle), II (adult form), IXa2, IXc, type 1 A; Osteopathia striata with cranial sclerosis; Gluthathione synthetase deficiency: Brugada syndrome and Brugada syndrome 4: Endometrial carcinoma; Hypohidrotic ectodermal dysplasia with immune deficiency, Cholestasis, intrahepatic, of pregnancy 3, Bemard-Soulier syndrome, types A1 and A2 (autosomal dominant); Salla disease, Ornithine aminotransferase deficiency; PTEN hamartoma tumor syndrome; Distichiasis-lymphedema syndrome; Corticosteroid-binding globulin deficiency; Adult neuronal ceroid lipofuscinosis; Dejerine-Sottas disease; Tetraamelia, autosomal recessive; Senior-Loken syndrome 4 and 5, Glutaric acidemia 11A and 11B; Aortic aneurysm, familial thoracic 4, 6, and 9; Hyperphosphatasia with mental retardation syndrome 2, 3, and 4; Dyskeratosis congenita X-linked; Arthrogryposis, renal dysfunction, and cholestasis 2, Bannavan-Riley-Ruvalcaba syndrome; 3-Methylglutaconic aciduria; Isolated 17,20-lyase deficiency, Gorlin syndrome; Hand foot uterus syndrome; Tay-Sachs disease, B1 variant, Gm2-gangliosidosis (adult). Gm2-gangliosidosis (adult-onset); Dowling-degos disease 4; Parkinson disease 14, 15, 19 (juvenile-onset), 2, 20 (early-onset), 6, (autosomal recessive early-onset, and 9; Ataxia, sensory, autosomal dominant; Congenital microvillous atrophy; Myoclonic-Atonic Epilepsy; Tangier disease; 2-methyl-3-hydroxybutyric aciduria; Familial renal hypouricemia; Schizencephaly; Mitochondrial DNA depletion syndrome 4B, MNGIE type; Feingold syndrome 1; Renal carnitine transport defect; Familial hypercholesterolemia; Townes-Brocks-branchiootorenal-like syndrome; Griscelli syndrome type 3; Meckel-Gruber syndrome; Bullous ichthyosiform erythroderma; Neutrophil immunodeficiency syndrome; Myasthenic Syndrome, Congenital, 17, 2A (slow-channel), 4B (fast-channel), and without tubular aggregates; Microvascular complications of diabetes 7; McKusick Kaufman syndrome; Chronic granulomatous disease, autosomal recessive cytochrome b-positive, types I and 2; Arginino succinate lyase deficiency; Mitochondrial phosphate carrier and pyruvate carrier deficiency; Lattice corneal dystrophy Type II, Ectodermal dysplasia-syndactyly syndrome 1, Hypomyelinating leukodystrophy 7; Mental retardation, autosomal dominant 12, 13, 15, 24, 3, 30, 4, 5, 6, and 9; Generalized epilepsy with febrile seizures plus, types 1 and 2; Psoriasis susceptibility 2; Frank Ter Haar syndrome; Thoracic aortic aneurysms and aortic dissections; Crouzon syndrome; Granulosa cell tumor of the ovary; Epidermolytic palmoplantar keratoderma; Leri Weill dyschondrosteosis; 3 beta-Hydroxysteroid dehydrogenase deficiency; Familial restrictive cardiomyopathy 1; Autosomal dominant progressive external ophthalmoplegia with mitochondrial DNA deletions 1 and 3; Antley-Bixler syndrome with genital anomalies and disordered steroidogenesis; Hajdu-Cheney syndrome; Pigmented nodular adrenocortical disease, primary, 1, Episodic pain syndrome, familial, 3; Dejerine-Sottas syndrome, autosomal dominant, FG syndrome and FG syndrome 4; Dendritic cell, monocyte, B lymphocyte, and natural killer lymphocyte deficiency; Hypothyroidism, congenital, nongoitrous, 1; Miller syndrome; Nemaline myopathy 3 and 9, Oligodontia-colorectal cancer syndrome; Cold-induced sweating syndrome 1; Van Buchem disease type 2: Glaucoma 3, primary congenital, d; Citrullinemia type 1 and II; Nonaka myopathy; Congenital muscular dystrophy due to partial LAMA2 deficiency; Myoneural gastrointestinal encephalopathy syndrome; Leigh syndrome due to mitochondrial complex I deficiency; Medulloblastoma; Pyruvate dehydrogenase El-alpha deficiency; Carcinoma of colon; Nance-Horan syndrome, Sandhotf disease, adult and infantil types; Arthrogryposis renal dysfunction cholestasis syndrome; Autosomal recessive hypophosphatemic bone disease; Doyne honeycomb retinal dystrophy; Spinocerebellar ataxia 14, 21, 35, 40, and 6; Lewy body dementia; RRM2B-related mitochondrial disease; Brody myopathy; Megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome 2, Usher syndrome, types 1, 1B, ID, 1G, 2A, 2C, and 2D; hypocalcification type and hypomaturation type, 1IA1 Amelogenesis imperfecta; Pituitary hormone deficiency, combined 1, 2, 3, and 4; Cushing symphalangism; Renal tubular acidosis, distal, autosomal recessive, with late-onset sensorineural hearing loss, or with hemolytic anemia, Infantile nephronophthisis; Juvenile polyposis syndrome; Sensory ataxic neuropathy, dysarthria, and ophthalmoparesis; Deficiency of 3-hydroxyacyl-CoA dehydrogenase, Parathyroid carcinoma; X-linked agammaglobulinemia; Megaloblastic anemia, thiamine-responsive, with diabetes mellitus and sensorineural deafness; Multiple sulfatase deficiency, Neurodegeneration with brain iron accumulation 4 and 6; Cholesterol monooxygenase (side-chain cleaving) deficiency, hemolytic anemia due to Adenylosuccinate lyase deficiency; Myoclonus with epilepsy with ragged red fibers; Pitt-Hopkins syndrome; Multiple pterygium syndrome Escobar type; Homocystinuria-Megaloblastic anemia due to defect in cobalamin metabolism. cblE complementation type; Cholecystitis; Spherocytosis types 4 and 5; Multiple congenital anomalies: Xeroderma pigmentosum, complementation group b, group D. group E. and group G; Leiner disease; Groenouw comeal dystrophy type I; Coenzyme Q10 deficiency, primary 1, 4, and 7; Distal spinal muscular atrophy, congenital nonprogressive: Warburg micro syndrome 2 and 4; Bile acid synthesis defect, congenital, 3; Acth-independent macronodular adrenal hyperplasia 2, Acrocapitofemoral dysplasia; Paget disease of bone, familial; Severe neonatal-onset encephalopathy with microcephaly; Zimmermann-Laband syndrome and Zimmermann-Laband syndrome 2; Reifenstein syndrome; Familial hypokalemia-hypomagnesemia; Photosensitive trichothiodystrophy: Adult junctional epidermolysis bullosa; Lung cancer; Freeman-Sheldon syndrome; Hyperinsulinism-hyperammonemia syndrome, Posterior polar cataract type 2: Sclerocornea, autosomal recessive: Juvenile GM>1<gangliosidosis; Cohen syndrome, Hereditary Paraganglioma-Pheochromocytoma Syndromes; Neonatal insulin-dependent diabetes mellitus; Flypochondrogenesis; Floating-Harbor syndrome; Cutis laxa with osteodystrophy and with severe pulmonary, gastrointestinal, and urinary abnormalities; Congenital contractures of the limbs and face, hypotonia, and developmental delay; Dyskeratosis congenita autosomal dominant and autosomal dominant, 3: Histiocytic medullary reticulosis: Costello syndrome; Immunodeficiency 15, 16, 19, 30, 3 IC, 38, 40, 8, due to defect in cd3-zeta, with hyper IgM type 1 and 2, and X-Linked, with magnesium defect, Epstein-Barr vims infection, and neoplasia; Atrial septal defects 2, 4, and 7 (with or without atrioventricular conduction defects); GTP cyclohydrolase I deficiency; Talipes equinovarus: Phosphoglycerate kinase 1 deficiency; Tuberous sclerosis 1 and 2; Autosomal recessive congenital ichthyosis 1, 2, 3, 4A, and 4B; and Familial hypertrophic cardiomyopathy 1, 2, 3, 4, 7, 10, 23 and 24.


Indications by Tissue

Additional suitable diseases and disorders that can be treated by the systems and methods provided herein include, without limitation, diseases of the central nervous system (CNS) (see exemplary diseases and affected genes in Table 13), diseases of the eye (see exemplary diseases and affected genes in Table 14), diseases of the heart (see exemplary diseases and affected genes in Table 15), diseases of the hematopoietic stem cells (HSC) (see exemplary diseases and affected genes in Table 16), diseases of the kidney (see exemplary diseases and affected genes in Table 17), diseases of the liver (see exemplary diseases and affected genes in Table 18), diseases of the lung (see exemplary diseases and affected genes in Table 19), diseases of the skeletal muscle (see exemplary diseases and affected genes in Table 20), and diseases of the skin (see exemplary diseases and affected genes in Table 21). Table 22 provides exemplary protective mutations that reduce risks of the indicated diseases. In some embodiments, a Gene Writer system described herein is used to treat an indication of any of Tables 13-21. In some embodiments, the GeneWriter system modifies a target site in genomic DNA in a cell, wherein the target site is in a gene of any of Tables 13-21, e.g., in a subject having the corresponding indication listed in any of Tables 13-21. In some embodiments, the GeneWriter corrects a mutation in the gene. In some embodiments, the GeneWriter inserts a sequence that had been deleted from the gene (e.g., through a disease-causing mutation). In some embodiments, the GeneWriter deletes a sequence that had been duplicated in the gene (e.g., through a disease-causing mutation). In some embodiments, the GeneWriter replaces a mutation (e.g., a disease-causing mutation) with the corresponding wild-type sequence. In some embodiments, the mutation is a substitution, insertion, deletion, or inversion.









TABLE 13







CNS diseases and genes affected.









Gene


Disease
Affected





Alpha-mannosidosis
MAN2B1


Ataxia-telangiectasia
ATM


CADASIL
NOTCH3


Canavan disease
ASPA


Carbamoyl-phosphate synthetase 1 deficiency
CPS1


CLN1 disease
PPT1


CLN2 Disease
TPP1


CLN3 Disease (Juvenile neuronal ceroid lipofuscinosis,
CLN3


Batten Disease)



Coffin-Lowry syndrome
RPS6KA3


Congenital myasthenic syndrome 5
COLQ


Cornelia de Lange syndrome (NIPBL)
NIPBL


Cornelia de Lange syndrome (SMC1A)
SMC1A


Dravet syndrome (SCN1A)
SCN1A


Glycine encephalopathy (GLDC)
GLDC


GM1 gangliosidosis
GLB1


Huntington's Disease
HTT


Hydrocephalus with stenosis of the aqueduct of Sylvius
L1CAM


Leigh Syndrome
SURF1


Metachromatic leukodystrophy (ARSA)
ARSA


MPS type 2
IDS


MPS type 3
SGSH


Mucolipidosis IV
MCOLN1


Neurofibromatosis Type 1
NF1


Neurofibromatosis type 2
NF2


Pantothenate kinase-associated neurodegeneration
PANK2


Pyridoxine-dependent epilepsy
ALDH7A1


Rett syndrome (MECP2)
MECP2


Sandhoff disease
HEXB


Semantic dementia (Frontotemporal dementia)
MAPT


Spinocerebellar ataxia with axonal neuropathy (Ataxia with
SETX


Oculomotor Apraxia)



Tay-Sachs disease
HEXA


X-linked Adrenoleukodystrophy
ABCD1
















TABLE 14







Eye diseases and genes affected.










Disease
Gene Affected







Achromatopsia
CNGB3



Amaurosis Congenita (LCA1)
GUCY2D



Amaurosis Congenita (LCA10)
CEP290



Amaurosis Congenita (LCA2)
RPE65



Amaurosis Congenita (LCA8)
CRB1



Choroideremia
CHM



Cone Rod Dystrophy (ABCA4)
ABCA4



Cone Rod Dystrophy (CRX)
CRX



Cone Rod Dystrophy (GUCY2D)
GUCY2D



Cystinosis, Ocular Nonnephropathic
CTNS



Lattice corneal dystrophy type I
TGFBI



Macular Corneal Dystrophy (MCD)
CHST6



Optic Atrophy
OPA1



Retinitis Pigmentosa (AR)
USH2A



Retinitis Rigmentosa (AD)
RHO



Stargardt Disease
ABCA4



Vitelliform Macular Dystrophy
BEST1; PRPH2

















TABLE 15







Heart diseases and genes affected.









Gene


Disease
Affected





Arrhythmogenic right ventricular cardiomyopathy (ARVC)
PKP2


Barth syndrome
TAZ


Becker muscular dystrophy
DMD


Brugada syndrome
SCN5A


Catecholaminergic polymorphic ventricular tachycardia
RYR2


(RYR2)



Dilated cardiomyopathy (LMNA)
LMNA


Dilated cardiomyopathy (TTN)
TTN


Duchenne muscular dystrophy
DMD


Emery-Dreifuss Muscular Dystrophy Type I
EMD


Familial hypertrophic cardiomyopathy
MYH7


Familial hypertrophic cardiomyopathy
MYBPC3


Jervell Lange-Nielsen syndrome
KCNQ1


LCHAD deficiency
HADHA


Limb-girdle muscular dystrophy type IB (Emery-Dreifuss
LMNA


EDMD2)



Limb-girdle muscular dystrophy, type 2D
SGCA


Long QT syndrome 1 (Romano Ward)
KCNQ1
















TABLE 16







HSC diseases and genes affected.









Gene


Disease
Affected





ADA-SCID
ADA


Adrenoleukodystrophy (CALD)
ABCD1


Alpha-mannosidosis
MAN2B1


Chronic granulomatous disease
CYBB; CYBA;



NCF1; NCF2;



NCF4


Common variable immunodeficiency
TNFRSF13B


Fanconi anemia
FANCA; FANCC;



FANCG


Gaucher disease
GBA


Globoid cell leukodystrophy (Krabbe disease)
GALC


Hemophagocytic lymphohistiocytosis
PRF1; STX11;



STXBP2; UNC13D


IL-7R SCID
IL7R


JAK-3 SCID
JAK3


Malignant infantile osteopetrosis—autosomal
TCIRG1;


recessive osteopetrosis
Many genes



implicated


Metachromatic leukodystrophy
ARSA; PSAP


MPS 1S (Scheie syndrome)
IDUA


MPS2
IDS


MPS7
GUSB


Mucolipidosis II
GNPTAB


Niemann-Pick disease A and B
SMPD1


Niemann-Pick disease C
NPC1


Paroxysmal Nocturnal Hemoglobinuria
PIGA


Pompe disease
GAA


Pyruvate kinase deficiency (PKD)
PKLR


RAG 1/2 Deficiency (SCID with granulomas)
RAG1/RAG2


Severe Congenital Neutropenia
ELANE; HAX1


Sickle cell disease (SCD)
HBB


Tay Sachs
HEXA


Thalassemia
HBB


Wiskott-Aldrich Syndrome
WAS


X-linked agammaglobulinemia
BTK


X-linked SCID
IL2RG
















TABLE 17







Kidney diseases and genes affected.









Gene


Disease
Affected





Alport syndrome
COL4A5


Autosomal dominant polycystic kidney disease (PKD1)
PKD1


Autosomal dominant polycystic kidney disease (PKD2)
PDK2


Autosomal dominant tubulointerstitial kidney disease
MUC1


(MUC1)



Autosomal dominant tubulointerstitial kidney disease
UMOD


(UMOD)



Autosomal recessive polycystic kidney disease
PKHD1


Congenital nephrotic syndrome
NPHS2


Cystinosis
CTNS
















TABLE 18







Liver diseases and genes affected.









Gene


Disease
Affected





Acute intermittent porphyria
HMBS


Alagille syndrome
JAG1


Alpha-1-antitrypsin deficiency
SERPINA1


Carbamoyl phosphate synthetase I deficiency
CPS1


Citrullinemia I
ASS1


Crigler-Najjar
UGT1A1


Fabry
LPL


Familial chylomicronemia syndrome
GLA


Gaucher
GBE1


GSD IV
GBA


Heme A
F8


Heme B
F9


Hereditary amyloidosis (hTTR)
TTR


Hereditary angioedema
SERPING1



(KLKB1



for CRISPR)


HoFH
LDLRAP1


Hypercholesterolemia
PCSK9


Methylmalonic acidemia
MMUT


MPS II
IDS


MPS III
Type IIIa: SGSH



Type IIIb: NAGLU



Type IIIc: HGSNAT



Type IIId: GNS


MPS IV
Type IVA: GALNS



Type IVB: GLB1


MPS VI
ARSB


MSUD
Type Ia: BCKDHA



Type Ib: BCKDHB



Type II: DBT


OTC Deficiency
OTC


Polycystic Liver Disease
PRKCSH


Pompe
GAA


Primary Hyperoxaluria 1
AGXT (HAO1 or



LDHA for CRISPR)


Progressive familial intrahepatic cholestasis type 1
ATP8B1


Progressive familial intrahepatic cholestasis type 2
ABCB11


Progressive familial intrahepatic cholestasis type 3
ABCB4


Propionic acidemia
PCCB; PCCA


Wilson's Disease
ATP7B


Glycogen storage disease, Type 1a
G6PC


Glycogen storage disease, Type IIIb
AGL


Isovaleric acidemia
IVD


Wolman disease
LIPA
















TABLE 19







Lung diseases and genes affected.











Gene



Disease
Affected







Alpha-1 antitrypsin deficiency
SERPINA1



Cystic fibrosis
CFTR



Primary ciliary dyskinesia
DNAI1



Primary ciliary dyskinesia
DNAH5



Primary pulmonary hypertension I
BMPR2



Surfactant Protein B (SP-B) Deficiency (pulmonary
SFTPB



surfactant metabolism dysfunction 1)

















TABLE 20







Skeletal muscle diseases and genes affected.









Gene


Disease
Affected





Becker muscular dystrophy
DMD


Becker myotonia
CLCN1


Bethlem myopathy
COL6A2


Centronuclear myopathy, X-linked (myotubular)
MTM1


Congenital myasthenic syndrome
CHRNE


Duchenne muscular dystrophy
DMD


Emery-Dreifuss muscular dystrophy, AD
LMNA


Facioscapulohumeral Muscular Dystrophy
DUX4-D4Z4



chromosomal



region


Hyperkalemic periodic paralysis
SCN4A


Hypokalemic periodic paralysis
CACNA1S


Limb-girdle muscular dystrophy 2A
CAPN3


Limb-girdle muscular dystrophy 2B
DYSF


Limb-girdle muscular dystrophy, type 2D
SGCA


Miyoshi muscular dystrophy 1
DYSF


Paramyotonia congenita
SCN4A


Thomsen myotonia
CLCN1


VCP myopathy (IBMPFD) 1
VCP
















TABLE 21







Skin diseases and genes affected.









Gene


Disease
Affected





Epidermolysis Bullosa Dystrophica Dominant
COL7A1


Epidermolysis Bullosa Dystrophica Recessive
COL7A1


(Hallopeau-Siemens)



Epidermolysis Bullosa Junctional
LAMB3


Epidermolysis Bullosa Simplex
KRT5; KRT14


Epidermolytic Ichthyosis
KRT1; KRT10


Hailey-Hailey Disease
ATP2C1


Lamellar Ichthyosis/Nonbullous Congenital
TGM1


Ichthyosiform Erythroderma (ARCI)



Netherton Syndrome
SPINK5
















TABLE 22







Exemplary protective mutations that reduce disease risk.









Disease
Gene
Exemplary Protective Mutation





Alzheimer's
APP
A673T


Parkinson's
SGK1



Diabetes (Type
SLC30A8
p.Arg138X; p.Lys34SerfsX50


II)




Cardiovascular
PCSK9
R46L


Disease




Cardiovascular
ASGR1
NM_001671.4, c.284-36_283 +


Disease

33delCTGGGGCTGGGG




(SEQ ID NO: 1605); NP_001662.1,




p.W158X


Cardiovascular
NPC1L1
p.Arg406X


Disease




Cardiovascular
APOC3
R19X; IVS2 + 1G→A; A43T


Disease




Cardiovascular
LPA



Disease




Cardiovascular
ANGPTL4
E40K


Disease




Cardiovascular
ANGPTL3
p.Ser17Ter; p.Asn121fs; p.Asn147fs;


Disease

c.495 + 6T→C


HIV infection
CCR5
CCR5-delta32









Pathogenic Mutations

In some embodiments, the systems or methods provided herein can be used to correct a pathogenic mutation. The pathogenic mutation can be a genetic mutation that increases an individual's susceptibility or predisposition to a certain disease or disorder. In some embodiments, the pathogenic mutation is a disease-causing mutation in a gene associated with a disease or disorder. In some embodiments, the systems or methods provided herein can be used to revert the pathogenic mutation to its wild-type counterpart. In some embodiments, the systems or methods provided herein can be used to change the pathogenic mutation to a sequence not causing the disease or disorder.


Table 23 provides exemplary indications (column 1), underlying genes (column 2), and pathogenic mutations that can be corrected using the systems or methods described herein (column 3).









TABLE 23







Indications, genes, and causitive pathogenic mutations.









Disease
Gene
Pathogenic Mutation#





Achromatopsia
CNGB3
1148delC


Alpha-1 Antitrypsin Deficiency
SERPINA1
E342K


Alpha-1 Antitrypsin Deficiency
SERPINA1
E342K


Alpha-1 Antitrypsin Deficiency
SERPINA1
R48C (R79C)


Amaurosis Congenita (LCA10)
CEP290
2991 + 1655A > G


Andersen- Tawil syndrome
KCNJ2
R218W


Arrhythmogenic right
PKP2
c.235C > T


ventricular cardiomyopathy




(ARVC)




associated with congenital factor
F11
E117*


XI deficiency




associated with congenital factor
F11
F283L


XI deficiency




ATTR amyloidosis
TTR
V50M/N30M


autosomal dominant deafness
COCH
G88E


autosomal dominant deafness
TECTA
Y1870C


autosomal dominant Parkinson's
SNCA
A53T


disease




autosomal dominant Parkinson's
SNCA
A30P


disease




Autosomal dominant rickets
FGF23
R176Q


autosomal recessive deafness
CX30
T5M


autosomal recessive deafness
DFNB59
R183W


autosomal recessive deafness
TMC1
Y182C


autosomal recessive
ARH
Q136*


hypercholesterolemia




Blackfan-Diamond anemia
RPS19
R62Q


blue-cone monochromatism
OPN.ILW
C203R


Brugada syndrome
SCN5A
E1784K


CADASIL syndrome
NOTCH3
R90C



gene



CADASIL syndrome
NOTCH3
R141C



gene



Canavan disease
ASPA
E285A


Canavan disease
ASPA
Y231X


Canavan disease
ASPA
A305E


carnitine palmitoyltransferase II
CPT2
S113L


deficiency




choroideremia
CHM
R293*


choroideremia
CHM
R270*


choroideremia
CHM
A117A


Citrullinemia Type I
ASS
G390R


classic galactosemia
GALT
Q188R


classic horoocystoinuria
CBS
T191M


classic homocystemuria
CBS
G307S


CLN2 Disease
TPP1
c.509 − 1 G > C


CLN2 Disease
TPP1
c.622 C < T


CLN2 Disease
TPP1
c.851 G > T


cone-rod dystrophy
GUCY2D
R838C


congenital factor V deficiency
F5
R506Q


congenital factor V deficiency
F5
R534Q


congenital factor VII deficiency
F7
A294V


congenital factor VII deficiency
F7
C310F


congenital factor VII deficiency
F7
R304Q


congenital factor VII deficiency
F7
QI00R


Creutzfeldt-Jakob disease (CJD)
PRNP
E200K


Creutzfeldt-Jakob disease (CJD)
PRNP
M129V


Creutzfeldt-Jakob disease (CJD)
PRNP
P102L


Creutzfeldt-Jakob disease (CJD)
PRNP
D178N


cystic fibrosis
CFTR
G551D


cystic fibrosis
CFTR
W1282*


cystic fibrosis
CFTR
R553*


cystic fibrosis
CFTR
R117H


cystic fibrosis
CFTR
delta F508


eystinosis
CTNS
W138*


Darier disease
ATP2A2
N767S


Darier disease
ATP2A2
N767S


Darier disease
ATP2A2
N767S


Epidermolysis Bullosa
LAMB3
R42X


Junctional




Epidermolysis Bullosa
LAMB3
R635X


Junctional




familial amyotrophic lateral
SOD1
A4V


sclerosis (ALS)




familial amyotrophic lateral
SOD1
H46R


sclerosis (ALS)




familial amyotrophic lateral
SOD1
G37R


sclerosis (ALS)




Gaucher disease
GBA
N370S


Gaucher disease
GBA
N370S


Gaucher disease
GBA
L444P


Gaucher disease
GBA
L444P


Gaucher disease
GBA
L483P


glutarvl-CoA dehydrogenase
GCDH
R138G


deficiency




glutaryl-CoA dehydrogenase
GCDH
M263V


deficiency




glutaryl-Co A dehydrogenase
GCDH
R402W


deficiency




glycine encephalopathy
GLDC
A389V


glycine encephalopathy
GLDC
G771R


glycine encephalopathy
GLDC
T269M


hemophilia A
F8
R2178C


hemophilia A
F8
R550C


hemophilia A
F8
R2169H


hemophilia A
F8
R1985Q


hemophilia B
F9
T342M


hemophilia B
F9
R294Q


hemophilia B
F9
R43Q


hemophilia B
F9
R191H


hemophilia B
F9
G106S


hemophilia B
F9
A279T


hemophilia B
F9
P75*


hemophilia B
F9
R294*


hemophilia B
F9
R379Q


Hereditary antithrombin
SERPINCI
R48C (R79C)


deficiency type I




hereditary chronic pancreatitis
PRSS1
R122H


Hunter syndrome
IDS
R88C


Hunter syndrome
IDS
G374G


Hurler syndrome (MPS1)
IDUA
Q70*


Hurler syndrome (MPS1)
IDUA
W402*


Hyperkalemic periodic paralysis
SCN4A
T704M


Hyperkalemic periodic paralysis
SCN4A
M1592V


Hyperkalemic periodic paralysis
CACNA1S
p.Arg528X


Hyperkalemic periodic paralysis
CACNA1S
p.Arg1239


intermittent porphyria
HMBS
R173W


isolated agammaglobulinemia
E47
E555K


Lattice corneal dystrophy type I
TGFBI
Arg124Cys


LCHAD deficiency
HADHA
Glu474Gln


Leber congenital amaurosis 2
RPE65
R44*


Leber congenital amaurosis 2
RPE65
IVS1


Leber congenital amaurosis 2
RPE65
G-A, + 5


Lesch-Nyhan syndrome
HPRTI
R51*


Lesch-Nyhan syndrome
HPRTI
R170*


Limb-girdle muscular dystrophy,
SGCA
Arg77Cys


type 2D




Marteauz-Lamy Syndrome
ARSB
Y210C


(MSPVI)




Mediterranean G6PD deficiency
G6PD
S188D


medium-chain acyl-CoA
ACADM
K329E


dehydrogenase deficiency




medium-chain acyl-CoA
ACADM
K329E


dehydrogenase deficiency




medium-chain acyl-CoA
ACADM
K329E


dehydrogenase deficiency




Meesmann epithelial corneal
KRT12
L132P


dystrophy




metachfoniatic leukodystrophy
ARSA
P426L


metachromatic leukodystrophy
ARSA
c.459 + 1G > A


Morquio Syndrome (MPSIVA)
GALNS
R386C


Mucolipidosis IV
MCOLN1
406-2A > G


Mucolipidosis IV
MCOLN1
511_6943del


Neimann-Pick disease type A
SMPDI
L302P


Neuronal ceroid lipofuscinosis
CLN2
R208*


(NCL)




neuronal ceroid lipofuscinosis 1
PPT1
R151*


Parkinsons disease
LRRK2
G2019S


Pendred syndrome
PDS
T461P


Pendred syndrome
PDS
L236P


Pendred syndrome
PDS
c.1001 + 1G > A


Pendred syndrome
PDS
IVS8, + 1 G > A,


phenylketonuria
PAH
R408W


phenylketonuria
PAH
165T


phenylketonuria
PAH
R261Q


phenylketonuria
PAH
IVS10-HG > A


phenylketonuria
PCDH15
R245*


phenylketonuria
PCDH15
R245*


Pompe disease
GAA
c.−32 − 13T > G


Primary ciliary dyskinesia
DNAI1
IVS1 + 2_3insT


Primary ciliary dyskinesia
DNAH5
10815delT


primary' hypoxalimia
AGXT
G170R


Progressive familial intrahepatic
ABCB11
D482G (c.1445A > G)


cholestasis type 2




Progressive familial intrahepatic
ABCB11
E297G


cholestasis type 2




Propionic acidemia
PCCB;PCCA
c.1218_1231del14ins12


pseudoxanthoma, eiasticum
ABCC6
R1141*


Pyruvate kinase deficiency
PKLR
c.1456c −> T


(PKD)




retinitis pignientos
USH2a
C759F


retinitis pigmentosa
IMPDHI
D226N


retinitis pigmentosa
PDE6A
V685M


retinitis pigmentosa
PDE6A
D670G


retinitis pigmentosa
PRPF3
T494M


retinitis pigmentosa
PRPF8
H2309R


retinitis pigmentosa
RHO
P23H


retinitis pigmentosa
RHO
P347L


retinitis pigmentosa
RHO
P347L


retinitis pigmentosa
RHO
D190N


retinitis pigmentosa
RPI
R667*


retinitis pigmentosa/Usher
USH1C
V72V


syndrome type 1C




Rett syndrome
MECP2
R106W


Rett syndrome
MECP2
R133C


Rett syndrome
MECP2
R306C


Rett syndrome
MECP2
R168*


Rett syndrome
MECP2
R255*


Sanfilippo syndrome A
SGSH
R74C


(MPSIIIA)




Sanfilippo syndrome A
SGSH
R245H


(MPSIIIA)




Sanfilippo syndrome B
NAGLU
R297*


(MPSIIIB)




Sanfilippo syndrome B
NAGLU
Y140C


(MPSIIIB)




severe combined
ADA
G216R


immunodeficiency




severe combined
ADA
G216R


immunodeficiency




severe combined
ADA
Q3*


immunodeficiency




sickle cell disease
HBB
E6V


sickle cell disease
HBB
E6V


sickle cell disease
HBB
E6V


sickle cell disease
HBB
E26K


sickle cell disease
HBB
E26K


sickle cell disease
HBB
E7K


sickle cell disease
HBB
c.−138C > T


sickle cell disease
HBB
IVS2


sickle cell disease
HBB
654 C > T


Sly Syndrome (MPSVH)
GUSB
L175F


Stargardt disease
ABCA4
A1038V


Stargardt disease
ABCA4
A1038V


Stargardt disease
ABCA4
L541P


Stargardt disease
ABCA4
G1961E


Stargardt disease
ABCA4
G1961E


Stargardt disease
ABCA4
G1961E


Stargardt disease
ABCA4
G1961E


Stargardt disease
ABCA4
c.2588G > C


Stargardt disease
ABCA4
c.5461 − 10 T > C


Stargardt disease
ABCA4
c.5714 + 5G > A


Tay Sachs
HEXA
InsTATC1278


tyrosinemia type 1
FAH
P261L


Usher syndrome type 1F
PCDH15
R245*


variegate porphyria
PPOX
R59W


VCP myopathy (IBMPFD) 1
VCP
R1555X


von Gierke disease
G6PC
Q347*


von Gierke disease
G6PC
Q347*


von Gierke disease
G6PC
Q347*


von Gierke disease
G6PC
R83C


Wilson's Disease
ATP7B
E297G


X-linked myotubular myopathy
MTMI
c.1261 − 10A > G


X-linked retinoschisis
RS1
R102W


X-linked retinoschisis
RS1
R141C






#See J T den Dunnen and S E Antonarakis, Hum Mutat. 2000; 15(1): 7-12, herein incorporated by reference in its entirety, for details of the nomenclatures of gene mutations.


*means a stop codon.






Compensatory Edits

In some embodiments, the systems or methods provided herein can be used to introduce a compensatory edit. In some embodiments, the compensatory edit is at a position of a gene associated with a disease or disorder, which is different from the position of a disease-causing mutation. In some embodiments, the compensatory mutation is not in the gene containing the causative mutation. In some embodiments, the compensatory edit can negate or compensate for a disease-causing mutation. In some embodiments, the compensatory edit can be introduced by the systems or methods provided herein to suppress or reverse the mutant effect of a disease-causing mutation.


Table 24 provides exemplary indications (column 1), genes (column 2), and compensatory edits that can be introduced using the systems or methods described herein (column 3). In some embodiments, the compensatory edits provided in Table 24 can be introduced to suppress or reverse the mutant effect of a disease-causing mutation.









TABLE 24







Indications, genes, compensatory edits,


and exemplary design features.









Disease
Gene
Nucleotide Change#





Alpha-1 Antitrypsin Deficiency
SERPINAI
F51L


Alpha-1 Antitrypsin Deficiency
SERPINAI
M3741


Alpha-1 Antitrypsin Deficiency
SERPINAI
A348V/A347V


Alpha-1 Antitrypsin Deficiency
SERPINAI
K387R


Alpha-1 Antitrypsin Deficiency
SERPINAI
T59A


Alpha-1 Antitiypsin Deficiency
SERPINAI
T68A


ATTR amyloidosis
TTR
A108V


ATTR amyloidosis
TTR
R104H


ATTR amyloidosis
TTR
T119M


Cystic fibroses
CFTR
R555K


Cystic fibrosis
CFTR
F409L


Cystic fibrosis
CFTR
F433L


Cystic fibrosis
CFTR
H667R


Cystic fibrosis
CFTR
R1070W


Cystic fibrosis
CFTR
R29K


Cystic fibrosis
CFTR
R553Q


Cystic fibrosis
CFTR
1539T


Cystic fibrosis
CFTR
G550E


Cystic fibroses
CFTR
F429S


Cystic fibrosis
CFTR
Q637R


Sickle cell disease
HBB
A70T


Sickle cell disease
HBB
A70V


Sickle cell disease
HBB
L88P


Sickle cell disease
HBB
F85L and/or F85P


Sickle cell disease
HBB
E22G


Sickle cell disease
HBB
G16D and/or G16N






#See J T den Dunnen and S E Antonarakis, Hum Mutat. 2000; 15(1): 7-12, herein incorporated by reference in its entirety, for details of the nomenclatures of gene mutations.






Regulatory Edits

In some embodiments, the systems or methods provided herein can be used to introduce a regulatory edit. In some embodiments, the regulatory edit is introduced to a regulatory sequence of a gene, for example, a gene promoter, gene enhancer, gene repressor, or a sequence that regulates gene splicing. In some embodiments, the regulatory edit increases or decreases the expression level of a target gene. In some embodiments, the target gene is the same as the gene containing a disease-causing mutation. In some embodiment, the target gene is different from the gene containing a disease-causing mutation. For example, the systems or methods provided herein can be used to upregulate the expression of fetal hemoglobin by introducing a regulatory edit at the promoter of bcl11a, thereby treating sickle cell disease.


Table 25 provides exemplary indications (column 1), genes (column 2), and regulatory edits that can be introduced using the systems or methods described herein (column 3).









TABLE 25







Indications, genes, and compensatory regulatory edits.









Disease
Gene
Nucleotide Change#





homozygous familial
LDLR
c.81C > T


hypercholesterolaemia




Porphyrias
ALAS1
c.3G > A


Porphyrias
ALAS1
c.2T > C


Porphyrias
ALAS1
c.46C > T


Porphyrias
ALAS1
c.91C > T


Porphyrias
ALAS1
c.91C > T


Porphyrias
ALAS1
c.226C > T


Porphyrias
ALAS1
c.226C > T


Porphyrias
ALAS1
c.226C > T


Porphyrias
ALAS1
c.229C > T


Porphyrias
ALAS1
c.247C > T


Porphyrias
ALAS1
c.247C > T


Porphyrias
ALAS1
c.250C > T


Porphyrias
ALAS1
c.250C > T


Porphyrias
ALAS1
c.340C > T


Porphyrias
ALAS1
c.340C > T


Porphyrias
ALAS1
c.349C > T


Porphyrias
ALAS1
c.391C > T


Porphyrias
ALAS1
c.391C > T


Porphyrias
ALAS1
c.403C > T


Porphyrias
ALAS1
c.403C > T


Porphyrias
ALAS1
c.199 + 1G > A


Porphyrias
ALAS1
c.199 + 1G > A


Porphyrias
ALAS1
c.199 + 1G > A


Porphyrias
ALAS1
c.199 + 1G > A


Porphyrias
ALAS1
c.199 + 2T > C


Porphyrias
ALAS1
c.199 + 2T > C


Porphyrias
ALAS1
c.199 + 2T > C


Porphyrias
ALAS1
c.199 + 2T > C


Porphyrias
ALAS1
c.200 − 2A > G


Porphyrias
ALAS1
C.427 + 1G > A


Porphyrias
ALAS1
c.427 + 2T > C


Porphyrias
ALAS1
c.1165 + 1G > A


Porphyrias
ALAS1
c.1165 + 2T > C


Porphyrias
ALAS1
c.1166 − 1A > G


Porphyrias
ALAS1
c.1331 − 2A > G


sickle cell disease
BCL11A
c.386-24278G > A


sickle cell disease
BCL11A
c.386-24983T > C


sickle cell disease
HBG1
c.−167C > T


sickle cell disease
HBG1
c.−170G > A


sickle cell disease
HBG1
c.−249C > T


sickle cell disease
HBG2
c.−211C > T


sickle cell disease
HBG2
c.−228T > C


sickle cell disease
HBG1/2
C. −198 T > C


sickle cell disease
HBG1/2
C. −198 T > C


sickle cell disease
HBG1/2
C. −198 T > C


sickle cell disease
HBG1/2
C. −198 T > C


sickle cell disease
HBG1/2
C. −198 T > C


sickle cell disease
HBG1/2
C. −198 T > C


sickle cell disease
HBG1/2
C. −198 T > C


sickle cell disease
HBG1/2
C. −175 T > C


sickle cell disease
HBG1/2
C. −175 T > C


sickle cell disease
HBG1/2
C. −175 T > C


sickle cell disease
HBG1/2
C. −175 T > C


sickle cell disease
HBG1/2
C. −175 T > C


sickle cell disease
HBG1/2
C. −114~−102 deletion


sickle cell disease
HBG1/2
C. −114~−102 deletion


sickle cell disease
HBG1/2
C. −114~−102 deletion


sickle cell disease
HBG1/2
C. −114~−102 deletion


sickle cell disease
HBG1/2
C. −114~−102 deletion


sickle cell disease
HBG1/2
C. −114~−102 deletion


sickle cell disease
HBG1/2
C. −114~−102 deletion


sickle cell disease
HBG1/2
C. −114~−102 deletion


sickle cell disease
HBG1/2
C. −114~−102 deletion


sickle cell disease
HBG1/2
C. −114~−102 deletion


sickle cell disease
HBG1/2
C. −114~−102 deletion


sickle cell disease
HBG1/2
c. −90 BCLllA Binding


sickle cell disease
HBG1/2
c. −90 BCLllA Binding


sickle cell disease
HBG1/2
C. −202 C > T, −201 C >




T, −198 T > C, −197 C >




T, −196 C > T, −195 C > G


sickle cell disease
HBG1/2
C. −197 C > T, −196 C >




T, −195 C > G






#See J T den Dunnen and S E Antonarakis, Hum Mutat. 2000; 15(1): 7-12, herein incorporated by reference in its entirety, for details of the nomenclatures of gene mutations.






Repeat Expansion Diseases

In some embodiments, the systems or methods provided herein can be used to treat a repeat expansion disease, for example, a repeat expansion disease provided in Table 26. Table 26 provides the indication (column 1), the gene (column 2), minimal repeat sequence of the repeat that is expanded in the condition (column 3), and the location of the repeat relative to the listed gene for each indication (column 4). In some embodiments, the systems or methods provided herein, for example, those comprising Gene Writers, can be used to treat repeat expansion diseases by resetting the number of repeats at the locus according to a customized RNA template (see, e.g., Example 24).









TABLE 26







Exemplary repeat expansion diseases, genes,


 causal repeats, and repeat locations.












Causal
Repeat


Disease
Gene
repeat
location





myotonic
DMPK/DM1
CTG
3′ UTR


dystrophy 1








myotonic
ZNF9/CNBP
CCTG
Intron 1


dystrophy 2








dentatorubral-
ATN1
CAG
Coding


pallidoluysian





atrophy








fragile X mental
FMR1
CGG
5′ UTR


retardation





syndrome








fragile X E mental
FMR2
GCC
5′ UTR


retardation








Friedreich's ataxia
FXN
GAA
Intron





fragile X tremor
FMR1
CGG
5′ UTR


ataxia





syndrome








Huntington's
HTT
CAG
Coding


disease








Huntington's
JPH3
CTG
3′ UTR,


disease-like 2


coding





myoclonic
CSTB
CCCC
Promoter


epilepsy of

GCCC



Unverricht

CGCG



and Lundborg

(SEQ





ID





NO:





1606)






oculopharyngeal
PABPN1
GCG
Coding


muscular





dystrophy








spinal and bulbar
AR
CAG
Coding


muscular





atrophy








spinocerebellar
ATXN1
CAG
Coding


ataxia 1








spinocerebellar
ATXN2
CAG
Coding


ataxia 2








spinocerebellar
ATXN3
CAG
Coding


ataxia 3








spinocerebellar
CACNA1A
CAG
Coding


ataxia 6








spinocerebellar
ATXN7
CAG
Coding


 ataxia 7








spinocerebellar
ATXN8
CTG/CAG
CTG/CAG


ataxia 8


(ATXN8)





spinocerebellar
ATXN10
ATTCT
Intron


ataxia 10








spinocerebellar
PPP2R2B
CAG
Promoter,


ataxia 12


5′ UTR?





spinocerebellar
TBP
CAG
Coding


ataxia 17








Syndromic/non-
ARX
GCG
Coding


syndromic X-





linked





mental





retardation









Exemplary Templates

In some embodiments, the systems or methods provided herein use the template sequences listed in Table 27. Table 27 provides exemplary template RNA sequences (column 5) and optional second-nick gRNA sequences (column 6) designed to be paired with a Gene Writing polypeptide to correct the indicated pathogenic mutations (column 4). All the templates in Table 27 are meant to exemplify the total sequence of: (1) targeting gRNA for first strand nick, (2) polypeptide binding domain, (3) heterologous object sequence, and (4) target homology domain for setting up TPRT at first strand nick.









TABLE 27







Exemplary diseases, tissues, genes, pathogenic mutations, template RNA sequences,


and second nick gRNA sequences.

















Second







nick


Disease
Tissue
Gene
Mutation
Template RNA
gRNA





Alpha-1
Liver
SERP
PIZ
TCCCCTCCAGGCCGTGCATAGTTTT
TTTGTT


antitrypsin

INA1

AGAGCTAGAAATAGCAAGTTAAAA
GAACTT






TAAGGCTAGTCCGTTATCAACTTGA
GACCTC






AAAAGTGGGACCGAGTCGGTCCTcG
GG (SEQ






TCGATGGTCAGCACAGCCTTATGCA
ID NO:






CGGCCTGGA (SEQ ID NO: 1607)
1608)





Cystic
Lung
CFTR
deltaF508
ACCATTAAAGAAAATATCATGTTTT
AaagAT


fibrosis



AGAGCTAGAAATAGCAAGTTAAAA
GATATT






TAAGGCTAGTCCGTTATCAACTTGA
TTCTTT






AAAAGTGGGACCGAGTCGGTCCAC
AA (SEQ






CAaagATGATATTTTCTTTA (SEQ ID
ID NO:






NO: 1609)
1610)





Sickle cell
HSC
HBB
HbS
GTAACGGCAGACTTCTCCACGTTTT
TGGTGA






AGAGCTAGAAATAGCAAGTTAAAA
GGCCCT






TAAGGCTAGTCCGTTATCAACTTGA
GGGCA






AAAAGTGGGACCGAGTCGGTCCGA
GGT






CTCCTGaGGAGAAGTCTGCC (SEQ ID
(SEQ ID






NO: 1611)
NO:







1612)





Wilson's
Liver
ATP7
H1069Q
TTGGTGACTGCCACGCCCAAGTTTT
GGCCA


Disease

B

AGAGCTAGAAATAGCAAGTTAAAA
GCAGT






TAAGGCTAGTCCGTTATCAACTTGA
GAACAc






AAAAGTGGGACCGAGTCGGTCCAC
CCCT






AcCCCTTGGGCGTGGCAGTC (SEQ ID
(SEQ ID






NO: 1613)
NO:







1614)





ARVC
Heart
PKP2
235C>T
ACTCAGGAACACTGCTGGTTGTTTT
TTGGTT






AGAGCTAGAAATAGCAAGTTAAAA
GAAAA






TAAGGCTAGTCCGTTATCAACTTGA
TGATTT






AAAAGTGGGACCGAGTCGGTCCTTC
TGT






ACtGAACCAGCAGTGTTCC (SEQ ID
(SEQ ID






NO: 1615)
NO:







1616)





Long QT
Heart
KCN
P343S
CCAGGGAAAACGCACCCACGGTTTT
CTCCTT


syndrome 1

Q1

AGAGCTAGAAATAGCAAGTTAAAA
CTTTGC






TAAGGCTAGTCCGTTATCAACTTGA
GCTCcC






AAAAGTGGGACCGAGTCGGTCCTCc
AG (SEQ






CAGCGGTAGGTGCCCCGTGGGTGC
ID NO:






GTTTTC (SEQ ID NO: 1617)
1618)





Mucolipidosis
CNS
MCO
406-2A>G
GCCCTCCCCTTCTCTGCCCAGTTTTA
TCAGGC


IV

LN1

GAGCTAGAAATAGCAAGTTAAAAT
AACGC






AAGGCTAGTCCGTTATCAACTTGAA
CAGGT






AAAGTGGGACCGAGTCGGTCCGGT
ACtG






ACtGTGGGCAGAGAAGGGG (SEQ ID
(SEQ ID






NO: 1619)
NO:







1620)









In some embodiments, the systems or methods provided herein use the template sequences listed in Table 35. Table 35 provides exemplary template RNA sequences (column 5) and optional second-nick gRNA sequences (column 6) designed to be paired with a Gene Writing polypeptide to correct the indicated pathogenic mutations (column 4). All the templates in Table 35 are meant to exemplify the total sequence of: (1) targeting gRNA for first strand nick, (2) polypeptide binding domain, (3) heterologous object sequence, and (4) target homology domain for setting up TPRT at first strand nick.









TABLE 35







Exemplary Gene Writing templates and second nick gRNA sequences for the correction


of exemplary repeat expansion diseases. The region of the template spanning the


repeat(s) is indicated in lowercase.



















Second




Reference



-nick


Disease
Gene
Accession
Repeat
Location
Template RNA
gRNA





myotonic
DMPK
NC_00019.10
CTG
3′ UTR
CTCGAAGGGTC
ATCA


dystrophy

(45769709 . . .


CTTGTAGCCGTT
CAGG


1

45782490,


TTAGAGCTAGA
ACTG




complement)


AATAGCAAGTT
GAGC







AAAATAAGGCT
TGGG







AGTCCGTTATCA
(SEQ







ACTTGAAAAAG
ID NO:







TGGGACCGAGT
1622)







CGGTCCGTGAT








CCCCCcagcagcagc








agcagcagcagcagcag








cagcagcagcagcagca








gcagcagcagcagcag








CATTCCCGGCTA








CAAGGACCCT








(SEQ ID NO: 1621)






myotonic
CNBP
NC_00003.12
CCTG
Intron 1
ACCACTGCACT
GCCT


dystrophy

(129167827 . . .


CCAGCCTAGGT
CAGC


2

129183896,


TTTAGAGCTAG
CTCC




complement)


AAATAGCAAGT
TGAG







TAAAATAAGGC
TAGC







TAGTCCGTTATC
(SEQ







AACTTGAAAAA
ID NO:







GTGGGACCGAG
1624)







TCGGTCCGTGTC








TGTCTGTCTGTC








TGTCTGTCTGTC








TGTCTGTCTGTC








TGcctgcctgcctgcctg








cctgcctgcctgcctggct








gcctgtctgcctgtctgcct








gcctgcctgcctgcctgcc








tgcctgTCTGTCTC








ACTTTGTCCCCT








AGGCTGGAGTG








CA (SEQ ID NO:








1623)






fragile X
FMR1
NC_00023.11
CGG
5′ UTR
GGGGGCGTGCG
GCTC


mental

(147911919 . . .


GCAGCGCGGGT
AGAG


retardation

147951127)


TTTAGAGCTAG
GCGG


syndrome




AAATAGCAAGT
CCCT







TAAAATAAGGC
CCAC







TAGTCCGTTATC
(SEQ







AACTTGAAAAA
ID NO:







GTGGGACCGAG
1626)







TCGGTCCTGCG








GGCGCTCGAGG








CCCAGccgccgccgc








cgccgccgccgccgccg








cctccgccgccgccgcc








gccgccgccgccgccg








CGCTGCCGCAC








G (SEQ ID NO:








1625)






Friedreich's
FXN
NC_00009.12
GAA
Intron
CAGGCGCGCGA
CGCT


ataxia

(69035752 . . .


CACCACGCCGT
TGAG




69079076)


TTTAGAGCTAG
CCCG







AAATAGCAAGT
GGAG







TAAAATAAGGC
GCAG







TAGTCCGTTATC
(SEQ







AACTTGAAAAA
ID NO:







GTGGGACCGAG
1628)







TCGGTCCAACC








CAGTATCTACTA








AAAAATACAAA








AAAAAAAAAAA








AAgaagaagaagaaga








agaaAATAAAGA








AAAGTTAGCCG








GGCGTGGTGTC








GCGC (SEQ ID








NO: 1627)






Huntington
HTT
NC_00004.12
CAG
Coding
GGCGGCTGAGG
CGCT


disease

(3074681 . . .


AAGCTGAGGGT
GCAC




3243960)


TTTAGAGCTAG
CGAC







AAATAGCAAGT
CGTG







TAAAATAAGGC
AGTT







TAGTCCGTTATC
(SEQ







AACTTGAAAAA
ID NO:







GTGGGACCGAG
1630)







TCGGTCCAGTCC








CTCAAGTCCTTC








cagcagcagcagcagca








gcagcagcagcagcage








agcagcagcagcagcag








cagcagcaacagccgcc








accgccgccgccgccgc








cgccgcctcctCAGCT








TCCTCAG (SEQ








ID NO: 1629)






spinocerebellar
ATXN
NC_00006.12
CAG
Coding
TGAGCCCCGGA
TCCA


ataxia
1
(16299112 . . .


GCCCTGCTGGTT
GTTC




16761490,


TTAGAGCTAGA
TCCG




complement)


AATAGCAAGTT
CAGA







AAAATAAGGCT
ACAC







AGTCCGTTATCA
(SEQ







ACTTGAAAAAG
ID NO:







TGGGACCGAGT
1632)







CGGTCCACAAG








GCTGAGcagcagca








gcagcagcagcagcagc








agcagcagcagcatcag








catcagcagcagcagca








gcagcagcagcagcagc








agcagcagcagCACC








TCAGCAGGGCT








CCGGG (SEQ ID








NO: 1631)









Exemplary Heterologous Object Sequences

In some embodiments, the systems or methods provided herein comprise a heterologous object sequence, wherein the heterologous object sequence or a reverse complementary sequence thereof, encodes a protein (e.g., an antibody) or peptide. In some embodiments, the therapy is one approved by a regulatory agency such as FDA.


In some embodiments, the protein or peptide is a protein or peptide from the THPdb database (Usmani et al. PLoS One 12(7):e0181748 (2017), herein incorporated by reference in its entirety. In some embodiments, the protein or peptide is a protein or peptide disclosed in Table 28. In some embodiments, the systems or methods disclosed herein, for example, those comprising Gene Writers, may be used to integrate an expression cassette for a protein or peptide from Table 28 into a host cell to enable the expression of the protein or peptide in the host. In some embodiments, the sequences of the protein or peptide in the first column of Table 28 can be found in the patents or applications provided in the third column of Table 28, incorporated by reference in their entireties.


In some embodiments, the protein or peptide is an antibody disclosed in Table 1 of Lu et al. J Biomed Sci 27(1):1 (2020), herein incorporated by reference in its entirety. In some embodiments, the protein or peptide is an antibody disclosed in Table 29. In some embodiments, the systems or methods disclosed herein, for example, those comprising Gene Writers, may be used to integrate an expression cassette for an antibody from Table 29 into a host cell to enable the expression of the antibody in the host. In some embodiments, a system or method described herein is used to express an agent that binds a target of column 2 of Table 29 (e.g., a monoclonal antibody of column 1 of Table 29) in a subject having an indication of column 3 of Table 29.









TABLE 28







Exemplary protein and peptide therapeutics.









Therapeutic peptide
Category
Pat. No.





Lepirudin
Antithrombins and
CA1339104



Fibrinolytic Agents



Cetuximab
Antineoplastic Agents
CA1340417


Dor se alpha
Enzymes
CA2184581


Denileukin diftitox
Antineoplastic Agents



Etanercept
Immunosuppressive
CA2476934



Agents



Bivalirudin
Antithrombins
U.S. Pat. No.




7,582,727


Leuprolide
Antineoplastic Agents



Peginterferon alpha-2a
Immunosuppressive
CA2203480



Agents



Alteplase
Thrombolytic Agents



Interferon alpha-n1
Antiviral Agents



Darbepoetin alpha
Anti-anemic Agents
CA2165694


Reteplase
Fibrinolytic Agents
CA2107476


Epoetin alpha
Hematinics
CA1339047


Salmon Calcitonin
Bone Density
U.S. Pat. No.



Conservation Agents
6,440,392


Interferon alpha-n3
Immunosuppressive




Agents



Pegfilgrastim
Immunosuppressive
CA1341537



Agents



Sargramostim
Immunosuppressive
CA1341150



Agents



Secretin
Diagnostic Agents



Peginterferon alpha-2b
Immunosuppressive
CA1341567



Agents



Asparagi se
Antineoplastic Agents



Thyrotropin alpha
Diagnostic Agents
U.S. Pat. No.




5,840,566


Antihemophilic Factor
Coagulants and
CA2124690



Thrombotic agents



A kinra
Antirheumatic Agents
CA2141953


Gramicidin D
Anti-Bacterial Agents



Intravenous
Immunologic Factors



Immunoglobulin




Anistreplase
Fibrinolytic Agents



Insulin Regular
Antidiabetic Agents



Tenecteplase
Fibrinolytic Agents
CA2129660


Menotropins
Fertility Agents



Interferon gamma-1b
Immunosuppressive
U.S. Pat. No.



Agents
6,936,695


Interferon alpha-2a,

CA2172664


Recombi nt




Coagulation factor
Coagulants



VIIa




Oprelvekin
Antineoplastic Agents



Palifermin
Anti-Mucositis Agents



Glucagon recombi nt
Hypoglycemic Agents



Aldesleukin
Antineoplastic Agents



Botulinum Toxin Type
Antidystonic Agents



B




Omalizumab
Anti-Allergic Agents
CA2113813


Lutropin alpha
Fertility Agents
U.S. Pat. No.




5,767,251


Insulin Lispro
Hypoglycemic Agents
U.S. Pat. No.




5,474,978


Insulin Glargine
Hypoglycemic Agents
U.S. Pat. No.




7,476,652


Collage se




Rasburicase
Gout Suppressants
CA2175971


Adalimumab
Antirheumatic Agents
CA2243459


Imiglucerase
Enzyme Replacement
U.S. Pat. No.



Agents
5,549,892


Abciximab
Anticoagulants
CA1341357


Alpha-1-protei se
Serine Protei se



inhibitor
Inhibitors



Pegaspargase
Antineoplastic Agents



Interferon beta-1a
Antineoplastic Agents
CA1341604


Pegademase bovine
Enzyme Replacement




Agents



Human Serum
Serum substitutes
U.S. Pat. No.


Albumin

6,723,303


Eptifibatide
Platelet Aggregation
U.S. Pat. No.



Inhibitors
6,706,681


Serum albumin iodo
Diagnostic Agents



ted




Infliximab
Antirheumatic Agents,
CA2106299



Anti-Inflammatory




Agents, Non-Steroidal,




Dermatologic Agents,




Gastrointesti 1 Agents




and Immunosuppressive




Agents



Follitropin beta
Fertility Agents
U.S. Pat. No.




7,741,268


Vasopressin
Anti diuretic Agents



Interferon beta-1b
Adjuvants, Immunologic
CA1340861



and Immunosuppressive




Agents



Interferon alphacon-1
Antiviral Agents and
CA1341567



Immunosuppressive




Agents



Hyaluronidase
Adjuvants, Anesthesia




and Permeabilizing




Agents



Insulin, porcine
Hypoglycemic Agents



Trastuzumab
Antineoplastic Agents
CA2103059


Rituximab
Antineoplastic Agents,
CA2149329



Immunologic Factors and




Antirheumatic Agents



Basiliximab
Immunosuppressive
CA2038279



Agents



Muromo b
Immunologic Factors and




Immunosuppressive




Agents



Digoxin Immune Fab
Antidotes



(Ovine)




Ibritumomab

CA2149329


Daptomycin

U.S. Pat. No.




6,468,967


Tositumomab




Pegvisomant
Hormone Replacement
U.S. Pat. No.



Agents
5,849,535


Botulinum Toxin Type
Neuromuscular Blocking
CA2280565


A
Agents, Anti-Wrinkle




Agents and Anti dystonic




Agents



Pancrelipase
Gastrointesti 1 Agents




and Enzyme




Replacement Agents



Streptoki se
Fibrinolytic Agents and




Thrombolytic Agents



Alemtuzumab

CA1339198


Alglucerase
Enzyme Replacement




Agents



Capromab
Indicators, Reagents and




Diagnostic Agents



Laronidase
Enzyme Replacement




Agents



Urofollitropin
Fertility Agents
U.S. Pat. No.




5,767,067


Efalizumab
Immunosuppressive




Agents



Serum albumin
Serum substitutes
U.S. Pat. No.




6,723,303


Choriogo dotropin
Fertility Agents and Go
U.S. Pat. No.


alpha
dotropins
6,706,681


Antithymocyte
Immunologic Factors and



globulin
Immunosuppressive




Agents



Filgrastim
Immunosuppressive
CA1341537



Agents, Antineutropenic




Agents and




Hematopoietic Agents



Coagulation factor ix
Coagulants and




Thrombotic Agents



Becaplermin
Angiogenesis Inducing
CA1340846



Agents



Agalsidase beta
Enzyme Replacement
CA2265464



Agents



Interferon alpha-2b
Immunosuppressive
CA1341567



Agents



Oxytocin
Oxytocics, Anti-tocolytic




Agents and Labor




Induction Agents



Enfuvirtide
HIV Fusion Inhibitors
U.S. Pat. No.




6,475,491


Palivizumab
Antiviral Agents
CA2197684


Daclizumab
Immunosuppressive




Agents



Bevacizumab
Angiogenesis Inhibitors
CA2286330


Arcitumomab
Diagnostic Agents
U.S. Pat. No.




8,420,081


Arcitumomab
Diagnostic Agents
U.S. Pat. No.




7,790,142


Eculizumab

CA2189015


Panitumumab




Ranibizumab
Ophthalmics
CA2286330


Idursulfase
Enzyme Replacement




Agents



Alglucosidase alpha
Enzyme Replacement
CA2416492



Agents



Exe tide
Hypoglycemic Agents
U.S. Pat. No.




6,872,700


Mecasermin

U.S. Pat. No.




5,681,814


Pramlintide

U.S. Pat. No.




5,686,411


Galsulfase
Enzyme Replacement




Agents



Abatacept
Antirheumatic Agents
CA2110518



and Immunosuppressive




Agents



Cosyntropin
Hormones and




Diagnostic Agents



Corticotropin




Insulin aspart
Hypoglycemic Agents
U.S. Pat. No.



and Antidiabetic Agents
5,866,538


Insulin detemir
Antidiabetic Agents
U.S. Pat. No.




5,750,497


Insulin glulisine
Antidiabetic Agents
U.S. Pat. No.




6,960,561


Pegaptanib
Intended for the




prevention of respiratory




distress syndrome (RDS)




in premature infants at




high risk for RDS.



Nesiritide




Thymalphasin




Defibrotide
Antithrombins



tural alpha interferon




OR multiferon




Glatiramer acetate




Preotact




Teicoplanin
Anti-Bacterial Agents



Ca kinumab
Anti-Inflammatory




Agents and Monoclo 1




antibodies



Ipilimumab
Antineoplastic Agents
CA2381770



and Monoclo 1




antibodies



Sulodexide
Antithrombins and




Fibrinolytic Agents and




Hypoglycemic Agents




and Anticoagulants and




Hypolipidemic Agents



Tocilizumab

CA2201781


Teriparatide
Bone Density
US6977077



Conservation Agents



Pertuzumab
Monoclo 1 antibodies
CA2376596


Rilo cept
Immunosuppressive
U.S. Pat. No.



Agents
5,844,099


Denosumab
Bone Density
CA2257247



Conservation Agents and




Monoclo 1 antibodies



Liraglutide

U.S. Pat. No.




6,268,343


Golimumab
Antipsoriatic Agents and




Monoclo 1 antibodies




and TNF inhibitor



Belatacept
Antirheumatic Agents




and Immunosuppressive




Agents



Buserelin




Velaglucerase alpha
Enzymes
U.S. Pat. No.




7,138,262


Tesamorelin

U.S. Pat. No.




5,861,379


Brentuximab vedotin




Taliglucerase alpha
Enzymes



Belimumab
Monoclo 1 antibodies



Aflibercept
Antineoplastic Agents
U.S. Pat. No.



and Ophthalmics
7,306,799


Asparagi se erwinia
Enzymes



chrysanthemi




Ocriplasmin
Ophthalmics



Glucarpidase
Enzymes



Teduglutide

U.S. Pat. No.




5,789,379


Raxibacumab
Anti-Infective Agents




and Monoclo 1




antibodies



Certolizumab pegol
TNF inhibitor
CA2380298


Insulin, isophane
Hypoglycemic Agents




and Antidiabetic Agents



Epoetin zeta




Obinutuzumab
Antineoplastic Agents



Fibrinolysin aka

U.S. Pat. No.


plasmin

3,234,106


Follitropin alpha




Romiplostim
Colony-Stimulating




Factors and




Thrombopoietic Agents



Luci ctant
Pulmo ry surfactants
U.S. Pat. No.




5,407,914


talizumab
Immunosuppressive




agents



Aliskiren
Renin inhibitor



Ragweed Pollen




Extract




Secukinumab
Inhibitor
US20130202610


Somatotropin Recombi
Hormone Replacement
CA1326439


nt
Agents



Drotrecogin alpha
Antisepsis
CA2036894


Alefacept
Dermatologic and




Immunosupressive




agents



OspA lipoprotein
Vaccines



Uroki se

U.S. Pat. No.




4,258,030


Abarelix
Anti-Testosterone Agents
U.S. Pat. No.




5,968,895


Sermorelin
Hormone Replacement




Agents



Aprotinin

U.S. Pat. No.




5,198,534


Gemtuzumab
Antineoplastic agents and
U.S. Pat. No.


ozogamicin
Immunotoxins
5,585,089


Satumomab Pendetide
Diagnostic Agents



Albiglutide
Drugs used in diabetes;




alimentary tract and




metabolism; blood




glucose lowering drugs,




excl. insulins.



Alirocumab




Ancestim




Antithrombin alpha




Antithrombin III




human




Asfotase alpha
Enzymes Alimentary




Tract and Metabolism



Atezolizumab




Autologous cultured




chondrocytes




B er actant




Bli tumomab
Antineoplastic Agents
US20120328618



Immunosuppressive




Agents




Monoclo 1 antibodies




Antineoplastic and




Immunomodulating




Agents



C1 Esterase Inhibitor




(Human)




Coagulation Factor




XIIIA-Subunit




(Recombi nt)




Conestat alpha




Daratumumab
Antineoplastic Agents



Desirudin




Dulaglutide
Hypoglycemic Agents;




Drugs Used in Diabetes;




Alimentary Tract and




Metabolism; Blood




Glucose Lowering




Drugs, Excl. Insulins



Elosulfase alpha
Enzymes; Alimentary




Tract and Metabolism



Elotuzumab

US2014055370


Evolocumab
Lipid Modifying Agents,




Plain; Cardiovascular




System



Fibrinogen




Concentrate (Human)




Filgrastim-sndz




Gastric intrinsic factor




Hepatitis B immune




globulin




Human calcitonin




Human Clostridium




tetani toxoid immune




globulin




Human rabies virus




immune globulin




Human Rho(D)




immune globulin




Hyaluronidase (Human

U.S. Pat. No.


Recombi nt)

7,767,429


Idarucizumab
Anticoagulant



Immune Globulin
Immunologic Factors;



Human
Immunosuppressive




Agents; Anti-Infective




Agents



Vedolizumab
Immunosupressive agent,
US2012151248



Antineoplastic agent



Ustekinumab
Deramtologic agent,




Immunosuppressive




agent, antineoplastic




agent



Turoctocog alpha




Tuberculin Purified




Protein Derivative




Simoctocog alpha
Antihaemorrhagics:




blood coagulation factor




VIII



Siltuximab
Antineoplastic and
U.S. Pat. No.



Immunomodulating
7,612,182



Agents,




Immunosuppressive




Agents



Sebelipase alpha
Enzymes



Sacrosidase
Enzymes



Ramucirumab
Antineoplastic and
US2013067098



Immunomodulating




Agents



Prothrombin complex




concentrate




Poractant alpha
Pulmo ry Surfactants



Pembrolizumab
Antineoplastic and
US2012135408



Immunomodulating




Agents



Peginterferon beta-1a




Ofatumumab
Antineoplastic and
U.S. Pat. No.



Immunomodulating
8,337,847



Agents



Obiltoxaximab




Nivolumab
Antineoplastic and
US2013173223



Immunomodulating




Agents



Necitumumab




Metreleptin

US20070099836


Methoxy polyethylene




glycol-epoetin beta




Mepolizumab
Antineoplastic and
US2008134721



Immunomodulating




Agents,




Immunosuppressive




Agents,




Interleukin Inhibitors



Ixekizumab




Insulin Pork
Hypoglycemic Agents,




Antidiabetic Agents



Insulin Degludec




Insulin Beef




Thyroglobulin
Hormone therapy
U.S. Pat. No.




5,099,001


Anthrax immune
Plasma derivative



globulin human




Anti-inhibitor
Blood Coagulation



coagulant complex
Factors, Antihemophilic




Agent



Anti-thymocyte
Antibody



Globulin (Equine)




Anti-thymocyte
Antibody



Globulin (Rabbit)




Brodalumab
Antineoplastic and




Immunomodulating




Agents



C1 Esterase Inhibitor
Blood and Blood



(Recombi nt)
Forming Organs



Ca kinumab
Antineoplastic and




Immunomodulating




Agents



Chorionic Go dotropin
Hormones
U.S. Pat. No.


(Human)

6,706,681


Chorionic Go dotropin
Hormones
U.S. Pat. No.


(Recombi nt)

5,767,251


Coagulation factor X
Blood Coagulation



human
Factors



Dinutuximab
Antibody,
US20140170155



Immunosuppresive




agent, Antineoplastic




agent



Efmoroctocog alpha
Antihemophilic Factor



Factor IX Complex
Antihemophilic agent



(Human)




Hepatitis A Vaccine
Vaccine



Human Varicella-
Antibody



Zoster Immune




Globulin




Ibritumomab tiuxetan
Antibody,
CA2149329



Immunosuppressive




Agents



Lenograstim
Antineoplastic and




Immunomodulating




Agents



Pegloticase
Enzymes



Protamine sulfate
Heparin Antagonists,




Hematologic Agents



Protein S human
Anticoagulant plasma




protein



Sipuleucel-T
Antineoplastic and
U.S. Pat. No.



Immunomodulating
8,153,120



Agents



Somatropin recombi nt
Hormones, Hormone
CA1326439,



Substitutes, and
CA2252535, U.S.



Hormone Antagonists
Pat. No. 5,288,703,




U.S. Pat. No.




5,849,700, U.S. Pat.




No. 5,849,704, U.S.




Pat. No. 5,898,030,




U.S. Pat. No.




6,004,297, U.S. Pat.




No. 6,152,897, U.S.




Pat. No. 6,235,004,




U.S. Pat. No.




6,899,699


Susoctocog alpha
Blood coagulation




factors,




Antihaemorrhagics



Thrombomodulin
Anticoagulant agent,



alpha
Antiplatelet agent
















TABLE 29







Exemplary monoclonal antibody therapies.









mAb
Target
Indication





Muromonab-
CD3
Kidney transplant rejection


CD3




Abciximab
GPIIb/IIIa
Prevention of blood dots in




angioplasty


Rituximab
CD20
Non-Hodgkin lymphoma


Palivizumab
RSV
Prevention of respiratory syncytial




virus infection


Infliximab
TNFα
Crohn's disease


Trastuzumab
HER2
Breast cancer


Alemtuzumab
CD52
Chronic myeloid leukemia


Adalimumab
TNFα
Rheumatoid arthritis


Ibritumomab
CD20
Non-Hodgkin lymphoma


tiuxetan




Omalizumab
IgE
Asthma


Cetuximab
EGER
Colorectal cancer


Bevacizumab
VEGF-A
Colorectal cancer


Natalizumab
ITGA4
Multiple sclerosis


Panitumumab
EGFR
Colorectal cancer


Ranibizumab
VEGF-A
Macular degeneration


Eculizumab
C5
Paroxysmal nocturnal hemoglobinuria


Certolizumab
TNFα
Crohn's disease


pegol




Ustekinumab
IL-12/23
Psoriasis


Canakinumab
IL-1β
Muckle-Wells syndrome


Golimumab
TNFα
Rheumatoid and psoriatic arthritis,




ankylosing spondylitis


Ofatumumab
CD20
Chronic lymphocytic leukemia


Tocilizumab
IL-6R
Rheumatoid arthritis


Denosumab
RANKE
Bone loss


Belimumab
BLyS
Systemic lupus erythematosus


Ipilimumab
CTLA-4
Metastatic melanoma


Brentuximab
CD30
Hodgkin lymphoma, systemic


vedotin

anaplastic large cell lymphoma


Pertuzumab
HER2
Breast Cancer


Trastuzumab
HER2
Breast cancer


emtansine




Raxibacumab

B. anthrasis PA
Anthrax infection


Obinutuzumab
CD20
Chronic lymphocytic leukemia


Siltuximab
IL-6
Castleman disease


Ramucirumab
VEGFR2
Gastric cancer


Vedolizumab
α4β7 integrin
Ulcerative colitis, Crohn disease


Blinatumomab
CD19, CD3
Acute lymphoblastic leukemia


Nivolumab
PD-1
Melanoma, non-small cell lung cancer


Pembrolizumab
PD-1
Melanoma


Idarucizumab
Dabigatran
Reversal of dabigatran-induced




anticoagulation


Necitumumab
EGFR
Non-small cell lung cancer


Dinutuximab
GD2
Neuroblastoma


Secukinumab
IL-17α
Psoriasis


Mepolizumab
IL-5
Severe eosinophilic asthma


Alirocurnab
PCSK9
High cholesterol


Evoloeumab
PCSK9
High cholesterol


Daratumumab
CD38
Multiple myeloma


Elotuzumab
SLAMF7
Muitiple myeloma


Ixekizumab
IL-17α
Psoriasis


Reslizumab
IL-5
Asthma


Olaratumab
PDGFRα
Soft tissue sarcoma


Bezlotoxumab

Clostridium

Prevention of Clostridium difficile




difficile

infection recurrence



enterotoxin B



Atezoiizumab
PD-L1
Bladder cancer


Obiltoxaximab

B. anthrasis PA
Prevention of inhalational anthrax


Inotuzumab
CD22
Acute lymphoblastic leukemia


ozogamicin




Brodalumab
IL-17R
Plaque psoriasis


Guselkumab
IL-23 p19
Plaque psoriasis


Dupilumab
IL-4Rα
Atopic dermatitis


Sarilumab
IL-6R
Rheumatoid arthritis


Avelumab
PD-L1
Merkel cell carcinoma


Ocrelizumab
CD20
Multiple sclerosis


Emicizumab
Factor IXa, X
Hemophilia A


Benralizumab
IL-5Rα
Asthma


Gemtuzumab
CD33
Acute myeloid leukemia


ozogamicin




Durvalumab
PD-L1
Bladder cancer


Burosumab
FGF23
X-linked hypophosphatemia


Lanadelumab
Plasma
Hereditary angioedema attacks



kallikrein



Mogamulizumab
CCR4
Mycosis fungoides or Sézary




syndrome


Erenumab
CGRPR
Migraine prevention


Galcanezumab
CGRP
Migraine prevention


Tildrakizumab
IL-23 p19
Plaque psoriasis


Cemiplimab
PD-1
Cutaneous squamous cell carcinoma


Emapalumab
IFNγ
Primary hemophagocytic




lymphohistiocytosis


Fremanezumab
CGRP
Migraine prevention


Ibalizumab
CD4
HIV infection


Moxetumomab
CD22
Hairy cell leukemia


pasudodox




Ravulizuniab
C5
Paroxysmal nocturnal hemoglobinuria


Caplacizumab
von Willebrand
Acquired thrombotic



factor
thrombocytopenic purpura


Romosozurnab
Sclerostin
Osteoporosis in postmenopausal




women at increased risk of fracture


Risankizumab
IL-23 p19
Plaque psoriasis


Polatuzumab
CD79P
Diffuse large B-cell lymphoma


vedotin




Brolucizumab
VEGF-A
Macular degeneration


Crizanlizumab
P-selectin
Sickle cell disease









Plant-Modification Methods

Gene Writer systems described herein may be used to modify a plant or a plant part (e.g., leaves, roots, flowers, fruits, or seeds), e.g., to increase the fitness of a plant.


A. Delivery to a Plant

Provided herein are methods of delivering a Gene Writer system described herein to a plant. Included are methods for delivering a Gene Writer system to a plant by contacting the plant, or part thereof, with a Gene Writer system. The methods are useful for modifying the plant to, e.g., increase the fitness of a plant.


More specifically, in some embodiments, a nucleic acid described herein (e.g., a nucleic acid encoding a GeneWriter) may be encoded in a vector, e.g., inserted adjacent to a plant promoter, e.g., a maize ubiquitin promoter (ZmUBI) in a plant vector (e.g., pHUC411). In some embodiments, the nucleic acids described herein are introduced into a plant (e.g., japonica rice) or part of a plant (e.g., a callus of a plant) via agrobacteria. In some embodiments, the systems and methods described herein can be used in plants by replacing a plant gene (e.g., hygromycin phosphotransferase (HPT)) with a null allele (e.g., containing a base substitution at the start codon). Systems and methods for modifying a plant genome are described in Xu et. al. Development of plant prime-editing systems for precise genome editing, 2020, Plant Communications.


In one aspect, provided herein is a method of increasing the fitness of a plant, the method including delivering to the plant the Gene Writer system described herein (e.g., in an effective amount and duration) to increase the fitness of the plant relative to an untreated plant (e.g., a plant that has not been delivered the Gene Writer system).


An increase in the fitness of the plant as a consequence of delivery of a Gene Writer system can manifest in a number of ways, e.g., thereby resulting in a better production of the plant, for example, an improved yield, improved vigor of the plant or quality of the harvested product from the plant, an improvement in pre- or post-harvest traits deemed desirable for agriculture or horticulture (e.g., taste, appearance, shelf life), or for an improvement of traits that otherwise benefit humans (e.g., decreased allergen production). An improved yield of a plant relates to an increase in the yield of a product (e.g., as measured by plant biomass, grain, seed or fruit yield, protein content, carbohydrate or oil content or leaf area) of the plant by a measurable amount over the yield of the same product of the plant produced under the same conditions, but without the application of the instant compositions or compared with application of conventional plant-modifying agents. For example, yield can be increased by at least about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or more than 100%. In some instances, the method is effective to increase yield by about 2×-fold, 5×-fold, 10×-fold, 25×-fold, 50×-fold, 75×-fold, 100×-fold, or more than 100×-fold relative to an untreated plant. Yield can be expressed in terms of an amount by weight or volume of the plant or a product of the plant on some basis. The basis can be expressed in terms of time, growing area, weight of plants produced, or amount of a raw material used. For example, such methods may increase the yield of plant tissues including, but not limited to: seeds, fruits, kernels, bolls, tubers, roots, and leaves.


An increase in the fitness of a plant as a consequence of delivery of a Gene Writer system can also be measured by other means, such as an increase or improvement of the vigor rating, the stand (the number of plants per unit of area), plant height, stalk circumference, stalk length, leaf number, leaf size, plant canopy, visual appearance (such as greener leaf color), root rating, emergence, protein content, increased tillering, bigger leaves, more leaves, less dead basal leaves, stronger tillers, less fertilizer needed, less seeds needed, more productive tillers, earlier flowering, early grain or seed maturity, less plant verse (lodging), increased shoot growth, earlier germination, or any combination of these factors, by a measurable or noticeable amount over the same factor of the plant produced under the same conditions, but without the administration of the instant compositions or with application of conventional plant-modifying agents.


Accordingly, provided herein is a method of modifying a plant, the method including delivering to the plant an effective amount of any of the Gene Writer systems provided herein, wherein the method modifies the plant and thereby introduces or increases a beneficial trait in the plant (e.g., by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%) relative to an untreated plant. In particular, the method may increase the fitness of the plant (e.g., by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%) relative to an untreated plant.


In some instances, the increase in plant fitness is an increase (e.g., by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%) in disease resistance, drought tolerance, heat tolerance, cold tolerance, salt tolerance, metal tolerance, herbicide tolerance, chemical tolerance, water use efficiency, nitrogen utilization, resistance to nitrogen stress, nitrogen fixation, pest resistance, herbivore resistance, pathogen resistance, yield, yield under water-limited conditions, vigor, growth, photosynthetic capability, nutrition, protein content, carbohydrate content, oil content, biomass, shoot length, root length, root architecture, seed weight, or amount of harvestable produce.


In some instances, the increase in fitness is an increase (e.g., by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%) in development, growth, yield, resistance to abiotic stressors, or resistance to biotic stressors. An abiotic stress refers to an environmental stress condition that a plant or a plant part is subjected to that includes, e.g., drought stress, salt stress, heat stress, cold stress, and low nutrient stress. A biotic stress refers to an environmental stress condition that a plant or plant part is subjected to that includes, e.g. nematode stress, insect herbivory stress, fungal pathogen stress, bacterial pathogen stress, or viral pathogen stress. The stress may be temporary, e.g. several hours, several days, several months, or permanent, e.g. for the life of the plant.


In some instances, the increase in plant fitness is an increase (e.g., by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%) in quality of products harvested from the plant. For example, the increase in plant fitness may be an improvement in commercially favorable features (e.g., taste or appearance) of a product harvested from the plant. In other instances, the increase in plant fitness is an increase in shelf-life of a product harvested from the plant (e.g., by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%).


Alternatively, the increase in fitness may be an alteration of a trait that is beneficial to human or animal health, such as a reduction in allergen production. For example, the increase in fitness may be a decrease (e.g., by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%) in production of an allergen (e.g., pollen) that stimulates an immune response in an animal (e.g., human).


The modification of the plant (e.g., increase in fitness) may arise from modification of one or more plant parts. For example, the plant can be modified by contacting leaf, seed, pollen, root, fruit, shoot, flower, cells, protoplasts, or tissue (e.g., meristematic tissue) of the plant. As such, in another aspect, provided herein is a method of increasing the fitness of a plant, the method including contacting pollen of the plant with an effective amount of any of the plant-modifying compositions herein, wherein the method increases the fitness of the plant (e.g., by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%) relative to an untreated plant.


In yet another aspect, provided herein is a method of increasing the fitness of a plant, the method including contacting a seed of the plant with an effective amount of any of the Gene Writer systems disclosed herein, wherein the method increases the fitness of the plant (e.g., by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%) relative to an untreated plant.


In another aspect, provided herein is a method including contacting a protoplast of the plant with an effective amount of any of the Gene Writer systems described herein, wherein the method increases the fitness of the plant (e.g., by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%) relative to an untreated plant.


In a further aspect, provided herein is a method of increasing the fitness of a plant, the method including contacting a plant cell of the plant with an effective amount of any of the Gene Writer system described herein, wherein the method increases the fitness of the plant (e.g., by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%) relative to an untreated plant.


In another aspect, provided herein is a method of increasing the fitness of a plant, the method including contacting meristematic tissue of the plant with an effective amount of any of the plant-modifying compositions herein, wherein the method increases the fitness of the plant (e.g., by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%) relative to an untreated plant.


In another aspect, provided herein is a method of increasing the fitness of a plant, the method including contacting an embryo of the plant with an effective amount of any of the plant-modifying compositions herein, wherein the method increases the fitness of the plant (e.g., by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%) relative to an untreated plant.


B. Application Methods

A plant described herein can be exposed to any of the Gene Writer system compositions described herein in any suitable manner that permits delivering or administering the composition to the plant. The Gene Writer system may be delivered either alone or in combination with other active (e.g., fertilizing agents) or inactive substances and may be applied by, for example, spraying, injection (e.g. microinjection), through plants, pouring, dipping, in the form of concentrated liquids, gels, solutions, suspensions, sprays, powders, pellets, briquettes, bricks and the like, formulated to deliver an effective concentration of the plant-modifying composition. Amounts and locations for application of the compositions described herein are generally determined by the habitat of the plant, the lifecycle stage at which the plant can be targeted by the plant-modifying composition, the site where the application is to be made, and the physical and functional characteristics of the plant-modifying composition.


In some instances, the composition is sprayed directly onto a plant, e.g., crops, by e.g., backpack spraying, aerial spraying, crop spraying/dusting etc. In instances where the Gene Writer system is delivered to a plant, the plant receiving the Gene Writer system may be at any stage of plant growth. For example, formulated plant-modifying compositions can be applied as a seed-coating or root treatment in early stages of plant growth or as a total plant treatment at later stages of the crop cycle. In some instances, the plant-modifying composition may be applied as a topical agent to a plant.


Further, the Gene Writer system may be applied (e.g., in the soil in which a plant grows, or in the water that is used to water the plant) as a systemic agent that is absorbed and distributed through the tissues of a plant. In some instances, plants or food organisms may be genetically transformed to express the Gene Writer system.


Delayed or continuous release can also be accomplished by coating the Gene Writer system or a composition with the plant-modifying composition(s) with a dissolvable or bioerodable coating layer, such as gelatin, which coating dissolves or erodes in the environment of use, to then make the plant-modifying com Gene Writer system position available, or by dispersing the agent in a dissolvable or erodable matrix. Such continuous release and/or dispensing means devices may be advantageously employed to consistently maintain an effective concentration of one or more of the plant-modifying compositions described herein.


In some instances, the Gene Writer system is delivered to a part of the plant, e.g., a leaf, seed, pollen, root, fruit, shoot, or flower, or a tissue, cell, or protoplast thereof. In some instances, the Gene Writer system is delivered to a cell of the plant. In some instances, the Gene Writer system is delivered to a protoplast of the plant. In some instances, the Gene Writer system is delivered to a tissue of the plant. For example, the composition may be delivered to meristematic tissue of the plant (e.g., apical meristem, lateral meristem, or intercalary meristem). In some instances, the composition is delivered to permanent tissue of the plant (e.g., simple tissues (e.g., parenchyma, collenchyma, or sclerenchyma) or complex permanent tissue (e.g., xylem or phloem)). In some instances, the Gene Writer system is delivered to a plant embryo.


C. Plants

A variety of plants can be delivered to or treated with a Gene Writer system described herein. Plants that can be delivered a Gene Writer system (i.e., “treated”) in accordance with the present methods include whole plants and parts thereof, including, but not limited to, shoot vegetative organs/structures (e.g., leaves, stems and tubers), roots, flowers and floral organs/structures (e.g., bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, cotyledons, and seed coat) and fruit (the mature ovary), plant tissue (e.g., vascular tissue, ground tissue, and the like) and cells (e.g., guard cells, egg cells, and the like), and progeny of same. Plant parts can further refer parts of the plant such as the shoot, root, stem, seeds, stipules, leaves, petals, flowers, ovules, bracts, branches, petioles, internodes, bark, pubescence, tillers, rhizomes, fronds, blades, pollen, stamen, and the like.


The class of plants that can be treated in a method disclosed herein includes the class of higher and lower plants, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, horsetails, psilophytes, lycophytes, bryophytes, and algae (e.g., multicellular or unicellular algae). Plants that can be treated in accordance with the present methods further include any vascular plant, for example monocotyledons or dicotyledons or gymnosperms, including, but not limited to alfalfa, apple, Arabidopsis, banana, barley, canola, castor bean, chrysanthemum, clover, cocoa, coffee, cotton, cottonseed, corn, crambe, cranberry, cucumber, dendrobium, dioscorea, eucalyptus, fescue, flax, gladiolus, liliacea, linseed, millet, muskmelon, mustard, oat, oil palm, oilseed rape, papaya, peanut, pineapple, ornamental plants, Phaseolus, potato, rapeseed, rice, rye, ryegrass, safflower, sesame, sorghum, soybean, sugarbeet, sugarcane, sunflower, strawberry, tobacco, tomato, turfgrass, wheat and vegetable crops such as lettuce, celery, broccoli, cauliflower, cucurbits; fruit and nut trees, such as apple, pear, peach, orange, grapefruit, lemon, lime, almond, pecan, walnut, hazel; vines, such as grapes (e.g., a vineyard), kiwi, hops; fruit shrubs and brambles, such as raspberry, blackberry, gooseberry; forest trees, such as ash, pine, fir, maple, oak, chestnut, popular; with alfalfa, canola, castor bean, corn, cotton, crambe, flax, linseed, mustard, oil palm, oilseed rape, peanut, potato, rice, safflower, sesame, soybean, sugarbeet, sunflower, tobacco, tomato, and wheat. Plants that can be treated in accordance with the methods of the present invention include any crop plant, for example, forage crop, oilseed crop, grain crop, fruit crop, vegetable crop, fiber crop, spice crop, nut crop, turf crop, sugar crop, beverage crop, and forest crop. In certain instances, the crop plant that is treated in the method is a soybean plant. In other certain instances, the crop plant is wheat. In certain instances, the crop plant is corn. In certain instances, the crop plant is cotton. In certain instances, the crop plant is alfalfa. In certain instances, the crop plant is sugarbeet. In certain instances, the crop plant is rice. In certain instances, the crop plant is potato. In certain instances, the crop plant is tomato.


In certain instances, the plant is a crop. Examples of such crop plants include, but are not limited to, monocotyledonous and dicotyledonous plants including, but not limited to, fodder or forage legumes, ornamental plants, food crops, trees, or shrubs selected from Acer spp., Allium spp., Amaranthus spp., Ananas comosus, Apium graveolens, Arachis spp, Asparagus officinalis, Beta vulgaris, Brassica spp. (e.g., Brassica napus, Brassica rapa ssp. (canola, oilseed rape, turnip rape), Camellia sinensis, Canna indica, Cannabis saliva, Capsicum spp., Castanea spp., Cichorium endivia, Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Coriandrum sativum, Corylus spp., Crataegus spp., Cucurbita spp., Cucumis spp., Daucus carota, Fagus spp., Ficus carica, Fragaria spp., Ginkgo biloba, Glycine spp. (e.g., Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthus spp. (e.g., Helianthus annuus), Hibiscus spp., Hordeum spp. (e.g., Hordeum vulgare), Ipomoea batatas, Juglans spp., Lactuca sativa, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Lycopersicon spp. (e.g., Lycopersicon esculenturn, Lycopersicon lycopersicum, Lycopersicon pyriforme), Malus spp., Medicago sativa, Mentha spp., Miscanthus sinensis, Morus nigra, Musa spp., Nicotiana spp., Olea spp., Oryza spp. (e.g., Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Petroselinum crispum, Phaseolus spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prunus spp., Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis spp., Solanum spp. (e.g., Solanum tuberosum, Solanum integrifolium or Solanum lycopersicum), Sorghum bicolor, Sorghum halepense, Spinacia spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Triticosecale rimpaui, Triticum spp. (e.g., Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum or Triticum vulgare), Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., and Zea mays. In certain embodiments, the crop plant is rice, oilseed rape, canola, soybean, corn (maize), cotton, sugarcane, alfalfa, sorghum, or wheat.


The plant or plant part for use in the present invention include plants of any stage of plant development. In certain instances, the delivery can occur during the stages of germination, seedling growth, vegetative growth, and reproductive growth. In certain instances, delivery to the plant occurs during vegetative and reproductive growth stages. In some instances, the composition is delivered to pollen of the plant. In some instances, the composition is delivered to a seed of the plant. In some instances, the composition is delivered to a protoplast of the plant. In some instances, the composition is delivered to a tissue of the plant. For example, the composition may be delivered to meristematic tissue of the plant (e.g., apical meristem, lateral meristem, or intercalary meristem). In some instances, the composition is delivered to permanent tissue of the plant (e.g., simple tissues (e.g., parenchyma, collenchyma, or sclerenchyma) or complex permanent tissue (e.g., xylem or phloem)). In some instances, the composition is delivered to a plant embryo. In some instances, the composition is delivered to a plant cell. The stages of vegetative and reproductive growth are also referred to herein as “adult” or “mature” plants.


In instances where the Gene Writer system is delivered to a plant part, the plant part may be modified by the plant-modifying agent. Alternatively, the Gene Writer system may be distributed to other parts of the plant (e.g., by the plant's circulatory system) that are subsequently modified by the plant-modifying agent.


Administration

The composition and systems described herein may be used in vitro or in vivo. In some embodiments the system or components of the system are delivered to cells (e.g., mammalian cells, e.g., human cells), e.g., in vitro or in vivo. In some embodiments, the cells are eukaryotic cells, e.g., cells of a multicellular organism, e.g., an animal, e.g., a mammal (e.g., human, swine, bovine) a bird (e.g., poultry, such as chicken, turkey, or duck), or a fish. In some embodiments, the cells are non-human animal cells (e.g., a laboratory animal, a livestock animal, or a companion animal). In some embodiments, the cell is a stem cell (e.g., a hematopoietic stem cell), a fibroblast, or a T cell. In some embodiments, the cell is a non-dividing cell, e.g., a non-dividing fibroblast or non-dividing T cell. In some embodiments, the cell is an HSC and p53 is not upregulated or is upregulated by less than 10%, 5%, 2%, or 1%, e.g., as determined according to the method described in Example 30 of PCT/US2019/048607 which is hereby incorporated by reference. The skilled artisan will understand that the components of the Gene Writer™ system may be delivered in the form of polypeptide, nucleic acid (e.g., DNA, RNA), and combinations thereof.


For instance, delivery can use any of the following combinations for delivering the retrotransposase (e.g., as DNA encoding the retrotransposase protein, as RNA encoding the retrotransposase protein, or as the protein itself) and the template RNA (e.g., as DNA encoding the RNA, or as RNA):

    • 1. Retrotransposase DNA+template DNA
    • 2. Retrotransposase RNA+template DNA
    • 3. Retrotransposase DNA+template RNA
    • 4. Retrotransposase RNA+template RNA
    • 5. Retrotransposase protein+template DNA
    • 6. Retrotransposase protein+template RNA
    • 7. Retrotransposase virus+template virus
    • 8. Retrotransposase virus+template DNA
    • 9. Retrotransposase virus+template RNA
    • 10. Retrotransposase DNA+template virus
    • 11. Retrotransposase RNA+template virus
    • 12. Retrotransposase protein+template virus


As indicated above, in some embodiments, the DNA or RNA that encodes the retrotransposase protein is delivered using a virus, and in some embodiments, the template RNA (or the DNA encoding the template RNA) is delivered using a virus.


In one embodiments the system and/or components of the system are delivered as nucleic acid. For example, the Gene Writer™ polypeptide may be delivered in the form of a DNA or RNA encoding the polypeptide, and the template RNA may be delivered in the form of RNA or its complementary DNA to be transcribed into RNA. In some embodiments the system or components of the system are delivered on 1, 2, 3, 4, or more distinct nucleic acid molecules. In some embodiments the system or components of the system are delivered as a combination of DNA and RNA. In some embodiments the system or components of the system are delivered as a combination of DNA and protein. In some embodiments the system or components of the system are delivered as a combination of RNA and protein. In some embodiments the Gene Writer™ genome editor polypeptide is delivered as a protein.


In some embodiments the system or components of the system are delivered to cells, e.g. mammalian cells or human cells, using a vector. The vector may be, e.g., a plasmid or a virus. In some embodiments delivery is in vivo, in vitro, ex vivo, or in situ. In some embodiments the virus is an adeno associated virus (AAV), a lentivirus, an adenovirus. In some embodiments the system or components of the system are delivered to cells with a viral-like particle or a virosome.


In some embodiments the delivery uses more than one virus, viral-like particle or virosome. In one embodiment, the compositions and systems described herein can be formulated in liposomes or other similar vesicles. Liposomes are spherical vesicle structures composed of a uni- or multilamellar lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes may be anionic, neutral or cationic. Liposomes are biocompatible, nontoxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes and the blood brain barrier (BBB) (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679 for review).


Vesicles can be made from several different types of lipids; however, phospholipids are most commonly used to generate liposomes as drug carriers. Methods for preparation of multilamellar vesicle lipids are known in the art (see for example U.S. Pat. No. 6,693,086, the teachings of which relating to multilamellar vesicle lipid preparation are incorporated herein by reference). Although vesicle formation can be spontaneous when a lipid film is mixed with an aqueous solution, it can also be expedited by applying force in the form of shaking by using a homogenizer, sonicator, or an extrusion apparatus (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679 for review). Extruded lipids can be prepared by extruding through filters of decreasing size, as described in Templeton et al., Nature Biotech, 15:647-652, 1997, the teachings of which relating to extruded lipid preparation are incorporated herein by reference.


A variety of nanoparticles can be used for delivery, such as a liposome, a lipid nanoparticle, a cationic lipid nanoparticle, an ionizable lipid nanoparticle, a polymeric nanoparticle, a gold nanoparticle, a dendrimer, a cyclodextrin nanoparticle, a micelle, or a combination of the foregoing.


Lipid nanoparticles are an example of a carrier that provides a biocompatible and biodegradable delivery system for the pharmaceutical compositions described herein. Nanostructured lipid carriers (NLCs) are modified solid lipid nanoparticles (SLNs) that retain the characteristics of the SLN, improve drug stability and loading capacity, and prevent drug leakage. Polymer nanoparticles (PNPs) are an important component of drug delivery. These nanoparticles can effectively direct drug delivery to specific targets and improve drug stability and controlled drug release. Lipid-polymer nanoparticles (PLNs), a new type of carrier that combines liposomes and polymers, may also be employed. These nanoparticles possess the complementary advantages of PNPs and liposomes. A PLN is composed of a core-shell structure; the polymer core provides a stable structure, and the phospholipid shell offers good biocompatibility. As such, the two components increase the drug encapsulation efficiency rate, facilitate surface modification, and prevent leakage of water-soluble drugs. For a review, see, e.g., Li et al. 2017, Nanomaterials 7, 122; doi:10.3390/nano7060122.


Exosomes can also be used as drug delivery vehicles for the compositions and systems described herein. For a review, see Ha et al. July 2016. Acta Pharmaceutica Sinica B. Volume 6, Issue 4, Pages 287-296; doi.org/10.1016/j.apsb.2016.02.001.


Fusosomes interact and fuse with target cells, and thus can be used as delivery vehicles for a variety of molecules. They generally consist of a bilayer of amphipathic lipids enclosing a lumen or cavity and a fusogen that interacts with the amphipathic lipid bilayer. The fusogen component has been shown to be engineerable in order to confer target cell specificity for the fusion and payload delivery, allowing the creation of delivery vehicles with programmable cell specificity (see for example Patent Application WO2020014209, the teachings of which relating to fusosome design, preparation, and usage are incorporated herein by reference).


In some embodiments, the protein component(s) of the Gene Writing™ system may be pre-associated with the template nucleic acid (e.g., template RNA). For example, in some embodiments, the Gene Writer™ polypeptide may be first combined with the template nucleic acid (e.g., template RNA) to form a ribonucleoprotein (RNP) complex. In some embodiments, the RNP may be delivered to cells via, e.g., transfection, nucleofection, virus, vesicle, LNP, exosome, fusosome.


A Gene Writer™ system can be introduced into cells, tissues and multicellular organisms. In some embodiments the system or components of the system are delivered to the cells via mechanical means or physical means.


Formulation of protein therapeutics is described in Meyer (Ed.), Therapeutic Protein Drug Products: Practical Approaches to formulation in the Laboratory, Manufacturing, and the Clinic, Woodhead Publishing Series (2012).


Tissue Specific Activity/Administration

In some embodiments, a system, template RNA, or polypeptide described herein is administered to or is active in (e.g., is more active in) a target tissue, e.g., a first tissue. In some embodiments, the system, template RNA, or polypeptide is not administered to or is less active in (e.g., not active in) a non-target tissue. In some embodiments, a system, template RNA, or polypeptide described herein is useful for modifying DNA in a target tissue, e.g., a first tissue, (e.g., and not modifying DNA in a non-target tissue).


In some embodiments, a system comprises (a) a polypeptide described herein or a nucleic acid encoding the same, (b) a template nucleic acid (e.g., template RNA) described herein, and (c) one or more first tissue-specific expression-control sequences specific to the target tissue, wherein the one or more first tissue-specific expression-control sequences specific to the target tissue are in operative association with (a), (b), or (a) and (b), wherein, when associated with (a), (a) comprises a nucleic acid encoding the polypeptide.


In some embodiments, the nucleic acid in (b) comprises RNA.


In some embodiments, the nucleic acid in (b) comprises DNA.


In some embodiments, the nucleic acid in (b): (i) is single-stranded or comprises a single-stranded segment, e.g., is single-stranded DNA or comprises a single-stranded segment and one or more double stranded segments; (ii) has inverted terminal repeats; or (iii) both (i) and (ii).


In some embodiments, the nucleic acid in (b) is double-stranded or comprises a double-stranded segment.


In some embodiments, (a) comprises a nucleic acid encoding the polypeptide.


In some embodiments, the nucleic acid in (a) comprises RNA.


In some embodiments, the nucleic acid in (a) comprises DNA.


In some embodiments, the nucleic acid in (a): (i) is single-stranded or comprises a single-stranded segment, e.g., is single-stranded DNA or comprises a single-stranded segment and one or more double stranded segments; (ii) has inverted terminal repeats; or (iii) both (i) and (ii).


In some embodiments, the nucleic acid in (a) is double-stranded or comprises a double-stranded segment.


In some embodiments, the nucleic acid in (a), (b), or (a) and (b) is linear.


In some embodiments, the nucleic acid in (a), (b), or (a) and (b) is circular, e.g., a plasmid or minicircle.


In some embodiments, the heterologous object sequence is in operative association with a first promoter.


In some embodiments, the one or more first tissue-specific expression-control sequences comprises a tissue specific promoter.


In some embodiments, the tissue-specific promoter comprises a first promoter in operative association with: i. the heterologous object sequence, ii. a nucleic acid encoding the transposase, or iii. (i) and (ii).


In some embodiments, the one or more first tissue-specific expression-control sequences comprises a tissue-specific microRNA recognition sequence in operative association with: i. the heterologous object sequence, ii. a nucleic acid encoding the transposase, or iii. (i) and (ii).


In some embodiments, a system comprises a tissue-specific promoter, and the system further comprises one or more tissue-specific microRNA recognition sequences, wherein: i. the tissue specific promoter is in operative association with: I. the heterologous object sequence, II. a nucleic acid encoding the transposase, or III. (I) and (II); and/or ii. the one or more tissue-specific microRNA recognition sequences are in operative association with: I. the heterologous object sequence, II. a nucleic acid encoding the transposase, or III.(I) and (II).


In some embodiments, wherein (a) comprises a nucleic acid encoding the polypeptide, the nucleic acid comprises a promoter in operative association with the nucleic acid encoding the polypeptide.


In some embodiments, the nucleic acid encoding the polypeptide comprises one or more second tissue-specific expression-control sequences specific to the target tissue in operative association with the polypeptide coding sequence.


In some embodiments, the one or more second tissue-specific expression-control sequences comprises a tissue specific promoter.


In some embodiments, the tissue-specific promoter is the promoter in operative association with the nucleic acid encoding the polypeptide.


In some embodiments, the one or more second tissue-specific expression-control sequences comprises a tissue-specific microRNA recognition sequence.


In some embodiments, the promoter in operative association with the nucleic acid encoding the polypeptide is a tissue-specific promoter, the system further comprising one or more tissue-specific microRNA recognition sequences.


In some embodiments, a nucleic acid component of a system provided by the invention a sequence (e.g., encoding the polypeptide or comprising a heterologous object sequence) is flanked by untranslated regions (UTRs) that modify protein expression levels. Various 5′ and 3′ UTRs can affect protein expression. For example, in some embodiments, the coding sequence may be preceded by a 5′ UTR that modifies RNA stability or protein translation. In some embodiments, the sequence may be followed by a 3′ UTR that modifies RNA stability or translation. In some embodiments, the sequence may be preceded by a 5′ UTR and followed by a 3′ UTR that modify RNA stability or translation. In some embodiments, the 5′ and/or 3′ UTR may be selected from the 5′ and 3′ UTRs of complement factor 3 (C3) (cactcctccccatcctctccctctgtccctctgtccctctgaccctgcactgtcccagcacc (SEQ ID NO: 1633)) or orosomucoid 1 (ORM1) (caggacacagccttggatcaggacagagacttgggggccatcctgcccctccaacccgacatgtgtacctcagctttttccctcacttgcat caataaagcttctgtgtttggaacagctaa (SEQ ID NO: 1634)) (Asrani et al. RNA Biology 2018). In certain embodiments, the 5′ UTR is the 5′ UTR from C3 and the 3′ UTR is the 3′ UTR from ORM1. In certain embodiments, a 5′ UTR and 3′ UTR for protein expression, e.g., mRNA (or DNA encoding the RNA) for a Gene Writer polypeptide or heterologous object sequence, comprise optimized expression sequences. In some embodiments, the 5′ UTR comprises GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC (SEQ ID NO: 1603) and/or the 3′ UTR comprising UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGA (SEQ ID NO: 1604), e.g., as described in Richner et al. Cell 168(6): P1114-1125 (2017), the sequences of which are incorporated herein by reference.


In some embodiments, a 5′ and/or 3′ UTR may be selected to enhance protein expression. In some embodiments, a 5′ and/or 3′ UTR may be selected to modify protein expression such that overproduction inhibition is minimized. In some embodiments, UTRs are around a coding sequence, e.g., outside the coding sequence and in other embodiments proximal to the coding sequence. In some embodiments additional regulatory elements (e.g., miRNA binding sites, cis-regulatory sites) are included in the UTRs.


In some embodiments, an open reading frame of a Gene Writer system, e.g., an ORF of an mRNA (or DNA encoding an mRNA) encoding a Gene Writer polypeptide or one or more ORFs of an mRNA (or DNA encoding an mRNA) of a heterologous object sequence, is flanked by a 5′ and/or 3′ untranslated region (UTR) that enhances the expression thereof. In some embodiments, the 5′ UTR of an mRNA component (or transcript produced from a DNA component) of the system comprises the sequence 5′-GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC-3′ (SEQ ID NO: 1603). In some embodiments, the 3′ UTR of an mRNA component (or transcript produced from a DNA component) of the system comprises the sequence 5′-UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGA-3′ (SEQ ID NO: 1604). This combination of 5′ UTR and 3′ UTR has been shown to result in desirable expression of an operably linked ORF by Richner et al. Cell 168(6): P1114-1125 (2017), the teachings and sequences of which are incorporated herein by reference. In some embodiments, a system described herein comprises a DNA encoding a transcript, wherein the DNA comprises the corresponding 5′ UTR and 3′ UTR sequences, with T substituting for U in the above-listed sequence). In some embodiments, a DNA vector used to produce an RNA component of the system further comprises a promoter upstream of the 5′ UTR for initiating in vitro transcription, e.g, a T7, T3, or SP6 promoter. The 5′ UTR above begins with GGG, which is a suitable start for optimizing transcription using T7 RNA polymerase. For tuning transcription levels and altering the transcription start site nucleotides to fit alternative 5′ UTRs, the teachings of Davidson et al. Pac Symp Biocomput 433-443 (2010) describe T7 promoter variants, and the methods of discovery thereof, that fulfill both of these traits.


Viral Vectors and Components Thereof

Viruses are a useful source of delivery vehicles for the systems described herein, in addition to a source of relevant enzymes or domains as described herein, e.g., as sources of polymerases and polymerase functions used herein, e.g., DNA-dependent DNA polymerase, RNA-dependent RNA polymerase, RNA-dependent DNA polymerase, DNA-dependent RNA polymerase, reverse transcriptase. Some enzymes, e.g., reverse transcriptases, may have multiple activities, e.g., be capable of both RNA-dependent DNA polymerization and DNA-dependent DNA polymerization, e.g., first and second strand synthesis. In some embodiments, the virus used as a Gene Writer delivery system or a source of components thereof may be selected from a group as described by Baltimore Bacteriol Rev 35(3):235-241 (1971).


In some embodiments, the virus is selected from a Group I virus, e.g., is a DNA virus and packages dsDNA into virions. In some embodiments, the Group I virus is selected from, e.g., Adenoviruses, Herpesviruses, Poxviruses.


In some embodiments, the virus is selected from a Group II virus, e.g., is a DNA virus and packages ssDNA into virions. In some embodiments, the Group II virus is selected from, e.g., Parvoviruses. In some embodiments, the parvovirus is a dependoparvovirus, e.g., an adeno-associated virus (AAV).


In some embodiments, the virus is selected from a Group III virus, e.g., is an RNA virus and packages dsRNA into virions. In some embodiments, the Group III virus is selected from, e.g., Reoviruses. In some embodiments, one or both strands of the dsRNA contained in such virions is a coding molecule able to serve directly as mRNA upon transduction into a host cell, e.g., can be directly translated into protein upon transduction into a host cell without requiring any intervening nucleic acid replication or polymerization steps.


In some embodiments, the virus is selected from a Group IV virus, e.g., is an RNA virus and packages ssRNA(+) into virions. In some embodiments, the Group IV virus is selected from, e.g., Coronaviruses, Picornaviruses, Togaviruses. In some embodiments, the ssRNA(+) contained in such virions is a coding molecule able to serve directly as mRNA upon transduction into a host cell, e.g., can be directly translated into protein upon transduction into a host cell without requiring any intervening nucleic acid replication or polymerization steps.


In some embodiments, the virus is selected from a Group V virus, e.g., is an RNA virus and packages ssRNA(−) into virions. In some embodiments, the Group V virus is selected from, e.g., Orthomyxoviruses, Rhabdoviruses. In some embodiments, an RNA virus with an ssRNA(−) genome also carries an enzyme inside the virion that is transduced to host cells with the viral genome, e.g., an RNA-dependent RNA polymerase, capable of copying the ssRNA(−) into ssRNA(+) that can be translated directly by the host.


In some embodiments, the virus is selected from a Group VI virus, e.g., is a retrovirus and packages ssRNA(+) into virions. In some embodiments, the Group VI virus is selected from, e.g., Retroviruses. In some embodiments, the retrovirus is a lentivirus, e.g., HIV-1, HIV-2, SIV, BIV. In some embodiments, the retrovirus is a spumavirus, e.g., a foamy virus, e.g., HFV, SFV, BFV. In some embodiments, the ssRNA(+) contained in such virions is a coding molecule able to serve directly as mRNA upon transduction into a host cell, e.g., can be directly translated into protein upon transduction into a host cell without requiring any intervening nucleic acid replication or polymerization steps. In some embodiments, the ssRNA(+) is first reverse transcribed and copied to generate a dsDNA genome intermediate from which mRNA can be transcribed in the host cell. In some embodiments, an RNA virus with an ssRNA(+) genome also carries an enzyme inside the virion that is transduced to host cells with the viral genome, e.g., an RNA-dependent DNA polymerase, capable of copying the ssRNA(+) into dsDNA that can be transcribed into mRNA and translated by the host. In some embodiments, the reverse transcriptase from a Group VI retrovirus is incorporated as the reverse transcriptase domain of a Gene Writer polypeptide.


In some embodiments, the virus is selected from a Group VII virus, e.g., is a retrovirus and packages dsRNA into virions. In some embodiments, the Group VII virus is selected from, e.g., Hepadnaviruses. In some embodiments, one or both strands of the dsRNA contained in such virions is a coding molecule able to serve directly as mRNA upon transduction into a host cell, e.g., can be directly translated into protein upon transduction into a host cell without requiring any intervening nucleic acid replication or polymerization steps. In some embodiments, one or both strands of the dsRNA contained in such virions is first reverse transcribed and copied to generate a dsDNA genome intermediate from which mRNA can be transcribed in the host cell. In some embodiments, an RNA virus with a dsRNA genome also carries an enzyme inside the virion that is transduced to host cells with the viral genome, e.g., an RNA-dependent DNA polymerase, capable of copying the dsRNA into dsDNA that can be transcribed into mRNA and translated by the host. In some embodiments, the reverse transcriptase from a Group VII retrovirus is incorporated as the reverse transcriptase domain of a Gene Writer polypeptide.


In some embodiments, virions used to deliver nucleic acid in this invention may also carry enzymes involved in the process of Gene Writing. For example, a retroviral virion may contain a reverse transcriptase domain that is delivered into a host cell along with the nucleic acid. In some embodiments, an RNA template may be associated with a Gene Writer polypeptide within a virion, such that both are co-delivered to a target cell upon transduction of the nucleic acid from the viral particle. In some embodiments, the nucleic acid in a virion may comprise DNA, e.g., linear ssDNA, linear dsDNA, circular ssDNA, circular dsDNA, minicircle DNA, dbDNA, ceDNA. In some embodiments, the nucleic acid in a virion may comprise RNA, e.g., linear ssRNA, linear dsRNA, circular ssRNA, circular dsRNA. In some embodiments, a viral genome may circularize upon transduction into a host cell, e.g., a linear ssRNA molecule may undergo a covalent linkage to form a circular ssRNA, a linear dsRNA molecule may undergo a covalent linkage to form a circular dsRNA or one or more circular ssRNA. In some embodiments, a viral genome may replicate by rolling circle replication in a host cell. In some embodiments, a viral genome may comprise a single nucleic acid molecule, e.g., comprise a non-segmented genome. In some embodiments, a viral genome may comprise two or more nucleic acid molecules, e.g., comprise a segmented genome. In some embodiments, a nucleic acid in a virion may be associated with one or proteins. In some embodiments, one or more proteins in a virion may be delivered to a host cell upon transduction. In some embodiments, a natural virus may be adapted for nucleic acid delivery by the addition of virion packaging signals to the target nucleic acid, wherein a host cell is used to package the target nucleic acid containing the packaging signals.


In some embodiments, a virion used as a delivery vehicle may comprise a commensal human virus. In some embodiments, a virion used as a delivery vehicle may comprise an anellovirus, the use of which is described in WO2018232017A1, which is incorporated herein by reference in its entirety.


AAV Administration

In some embodiments, an adeno-associated virus (AAV) is used in conjunction with the system, template nucleic acid, and/or polypeptide described herein. In some embodiments, an AAV is used to deliver, administer, or package the system, template nucleic acid, and/or polypeptide described herein. In some embodiments, the AAV is a recombinant AAV (rAAV).


In some embodiments, a system comprises (a) a polypeptide described herein or a nucleic acid encoding the same, (b) a template nucleic acid (e.g., template RNA) described herein, and (c) one or more first tissue-specific expression-control sequences specific to the target tissue, wherein the one or more first tissue-specific expression-control sequences specific to the target tissue are in operative association with (a), (b), or (a) and (b), wherein, when associated with (a), (a) comprises a nucleic acid encoding the polypeptide.


In some embodiments, a system described herein further comprises a first recombinant adeno-associated virus (rAAV) capsid protein; wherein the at least one of (a) or (b) is associated with the first rAAV capsid protein, wherein at least one of (a) or (b) is flanked by AAV inverted terminal repeats (ITRs).


In some embodiments, (a) and (b) are associated with the first rAAV capsid protein.


In some embodiments, (a) and (b) are on a single nucleic acid.


In some embodiments, the system further comprises a second rAAV capsid protein, wherein at least one of (a) or (b) is associated with the second rAAV capsid protein, and wherein the at least one of (a) or (b) associated with the second rAAV capsid protein is different from the at least one of (a) or (b) is associated with the first rAAV capsid protein.


In some embodiments, the at least one of (a) or (b) is associated with the first or second rAAV capsid protein is dispersed in the interior of the first or second rAAV capsid protein, which first or second rAAV capsid protein is in the form of an AAV capsid particle.


In some embodiments, the system further comprises a nanoparticle, wherein the nanoparticle is associated with at least one of (a) or (b).


In some embodiments, (a) and (b), respectively are associated with: a) a first rAAV capsid protein and a second rAAV capsid protein; b) a nanoparticle and a first rAAV capsid protein; c) a first rAAV capsid protein; d) a first adenovirus capsid protein; e) a first nanoparticle and a second nanoparticle; or f) a first nanoparticle.


Viral vectors are useful for delivering all or part of a system provided by the invention, e.g., for use in methods provided by the invention. Systems derived from different viruses have been employed for the delivery of polypeptides, nucleic acids, or transposons; for example: integrase-deficient lentivirus, adenovirus, adeno-associated virus (AAV), herpes simplex virus, and baculovirus (reviewed in Hodge et al. Hum Gene Ther 2017; Narayanavari et al. Crit Rev Biochem Mol Biol 2017; Boehme et al. Curr Gene Ther 2015).


Adenoviruses are common viruses that have been used as gene delivery vehicles given well-defined biology, genetic stability, high transduction efficiency, and ease of large-scale production (see, for example, review by Lee et al. Genes & Diseases 2017). They possess linear dsDNA genomes and come in a variety of serotypes that differ in tissue and cell tropisms. In order to prevent replication of infectious virus in recipient cells, adenovirus genomes used for packaging are deleted of some or all endogenous viral proteins, which are provided in trans in viral production cells. This renders the genomes helper-dependent, meaning they can only be replicated and packaged into viral particles in the presence of the missing components provided by so-called helper functions. A helper-dependent adenovirus system with all viral ORFs removed may be compatible with packaging foreign DNA of up to ˜37 kb (Parks et al. J Virol 1997). In some embodiments, an adenoviral vector is used to deliver DNA corresponding to the polypeptide or template component of the Gene Writing™ system, or both are contained on separate or the same adenoviral vector. In some embodiments, the adenovirus is a helper-dependent adenovirus (HD-AdV) that is incapable of self-packaging. In some embodiments, the adenovirus is a high-capacity adenovirus (HC-AdV) that has had all or a substantial portion of endogenous viral ORFs deleted, while retaining the necessary sequence components for packaging into adenoviral particles. For this type of vector, the only adenoviral sequences required for genome packaging are noncoding sequences: the inverted terminal repeats (ITRs) at both ends and the packaging signal at the 5′-end (Jager et al. Nat Protoc 2009). In some embodiments, the adenoviral genome also comprises stuffer DNA to meet a minimal genome size for optimal production and stability (see, for example, Hausl et al. Mol Ther 2010). Adenoviruses have been used in the art for the delivery of transposons to various tissues. In some embodiments, an adenovirus is used to deliver a Gene Writing™ system to the liver.


In some embodiments, an adenovirus is used to deliver a Gene Writing™ system to HSCs, e.g., HDAd5/35++. HDAd5/35++ is an adenovirus with modified serotype 35 fibers that de-target the vector from the liver (Wang et al. Blood Adv 2019). In some embodiments, the adenovirus that delivers a Gene Writing™ system to HSCs utilizes a receptor that is expressed specifically on primitive HSCs, e.g., CD46.


Adeno-associated viruses (AAV) belong to the parvoviridae family and more specifically constitute the dependoparvovirus genus. The AAV genome is composed of a linear single-stranded DNA molecule which contains approximately 4.7 kilobases (kb) and consists of two major open reading frames (ORFs) encoding the non-structural Rep (replication) and structural Cap (capsid) proteins. A second ORF within the cap gene was identified that encodes the assembly-activating protein (AAP). The DNAs flanking the AAV coding regions are two cis-acting inverted terminal repeat (ITR) sequences, approximately 145 nucleotides in length, with interrupted palindromic sequences that can be folded into energetically stable hairpin structures that function as primers of DNA replication. In addition to their role in DNA replication, the ITR sequences have been shown to be involved in viral DNA integration into the cellular genome, rescue from the host genome or plasmid, and encapsidation of viral nucleic acid into mature virions (Muzyczka, (1992) Curr. Top. Micro. Immunol. 158:97-129). In some embodiments, one or more Gene Writing™ nucleic acid components is flanked by ITRs derived from AAV for viral packaging. See, e.g., WO2019113310.


In some embodiments, one or more components of the Gene Writing™ system are carried via at least one AAV vector. In some embodiments, the at least one AAV vector is selected for tropism to a particular cell, tissue, organism. In some embodiments, the AAV vector is pseudotyped, e.g., AAV2/8, wherein AAV2 describes the design of the construct but the capsid protein is replaced by that from AAV8. It is understood that any of the described vectors could be pseudotype derivatives, wherein the capsid protein used to package the AAV genome is derived from that of a different AAV serotype. In some embodiments, an AAV to be employed for Gene Writing™ may be evolved for novel cell or tissue tropism as has been demonstrated in the literature (e.g., Davidsson et al. Proc Natl Acad Sci USA 2019).


In some embodiments, the AAV delivery vector is a vector which has two AAV inverted terminal repeats (ITRs) and a nucleotide sequence of interest (for example, a sequence coding for a Gene Writer™ polypeptide or a DNA template, or both), each of said ITRs having an interrupted (or noncontiguous) palindromic sequence, i.e., a sequence composed of three segments: a first segment and a last segment that are identical when read 5′-3′ but hybridize when placed against each other, and a segment that is different that separates the identical segments. Such sequences, notably the ITRs, form hairpin structures. See, for example, WO2012123430.


Conventionally, AAV virions with capsids are produced by introducing a plasmid or plasmids encoding the rAAV or scAAV genome, Rep proteins, and Cap proteins (Grimm et al, 1998). Upon introduction of these helper plasmids in trans, the AAV genome is “rescued” (i.e., released and subsequently recovered) from the host genome, and is further encapsidated to produce infectious AAV. In some embodiments, one or more Gene Writing™ nucleic acids are packaged into AAV particles by introducing the ITR-flanked nucleic acids into a packaging cell in conjunction with the helper functions.


In some embodiments, the AAV genome is a so called self-complementary genome (referred to as scAAV), such that the sequence located between the ITRs contains both the desired nucleic acid sequence (e.g., DNA encoding the Gene Writer™ polypeptide or template, or both) in addition to the reverse complement of the desired nucleic acid sequence, such that these two components can fold over and self-hybridize. In some embodiments, the self-complementary modules are separated by an intervening sequence that permits the DNA to fold back on itself, e.g., forms a stem-loop. An scAAV has the advantage of being poised for transcription upon entering the nucleus, rather than being first dependent on ITR priming and second-strand synthesis to form dsDNA. In some embodiments, one or more Gene Writing™ components is designed as an scAAV, wherein the sequence between the AAV ITRs contains two reverse complementing modules that can self-hybridize to create dsDNA.


In some embodiments, nucleic acid (e.g., encoding a polypeptide, or a template, or both) delivered to cells is closed-ended, linear duplex DNA (CELiD DNA or ceDNA). In some embodiments, ceDNA is derived from the replicative form of the AAV genome (Li et al. PLoS One 2013). In some embodiments, the nucleic acid (e.g., encoding a polypeptide, or a template DNA, or both) is flanked by ITRs, e.g., AAV ITRs, wherein at least one of the ITRs comprises a terminal resolution site and a replication protein binding site (sometimes referred to as a replicative protein binding site). In some embodiments, the ITRs are derived from an adeno-associated virus, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV 11, AAV12, or a combination thereof. In some embodiments, the ITRs are symmetric. In some embodiments, the ITRs are asymmetric. In some embodiments, at least one Rep protein is provided to enable replication of the construct. In some embodiments, the at least one Rep protein is derived from an adeno-associated virus, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or a combination thereof. In some embodiments, ceDNA is generated by providing a production cell with (i) DNA flanked by ITRs, e.g., AAV ITRs, and (ii) components required for ITR-dependent replication, e.g., AAV proteins Rep78 and Rep52 (or nucleic acid encoding the proteins). In some embodiments, ceDNA is free of any capsid protein, e.g., is not packaged into an infectious AAV particle. In some embodiments, ceDNA is formulated into LNPs (see, for example, WO2019051289A1).


In some embodiments, the ceDNA vector consists of two self complementary sequences, e.g., asymmetrical or symmetrical or substantially symmetrical ITRs as defined herein, flanking said expression cassette, wherein the ceDNA vector is not associated with a capsid protein. In some embodiments, the ceDNA vector comprises two self-complementary sequences found in an AAV genome, where at least one ITR comprises an operative Rep-binding element (RBE) (also sometimes referred to herein as “RBS”) and a terminal resolution site (trs) of AAV or a functional variant of the RBE. See, for example, WO2019113310.


In some embodiments, the AAV genome comprises two genes that encode four replication proteins and three capsid proteins, respectively. In some embodiments, the genes are flanked on either side by 145-bp inverted terminal repeats (ITRs). In some embodiments, the virion comprises up to three capsid proteins (Vp1, Vp2, and/or Vp3), e.g., produced in a 1:1:10 ratio. In some embodiments, the capsid proteins are produced from the same open reading frame and/or from differential splicing (Vp1) and alternative translational start sites (Vp2 and Vp3, respectively). Generally, Vp3 is the most abundant subunit in the virion and participates in receptor recognition at the cell surface defining the tropism of the virus. In some embodiments, Vp1 comprises a phospholipase domain, e.g., which functions in viral infectivity, in the N-terminus of Vp1.


In some embodiments, packaging capacity of the viral vectors limits the size of the base editor that can be packaged into the vector. For example, the packaging capacity of the AAVs can be about 4.5 kb (e.g., about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, or 6.0 kb), e.g., including one or two inverted terminal repeats (ITRs), e.g., 145 base ITRs.


In some embodiments, recombinant AAV (rAAV) comprises cis-acting 145-bp ITRs flanking vector transgene cassettes, e.g., providing up to 4.5 kb for packaging of foreign DNA. Subsequent to infection, rAAV can, in some instances, express a fusion protein of the invention and persist without integration into the host genome by existing episomally in circular head-to-tail concatemers. rAAV can be used, for example, in vitro and in vivo. In some embodiments, AAV-mediated gene delivery requires that the length of the coding sequence of the gene is equal or greater in size than the wild-type AAV genome.


AAV delivery of genes that exceed this size and/or the use of large physiological regulatory elements can be accomplished, for example, by dividing the protein(s) to be delivered into two or more fragments. In some embodiments, the N-terminal fragment is fused to a split intein-N. In some embodiments, the C-terminal fragment is fused to a split intein-C. In embodiments, the fragments are packaged into two or more AAV vectors.


In some embodiments, dual AAV vectors are generated by splitting a large transgene expression cassette in two separate halves (5 and 3 ends, or head and tail), e.g., wherein each half of the cassette is packaged in a single AAV vector (of <5 kb). The re-assembly of the full-length transgene expression cassette can, in some embodiments, then be achieved upon co-infection of the same cell by both dual AAV vectors. In some embodiments, co-infection is followed by one or more of: (1) homologous recombination (HR) between 5 and 3 genomes (dual AAV overlapping vectors); (2) ITR-mediated tail-to-head concatemerization of 5 and 3 genomes (dual AAV trans-splicing vectors); and/or (3) a combination of these two mechanisms (dual AAV hybrid vectors). In some embodiments, the use of dual AAV vectors in vivo results in the expression of full-length proteins. In some embodiments, the use of the dual AAV vector platform represents an efficient and viable gene transfer strategy for transgenes of greater than about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 kb in size. In some embodiments, AAV vectors can also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides. In some embodiments, AAV vectors can be used for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994); each of which is incorporated herein by reference in their entirety). The construction of recombinant AAV vectors is described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989) (incorporated by reference herein in their entirety).


In some embodiments, a Gene Writer described herein (e.g., with or without one or more guide nucleic acids) can be delivered using AAV, lentivirus, adenovirus or other plasmid or viral vector types, in particular, using formulations and doses from, for example, U.S. Pat. No. 8,454,972 (formulations, doses for adenovirus), U.S. Pat. No. 8,404,658 (formulations, doses for AAV) and U.S. Pat. No. 5,846,946 (formulations, doses for DNA plasmids) and from clinical trials and publications regarding the clinical trials involving lentivirus, AAV and adenovirus. For example, for AAV, the route of administration, formulation and dose can be as described in U.S. Pat. No. 8,454,972 and as in clinical trials involving AAV. For Adenovirus, the route of administration, formulation and dose can be as described in U.S. Pat. No. 8,404,658 and as in clinical trials involving adenovirus. For plasmid delivery, the route of administration, formulation and dose can be as described in U.S. Pat. No. 5,846,946 and as in clinical studies involving plasmids. Doses can be based on or extrapolated to an average 70 kg individual (e.g. a male adult human), and can be adjusted for patients, subjects, mammals of different weight and species. Frequency of administration is within the ambit of the medical or veterinary practitioner (e.g., physician, veterinarian), depending on usual factors including the age, sex, general health, other conditions of the patient or subject and the particular condition or symptoms being addressed. In some embodiments, the viral vectors can be injected into the tissue of interest. For cell-type specific Gene Writing, the expression of the Gene Writer and optional guide nucleic acid can, in some embodiments, be driven by a cell-type specific promoter.


In some embodiments, AAV allows for low toxicity, for example, due to the purification method not requiring ultracentrifugation of cell particles that can activate the immune response.


In some embodiments, AAV allows low probability of causing insertional mutagenesis, for example, because it does not substantially integrate into the host genome.


In some embodiments, AAV has a packaging limit of about 4.4, 4.5, 4.6, 4.7, or 4.75 kb.


In some embodiments, a Gene Writer, promoter, and transcription terminator can fit into a single viral vector. SpCas9 (4.1 kb) may, in some instances, be difficult to package into AAV. Therefore, in some embodiments, a Gene Writer is used that is shorter in length than other Gene Writers or base editors. In some embodiments, the Gene Writers are less than about 4.5 kb, 4.4 kb, 4.3 kb, 4.2 kb, 4.1 kb, 4 kb, 3.9 kb, 3.8 kb, 3.7 kb, 3.6 kb, 3.5 kb, 3.4 kb, 3.3 kb, 3.2 kb, 3.1 kb, 3 kb, 2.9 kb, 2.8 kb, 2.7 kb, 2.6 kb, 2.5 kb, 2 kb, or 1.5 kb.


An AAV can be AAV1, AAV2, AAV5 or any combination thereof. In some embodiments, the type of AAV is selected with respect to the cells to be targeted; e.g., AAV serotypes 1, 2, 5 or a hybrid capsid AAV1, AAV2, AAV5 or any combination thereof can be selected for targeting brain or neuronal cells; or AAV4 can be selected for targeting cardiac tissue. In some embodiments, AAV8 is selected for delivery to the liver. Exemplary AAV serotypes as to these cells are described, for example, in Grimm, D. et al, J. Virol.82: 5887-5911 (2008) (incorporated herein by reference in its entirety). In some embodiments, AAV refers all serotypes, subtypes, and naturally-occurring AAV as well as recombinant AAV. AAV may be used to refer to the virus itself or a derivative thereof. In some embodiments, AAV includes AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV6.2, AAV7, AAVrh.64R1, AAVhu.37, AAVrh.8, AAVrh.32.33, AAV8, AAV9, AAV-DJ, AAV2/8, AAVrh10, AAVLK03, AV10, AAV11, AAV 12, rhlO, and hybrids thereof, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, nonprimate AAV, and ovine AAV. The genomic sequences of various serotypes of AAV, as well as the sequences of the native terminal repeats (TRs), Rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as Genflank. Additional exemplary AAV serotypes are listed in Table 36.









TABLE 36







Exemplary AAV serotypes.









Target




Tissue
Vehicle
Reference





Liver
AAV (AAV81, AAVrh.81,
1. Wang et al., Mol. Ther. 18,



AAVhu.371, AAV2/8,
118-25 (2010)



AAV2/rh102, AAV9, AAV2,
2. Ginn et al., JHEP Reports,



NP403, NP592, 3, AAV3B5,
100065 (2019)



AAV-DJ4, AAV-LK014,
3. Paulk et al., Mol. Ther. 26,



AAV-LK024, AAV-LK034,
289-303 (2018).



AAV-LK194
4. L. Lisowski et al., Nature.



Adenovirus (Ad5, HC-AdV6)
506, 382-6 (2014).




5. L. Wang et al., Mol. Ther.




23, 1877-87 (2015).




6. Hausl Mol Ther (2010)


Lung
AAV (AAV4, AAV5,
1. Duncan et al., Mol Ther



AAV61, AAV9, H222)

Methods Clin Dev (2018)



Adenovirus (Ad5, Ad3,
2. Cooney et al., Am J Respir



Ad21, Ad14)3
Cell Mol Biol (2019)




3. Li et al., Mol Ther Methods





Clin Dev (2019)


Skin
AAV (AAV61, AAV-LK192)
1. Petek et al., Mol. Ther.




(2010)




2. L. Lisowski et al., Nature.




506, 382-6 (2014).


HSCs
Adenovirus (HDAd5/35++)
Wang et al. Blood Adv (2019)









In some embodiments, a pharmaceutical composition (e.g., comprising an AAV as dscribed herein) has less than 10% empty capsids, less than 8% empty capsids, less than 7% empty capsids, less than 5% empty capsids, less than 3% empty capsids, or less than 1% empty capsids. In some embodiments, the pharmaceutical composition has less than about 5% empty capsids. In some embodiments, the number of empty capsids is below the limit of detection. In some embodiments, it is advantageous for the pharmaceutical composition to have low amounts of empty capsids, e.g., because empty capsids may generate an adverse response (e.g., immune response, inflammatory response, liver response, and/or cardiac response), e.g., with little or no substantial therapeutic benefit.


In some embodiments, the residual host cell protein (rHCP) in the pharmaceutical composition is less than or equal to 100 ng/ml rHCP per 1×1013 vg/ml, e.g., less than or equal to 40 ng/ml rHCP per 1×1013 vg/ml or 1-50 ng/ml rHCP per 1×1013 vg/ml. In some embodiments, the pharmaceutical composition comprises less than 10 ng rHCP per 1.0×1013 vg, or less than 5 ng rHCP per 1.0×1013 vg, less than 4 ng rHCP per 1.0×1013 vg, or less than 3 ng rHCP per 1.0×1013 vg, or any concentration in between. In some embodiments, the residual host cell DNA (hcDNA) in the pharmaceutical composition is less than or equal to 5×106 pg/ml hcDNA per 1×1013 vg/ml, less than or equal to 1.2×106 pg/ml hcDNA per 1×1013 vg/ml, or 1×105 pg/ml hcDNA per 1×1013 vg/ml. In some embodiments, the residual host cell DNA in said pharmaceutical composition is less than 5.0×105 pg per 1×1013 vg, less than 2.0×105 pg per 1.0×1013 vg, less than 1.1×105 pg per 1.0×1013 vg, less than 1.0×105 pg hcDNA per 1.0×1013 vg, less than 0.9×105 pg hcDNA per 1.0×1013 vg, less than 0.8×105 pg hcDNA per 1.0×1013 vg, or any concentration in between.


In some embodiments, the residual plasmid DNA in the pharmaceutical composition is less than or equal to 1.7×105 pg/ml per 1.0×1013 vg/ml, or 1×105 pg/ml per 1×1.0×1013 vg/ml, or 1.7×106 pg/ml per 1.0×1013 vg/ml. In some embodiments, the residual DNA plasmid in the pharmaceutical composition is less than 10.0×105 pg by 1.0×1013 vg, less than 8.0×105 pg by 1.0×1013 vg or less than 6.8×105 pg by 1.0×1013 vg. In embodiments, the pharmaceutical composition comprises less than 0.5 ng per 1.0×1013 vg, less than 0.3 ng per 1.0×1013 vg, less than 0.22 ng per 1.0×1013 vg or less than 0.2 ng per 1.0×1013 vg or any intermediate concentration of bovine serum albumin (BSA). In embodiments, the benzonase in the pharmaceutical composition is less than 0.2 ng by 1.0×1013 vg, less than 0.1 ng by 1.0×1013 vg, less than 0.09 ng by 1.0×1013 vg, less than 0.08 ng by 1.0×1013 vg or any intermediate concentration. In embodiments, Poloxamer 188 in the pharmaceutical composition is about 10 to 150 ppm, about 15 to 100 ppm or about 20 to 80 ppm. In embodiments, the cesium in the pharmaceutical composition is less than 50 pg/g (ppm), less than 30 pg/g (ppm) or less than 20 pg/g (ppm) or any intermediate concentration.


In embodiments, the pharmaceutical composition comprises total impurities, e.g., as determined by SDS-PAGE, of less than 10%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or any percentage in between. In embodiments, the total purity, e.g., as determined by SDS-PAGE, is greater than 90%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or any percentage in between. In embodiments, no single unnamed related impurity, e.g., as measured by SDS-PAGE, is greater than 5%, greater than 4%, greater than 3% or greater than 2%, or any percentage in between. In embodiments, the pharmaceutical composition comprises a percentage of filled capsids relative to total capsids (e.g., peak 1+peak 2 as measured by analytical ultracentrifugation) of greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, greater than 90%, greater than 91%, greater than 91.9%, greater than 92%, greater than 93%, or any percentage in between. In embodiments of the pharmaceutical composition, the percentage of filled capsids measured in peak 1 by analytical ultracentrifugation is 20-80%, 25-75%, 30-75%, 35-75%, or 37.4-70.3%. In embodiments of the pharmaceutical composition, the percentage of filled capsids measured in peak 2 by analytical ultracentrifugation is 20-80%, 20-70%, 22-65%, 24-62%, or 24.9-60.1%.


In one embodiment, the pharmaceutical composition comprises a genomic titer of 1.0 to 5.0×1013 vg/mL, 1.2 to 3.0×1013 vg/mL or 1.7 to 2.3×1013 vg/ml. In one embodiment, the pharmaceutical composition exhibits a biological load of less than 5 CFU/mL, less than 4 CFU/mL, less than 3 CFU/mL, less than 2 CFU/mL or less than 1 CFU/mL or any intermediate contraction. In embodiments, the amount of endotoxin according to USP, for example, USP <85> (incorporated by reference in its entirety) is less than 1.0 EU/mL, less than 0.8 EU/mL or less than 0.75 EU/mL. In embodiments, the osmolarity of a pharmaceutical composition according to USP, for example, USP <785> (incorporated by reference in its entirety) is 350 to 450 mOsm/kg, 370 to 440 mOsm/kg or 390 to 430 mOsm/kg. In embodiments, the pharmaceutical composition contains less than 1200 particles that are greater than 25 m per container, less than 1000 particles that are greater than 25 m per container, less than 500 particles that are greater than 25 μm per container or any intermediate value. In embodiments, the pharmaceutical composition contains less than 10,000 particles that are greater than 10 m per container, less than 8000 particles that are greater than 10 m per container or less than 600 particles that are greater than 10 pm per container.


In one embodiment, the pharmaceutical composition has a genomic titer of 0.5 to 5.0×1013 vg/mL, 1.0 to 4.0×1013 vg/mL, 1.5 to 3.0×1013 vg/ml or 1.7 to 2.3×1013 vg/ml. In one embodiment, the pharmaceutical composition described herein comprises one or more of the following: less than about 0.09 ng benzonase per 1.0×1013 vg, less than about 30 pg/g (ppm) of cesium, about 20 to 80 ppm Poloxamer 188, less than about 0.22 ng BSA per 1.0×1013 vg, less than about 6.8×105 pg of residual DNA plasmid per 1.0×1013 vg, less than about 1.1×105 pg of residual hcDNA per 1.0×1013 vg, less than about 4 ng of rHCP per 1.0×1013 vg, pH 7.7 to 8.3, about 390 to 430 mOsm/kg, less than about 600 particles that are >25 m in size per container, less than about 6000 particles that are >10 m in size per container, about 1.7×1013-2.3×1013 vg/mL genomic titer, infectious titer of about 3.9×108 to 8.4×1010 IU per 1.0×1013 vg, total protein of about 100-300 pg per 1.0×1013 vg, mean survival of >24 days in A7SMA mice with about 7.5×1013 vg/kg dose of viral vector, about 70 to 130% relative potency based on an in vitro cell based assay and/or less than about 5% empty capsid. In various embodiments, the pharmaceutical compositions described herein comprise any of the viral particles discussed here, retain a potency of between ±20%, between ±15%, between ±10% or within ±5% of a reference standard. In some embodiments, potency is measured using a suitable in vitro cell assay or in vivo animal model.


Additional methods of preparation, characterization, and dosing AAV particles are taught in WO2019094253, which is incorporated herein by reference in its entirety.


Additional rAAV constructs that can be employed consonant with the invention include those described in Wang et al 2019, available at: //doi.org/10.1038/s41573-019-0012-9, including Table 1 thereof, which is incorporated by reference in its entirety.


Inteins


In some embodiments, as described in more detail below, Intein-N may be fused to the N-terminal portion of a first domain described herein, and and intein-C may be fused to the C-terminal portion of a second domain described herein for the joining of the N-terminal portion to the C-terminal portion, thereby joining the first and second domains. In some embodiments, the first and second domains are each independent chosen from a DNA binding domain, an RNA binding domain, an RT domain, and an endonuclease domain.


As used herein, “intein” refers to a self-splicing protein intron (e.g., peptide), e.g., which ligates flanking N-terminal and C-terminal exteins (e.g., fragments to be joined). An intein may, in some instances, comprise a fragment of a protein that is able to excise itself and join the remaining fragments (the exteins) with a peptide bond in a process known as protein splicing. Inteins are also referred to as “protein introns.” The process of an intein excising itself and joining the remaining portions of the protein is herein termed “protein splicing” or “intein-mediated protein splicing.” In some embodiments, an intein of a precursor protein (an intein containing protein prior to intein-mediated protein splicing) comes from two genes. Such intein is referred to herein as a split intein (e.g., split intein-N and split intein-C). For example, in cyanobacteria, DnaE, the catalytic subunit a of DNA polymerase III, is encoded by two separate genes, dnaE-n and dnaE-c. The intein encoded by the dnaE-n gene may be herein referred as “intein-N.” The intein encoded by the dnaE-c gene may be herein referred as “intein-C.”


Use of inteins for joining heterologous protein fragments is described, for example, in Wood et al., J. Biol. Chem. 289(21); 14512-9 (2014) (incorporated herein by reference in its entirety). For example, when fused to separate protein fragments, the inteins IntN and IntC may recognize each other, splice themselves out, and/or simultaneously ligate the flanking N- and C-terminal exteins of the protein fragments to which they were fused, thereby reconstituting a full-length protein from the two protein fragments.


In some embodiments, a synthetic intein based on the dnaE intein, the Cfa-N (e.g., split intein-N) and Cfa-C (e.g., split intein-C) intein pair, is used. Examples of such inteins have been described, e.g., in Stevens et al., J Am Chem Soc. 2016 Feb. 24; 138(7):2162-5 (incorporated herein by reference in its entirety). Non-limiting examples of intein pairs that may be used in accordance with the present disclosure include: Cfa DnaE intein, Ssp GyrB intein, Ssp DnaX intein, Ter DnaE3 intein, Ter ThyX intein, Rma DnaB intein and Cne Prp8 intein (e.g., as described in U.S. Pat. No. 8,394,604, incorporated herein by reference.


In some embodiments, Intein-N and intein-C may be fused to the N-terminal portion of the split Cas9 and the C-terminal portion of a split Cas9, respectively, for the joining of the N-terminal portion of the split Cas9 and the C-terminal portion of the split Cas9. For example, in some embodiments, an intein-N is fused to the C-terminus of the N-terminal portion of the split Cas9, i.e., to form a structure of N [N-terminal portion of the split Cas9]-[intein-N]˜ C. In some embodiments, an intein-C is fused to the N-terminus of the C-terminal portion of the split Cas9, i.e., to form a structure of N-[intein-C]˜[C-terminal portion of the split Cas9]-C. The mechanism of intein-mediated protein splicing for joining the proteins the inteins are fused to (e.g., split Cas9) is described in Shah et al., Chem Sci. 2014; 5(1):446-461, incorporated herein by reference. Methods for designing and using inteins are known in the art and described, for example by WO2020051561, WO2014004336, WO2017132580, US20150344549, and US20180127780, each of which is incorporated herein by reference in their entirety.


In some embodiments, a split refers to a division into two or more fragments. In some embodiments, a split Cas9 protein or split Cas9 comprises a Cas9 protein that is provided as an N-terminal fragment and a C-terminal fragment encoded by two separate nucleotide sequences. The polypeptides corresponding to the N-terminal portion and the C-terminal portion of the Cas9 protein may be spliced to form a reconstituted Cas9 protein. In embodiments, the Cas9 protein is divided into two fragments within a disordered region of the protein, e.g., as described in Nishimasu et al., Cell, Volume 156, Issue 5, pp. 935-949, 2014, or as described in Jiang et al. (2016) Science 351: 867-871 and PDB file: 5F9R (each of which is incorporated herein by reference in its entirety). A disordered region may be determined by one or more protein structure determination techniques known in the art, including, without limitation, X-ray crystallography, NMR spectroscopy, electron microscopy (e.g., cryoEM), and/or in silico protein modeling. In some embodiments, the protein is divided into two fragments at any C, T, A, or S, e.g., within a region of SpCas9 between amino acids A292-G364, F445-K483, or E565-T637, or at corresponding positions in any other Cas9, Cas9 variant (e.g., nCas9, dCas9), or other napDNAbp. In some embodiments, protein is divided into two fragments at SpCas9 T310, T313, A456, S469, or C574. In some embodiments, the process of dividing the protein into two fragments is referred to as splitting the protein.


In some embodiments, a protein fragment ranges from about 2-1000 amino acids (e.g., between 2-10, 10-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 amino acids) in length. In some embodiments, a protein fragment ranges from about 5-500 amino acids (e.g., between 5-10, 10-50, 50-100, 100-200, 200-300, 300-400, or 400-500 amino acids) in length. In some embodiments, a protein fragment ranges from about 20-200 amino acids (e.g., between 20-30, 30-40, 40-50, 50-100, or 100-200 amino acids) in length.


In some embodiments, a portion or fragment of a Gene Writer (e.g., Cas9-R2Tg) is fused to an intein. The nuclease can be fused to the N-terminus or the C-terminus of the intein. In some embodiments, a portion or fragment of a fusion protein is fused to an intein and fused to an AAV capsid protein. The intein, nuclease and capsid protein can be fused together in any arrangement (e.g., nuclease-intein-capsid, intein-nuclease-capsid, capsid-intein-nuclease, etc.). In some embodiments, the N-terminus of an intein is fused to the C-terminus of a fusion protein and the C-terminus of the intein is fused to the N-terminus of an AAV capsid protein.


In some embodiments, an endonuclease domain (e.g., a nickase Cas9 domain) is fused to intein-N and a polypeptide comprising an RT domain is fused to an intein-C. Exemplary nucleotide and amino acid sequences of interns are provided below:











DnaE Intein-N DNA:



(SEQ ID NO: 1637)



TGCCTGTCATACGAAACCGAGATACTGACAGTAGAA






TATGGCCTTCTGCCAATCGGGAAGATTGTGGAGAAA






CGGATAGAATGCACAGTTTACTCTGTCGATAACAAT






GGTAACATTTATACTCAGCCAGTTGCCCAGTGGCAC






GACCGGGGAGAGCAGGAAGTATTCGAATACTGTCTG






GAGGATGGAAGTCTCATTAGGGCCACTAAGGACCAC






AAATTTATGACAGTCGATGGCCAGATGCTGCCTATA






GACGAAATCTTTGAGCGAGAGTTGGACCTCATGCGA






GTTGACAACCTTCCTAAT






DnaE Intein-N Protein:



(SEQ ID NO: 1638)



CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNN






GNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDH






KFMTVDGQMLPIDEIFERELDLMRVDNLPN






DnaE Intein-C DNA:



(SEQ ID NO: 1639)



ATGATCAAGATAGCTACAAGGAAGTATCTTGGCAAA






CAAAACGTTTATGATATTGGAGTCGAAAGAGATCAC






AACTTTGCTCTGAAGAACGGATTCATAGCTTCTAAT






Intein-C:



(SEQ ID NO: 1640)



MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN






Cfa-N DNA:



(SEQ ID NO: 1641)



TGCCTGTCTTATGATACCGAGATACTTACCGTTGAA






TATGGCTTCTTGCCTATTGGAAAGATTGTCGAAGAG






AGAATTGAATGCACAGTATATACTGTAGACAAGAAT






GGTTTCGTTTACACACAGCCCATTGCTCAATGGCAC






AATCGCGGCGAACAAGAAGTATTTGAGTACTGTCTC






GAGGATGGAAGCATCATACGAGCAACTAAAGATCAT






AAATTCATGACCACTGACGGGCAGATGTTGCCAATA






GATGAGATATTCGAGCGGGGCTTGGATCTCAAACAA






GTGGATGGATTGCCA






Cfa-N Protein:



(SEQ ID NO: 1642)



CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKN






GFVYTQPIAQWHNRGEQEVFEYCLEDGSIIRATKDH






KFMTTDGQMLPIDEIFERGLDLKQVDGLP






Cfa-C DNA:



(SEQ ID NO: 1643)



ATGAAGAGGACTGCCGATGGATCAGAGTTTGAATCT






CCCAAGAAGAAGAGGAAAGTAAAGATAATATCTCGA






AAAAGTCTTGGTACCCAAAATGTCTATGATATTGGA






GTGGAGAAAGATCACAACTTCCTTCTCAAGAACGGT






CTCGTAGCCAGCAAC






Cfa-C Protein:



(SEQ ID NO: 1644)



MKRTADGSEFESPKKKRKVKIISRKSLGTQNVYDIG






VEKDHNFLLKNGLVASN






Lipid Nanoparticles

The methods and systems provided by the invention, may employ any suitable carrier or delivery modality, including, in certain embodiments, lipid nanoparticles (LNPs). Lipid nanoparticles, in some embodiments, comprise one or more ionic lipids, such as non-cationic lipids (e.g., neutral or anionic, or zwitterionic lipids); one or more conjugated lipids (such as PEG-conjugated lipids or lipids conjugated to polymers described in Table 5 of WO2019217941; incorporated herein by reference in its entirety); one or more sterols (e.g., cholesterol); and, optionally, one or more targeting molecules (e.g., conjugated receptors, receptor ligands, antibodies); or combinations of the foregoing.


Lipids that can be used in nanoparticle formations (e.g., lipid nanoparticles) include, for example those described in Table 4 of WO2019217941, which is incorporated by reference e.g., a lipid-containing nanoparticle can comprise one or more of the lipids in table 4 of WO2019217941. Lipid nanoparticles can include additional elements, such as polymers, such as the polymers described in table 5 of WO2019217941, incorporated by reference.


In some embodiments, conjugated lipids, when present, can include one or more of PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-O-(2′,3′-di(tetradecanoyloxy)propyl-1-O-(w-methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N-(carbonyl-methoxypoly ethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, and those described in Table 2 of WO2019051289 (incorporated by reference), and combinations of the foregoing.


In some embodiments, sterols that can be incorporated into lipid nanoparticles include one or more of cholesterol or cholesterol derivatives, such as those in WO2009/127060 or US2010/0130588, which are incorporated by reference. Additional exemplary sterols include phytosterols, including those described in Eygeris et al (2020), dx.doi.org/10.1021/acs.nanolett.0c01386, incorporated herein by reference.


In some embodiments, the lipid particle comprises an ionizable lipid, a non-cationic lipid, a conjugated lipid that inhibits aggregation of particles, and a sterol. The amounts of these components can be varied independently and to achieve desired properties. For example, in some embodiments, the lipid nanoparticle comprises an ionizable lipid is in an amount from about 20 mol % to about 90 mol % of the total lipids (in other embodiments it may be 20-70% (mol), 30-60% (mol) or 40-50% (mol); about 50 mol % to about 90 mol % of the total lipid present in the lipid nanoparticle), a non-cationic lipid in an amount from about 5 mol % to about 30 mol % of the total lipids, a conjugated lipid in an amount from about 0.5 mol % to about 20 mol % of the total lipids, and a sterol in an amount from about 20 mol % to about 50 mol % of the total lipids. The ratio of total lipid to nucleic acid (e.g., encoding the Gene Writer or template nucleic acid) can be varied as desired. For example, the total lipid to nucleic acid (mass or weight) ratio can be from about 10:1 to about 30:1.


In some embodiments, an ionizable lipid may be a cationic lipid, a ionizable cationic lipid, e.g., a cationic lipid that can exist in a positively charged or neutral form depending on pH, or an amine-containing lipid that can be readily protonated. In some embodiments, the cationic lipid is a lipid capable of being positively charged, e.g., under physiological conditions. Exemplary cationic lipids include one or more amine group(s) which bear the positive charge. In some embodiments, the lipid particle comprises a cationic lipid in formulation with one or more of neutral lipids, ionizable amine-containing lipids, biodegradable alkyn lipids, steroids, phospholipids including polyunsaturated lipids, structural lipids (e.g., sterols), PEG, cholesterol and polymer conjugated lipids. In some embodiments, the cationic lipid may be an ionizable cationic lipid. An exemplary cationic lipid as disclosed herein may have an effective pKa over 6.0. In embodiments, a lipid nanoparticle may comprise a second cationic lipid having a different effective pKa (e.g., greater than the first effective pKa), than the first cationic lipid. A lipid nanoparticle may comprise between 40 and 60 mol percent of a cationic lipid, a neutral lipid, a steroid, a polymer conjugated lipid, and a therapeutic agent, e.g., a nucleic acid (e.g., RNA) described herein (e.g., a template nucleic acid or a nucleic acid encoding a GeneWriter), encapsulated within or associated with the lipid nanoparticle. In some embodiments, the nucleic acid is co-formulated with the cationic lipid. The nucleic acid may be adsorbed to the surface of an LNP, e.g., an LNP comprising a cationic lipid. In some embodiments, the nucleic acid may be encapsulated in an LNP, e.g., an LNP comprising a cationic lipid. In some embodiments, the lipid nanoparticle may comprise a targeting moiety, e.g., coated with a targeting agent. In embodiments, the LNP formulation is biodegradable. In some embodiments, a lipid nanoparticle comprising one or more lipid described herein, e.g., Formula (i), (ii), (ii), (vii) and/or (ix) encapsulates at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% or 100% of an RNA molecule, e.g., template RNA and/or a mRNA encoding the Gene Writer polypeptide.


In some embodiments, the lipid to nucleic acid ratio (mass/mass ratio; w/w ratio) can be in the range of from about 1:1 to about 25:1, from about 10:1 to about 14:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. The amounts of lipids and nucleic acid can be adjusted to provide a desired N/P ratio, for example, N/P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or higher. Generally, the lipid nanoparticle formulation's overall lipid content can range from about 5 mg/ml to about 30 mg/mL.


Exemplary ionizable lipids that can be used in lipid nanoparticle formulations include, without limitation, those listed in Table 1 of WO2019051289, incorporated herein by reference. Additional exemplary lipids include, without limitation, one or more of the following formulae: X of US2016/0311759; I of US20150376115 or in US2016/0376224; I, II or III of US20160151284; I, IA, II, or IIA of US20170210967; I-c of US20150140070; A of US2013/0178541; I of US2013/0303587 or US2013/0123338; I of US2015/0141678; II, III, IV, or V of US2015/0239926; I of US2017/0119904; I or II of WO2017/117528; A of US2012/0149894; A of US2015/0057373; A of WO2013/116126; A of US2013/0090372; A of US2013/0274523; A of US2013/0274504; A of US2013/0053572; A of WO2013/016058; A of WO2012/162210; I of US2008/042973; I, II, III, or IV of US2012/01287670; I or II of US2014/0200257; I, II, or III of US2015/0203446; I or III of US2015/0005363; I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID, or III-XXIV of US2014/0308304; of US2013/0338210; I, II, III, or IV of WO2009/132131; A of US2012/01011478; I or XXXV of US2012/0027796; XIV or XVII of US2012/0058144; of US2013/0323269; I of US2011/0117125; I, II, or III of US2011/0256175; I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII of US2012/0202871; I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV, or XVI of US2011/0076335; I or II of US2006/008378; I of US2013/0123338; I or X-A-Y-Z of US2015/0064242; XVI, XVII, or XVIII of US2013/0022649; I, II, or III of US2013/0116307; I, II, or III of US2013/0116307; I or II of US2010/0062967; I-X of US2013/0189351; I of US2014/0039032; V of US2018/0028664; I of US2016/0317458; I of US2013/0195920; 5, 6, or 10 of U.S. Pat. No. 10,221,127; 111-3 of WO2018/081480; I-5 or 1-8 of WO2020/081938; 18 or 25 of U.S. Pat. No. 9,867,888; A of US2019/0136231; II of WO2020/219876; I of US2012/0027803; OF-02 of US2019/0240349; 23 of U.S. Pat. No. 10,086,013; cKK-E12/A6 of Miao et al (2020); C12-200 of WO2010/053572; 7C1 of Dahlman et al (2017); 304-O13 or 503-013 of Whitehead et al; TS-P4C2 of U.S. Pat. No. 9,708,628; I of WO2020/106946; I of WO2020/106946.


In some embodiments, the ionizable lipid is MC3 (6Z,9Z,28Z,3lZ)-heptatriaconta-6,9,28,3 1-tetraen-19-yl-4-(dimethylamino) butanoate (DLin-MC3-DMA or MC3), e.g., as described in Example 9 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is the lipid ATX-002, e.g., as described in Example 10 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is (13Z,16Z)-A,A-dimethyl-3-nonyldocosa-13, 16-dien-1-amine (Compound 32), e.g., as described in Example 11 of WO2019051289A9 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is Compound 6 or Compound 22, e.g., as described in Example 12 of WO2019051289A9 (incorporated by reference herein in its entirety) In some embodiments, the ionizable lipid is heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102); e.g., as described in Example 1 of U.S. Pat. No. 9,867,888 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is 9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate (LP01) e.g., as synthesized in Example 13 of WO2015/095340 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is Di((Z)-non-2-en-1-yl) 9-((4-dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g. as synthesized in Example 7, 8, or 9 of US2012/0027803 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is 1,1′-((2-(4-(2-((2-(Bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl) amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), e.g., as synthesized in Examples 14 and 16 of WO2010/053572 (incorporated by reference herein in its entirety). In some embodiments, the ionizable lipid is; Imidazole cholesterol ester (ICE) lipid (3S, 10R, 13R, 17R)-10, 13-dimethyl-17-((R)-6-methylheptan-2-yl)-2, 3, 4, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 3-(1H-imidazol-4-yl)propanoate, e.g., Structure (I) from WO2020/106946 (incorporated by reference herein in its entirety).


Some non-limiting example of lipid compounds that may be used (e.g., in combination with other lipid components) to form lipid nanoparticles for the delivery of compositions described herein, e.g., nucleic acid (e.g., RNA) described herein (e.g., a template nucleic acid or a nucleic acid encoding a GeneWriter) includes,




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In some embodiments an LNP comprising Formula (i) is used to deliver a GeneWriter composition described herein to the liver and/or hepatocyte cells.




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In some embodiments an LNP comprising Formula (ii) is used to deliver a GeneWriter composition described herein to the liver and/or hepatocyte cells.




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In some embodiments an LNP comprising Formula (iii) is used to deliver a GeneWriter composition described herein to the liver and/or hepatocyte cells.




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In some embodiments an LNP comprising Formula (v) is used to deliver a GeneWriter composition described herein to the liver and/or hepatocyte cells.




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In some embodiments an LNP comprising Formula (vi) is used to deliver a GeneWriter composition described herein to the liver and/or hepatocyte cells.




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In some embodiments an LNP comprising Formula (viii) is used to deliver a GeneWriter composition described herein to the liver and/or hepatocyte cells.




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In some embodiments an LNP comprising Formula (ix) is used to deliver a GeneWriter composition described herein to the liver and/or hepatocyte cells.




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    • wherein

    • X1 is O, NR, or a direct bond, X1 is C2-5 alkylene, X3 is C(═O) or a direct bond, R1 is H or Me, R3 is Ci-3 alkyl, R2 is Ci-3 alkyl, or R2 taken together with the nitrogen atom to which it is attached and 1-3 carbon atoms of X2 form a 4-, 5-, or 6-membered ring, or X1 is NR1, R1 and R2 taken together with the nitrogen atoms to which they are attached form a 5- or 6-membered ring, or R2 taken together with R and the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered ring, Y1 is C2-12 alkylene, Y2 is selected from







embedded image




    • n is 0 to 3, R4 is Ci-15 alkyl, Z1 is Ci-6 alkylene or a direct bond,

    • Z2 is







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    • (in either orientation) or absent. provided that if Z1 is a direct bond, Z2 is absent,

    • R5 is C5-9 alkyl or C6-10 alkoxy, R6 is C5-9 alkyl or C6-10 alkoxy, W is methylene or a direct bond, and R is H or Me, or a salt thereof provided that if R3 and R2 are C2 alkyls, X1 is O, X2 is linear C3 alkylene, X1 is C(═O), Y1 is linear Ce alkylene, (Y2)n-R4 is







embedded image




    •  R4 is linear C5 alkyl, Z1 is C2 alkylene, Z2 is absent, W is methylene, and R7 is H, then R5 and R6 are not Cx alkoxy.





In some embodiments an LNP comprising Formula (xii) is used to deliver a GeneWriter composition described herein to the liver and/or hepatocyte cells.




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In some embodiments an LNP comprising Formula (xi) is used to deliver a GeneWriter composition described herein to the liver and/or hepatocyte cells.




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where R




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In some embodiments a lipid of Formula (xii) can be represented by the following structure




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In some embodiments an LNP comprises a compound of Formula (xiii) and a compound of Formula (xiv).




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In some embodiments an LNP comprising Formula (xv) is used to deliver a GeneWriter composition described herein to the liver and/or hepatocyte cells.




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In some embodiments an LNP comprising a formulation of Formula (xvi) is used to deliver a GeneWriter composition described herein to the lung endothelial cells.




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where X=




text missing or illegible when filed


In some embodiments, a lipid compound used to form lipid nanoparticles for the delivery of compositions described herein, e.g., nucleic acid (e.g., RNA) described herein (e.g., a template nucleic acid or a nucleic acid encoding a GeneWriter) is made by one of the following reactions:




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Exemplary non-cationic lipids include, but are not limited to, distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidicacid, cerebrosides, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof. It is understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, paimitoyl, stearoyl, or oleoyl. Additional exemplary lipids, in certain embodiments, include, without limitation, those described in Kim et al. (2020) dx.doi.org/10.1021/acs.nanolett.0c01386, incorporated herein by reference. Such lipids include, in some embodiments, plant lipids found to improve liver transfection with mRNA (e.g., DGTS).


In some embodiments, the non-cationic lipid may have the following structure




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Other examples of non-cationic lipids suitable for use in the lipid nanopartieles include, without limitation, nonphosphorous lipids such as, e.g., stearylamine, dodeeylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyl dimethyl ammonium bromide, ceramide, sphingomyelin, and the like. Other non-cationic lipids are described in WO2017/099823 or US patent publication US2018/0028664, the contents of which is incorporated herein by reference in their entirety.


In some embodiments, the non-cationic lipid is oleic acid or a compound of Formula I, II, or IV of US2018/0028664, incorporated herein by reference in its entirety. The non-cationic lipid can comprise, for example, 0-30% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, the non-cationic lipid content is 5-20% (mol) or 10-15% (mol) of the total lipid present in the lipid nanoparticle. In embodiments, the molar ratio of ionizable lipid to the neutral lipid ranges from about 2:1 to about 8:1 (e.g., about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1).


In some embodiments, the lipid nanoparticles do not comprise any phospholipids.


In some aspects, the lipid nanoparticle can further comprise a component, such as a sterol, to provide membrane integrity. One exemplary sterol that can be used in the lipid nanoparticle is cholesterol and derivatives thereof. Non-limiting examples of cholesterol derivatives include polar analogues such as 5a-choiestanol, 53-coprostanol, choiesteryl-(2′-hydroxy)-ethyl ether, choiesteryl-(4′-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5p-cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analogue, e.g., choiesteryl-(4′-hydroxy)-butyl ether. Exemplary cholesterol derivatives are described in PCT publication WO2009/127060 and US patent publication US2010/0130588, each of which is incorporated herein by reference in its entirety.


In some embodiments, the component providing membrane integrity, such as a sterol, can comprise 0-50% (mol) (e.g., 0-10%, 10-20%, 20-30%, 30-40%, or 40-50%) of the total lipid present in the lipid nanoparticle. In some embodiments, such a component is 20-50% (mol) 30-40% (mol) of the total lipid content of the lipid nanoparticle.


In some embodiments, the lipid nanoparticle can comprise a polyethylene glycol (PEG) or a conjugated lipid molecule. Generally, these are used to inhibit aggregation of lipid nanoparticles and/or provide steric stabilization. Exemplary conjugated lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic-polymer lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, for example, a (methoxy polyethylene glycol)-conjugated lipid.


Exemplary PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-O-(2′,3′-di(tetradecanoyloxy)propyl-1-O-(w-methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, 1,2-dimyristoyl-sn-glycerol, methoxypoly ethylene glycol (DMG-PEG-2K), or a mixture thereof. Additional exemplary PEG-lipid conjugates are described, for example, in U.S. Pat. Nos. 5,885,613, 6,287,591, US2003/0077829, US2003/0077829, US2005/0175682, US2008/0020058, US2011/0117125, US2010/0130588, US2016/0376224, US2017/0119904, and US/099823, the contents of all of which are incorporated herein by reference in their entirety. In some embodiments, a PEG-lipid is a compound of Formula III, III-a-I, III-a-2, III-b-1, III-b-2, or V of US2018/0028664, the content of which is incorporated herein by reference in its entirety. In some embodiments, a PEG-lipid is of Formula II of US20150376115 or US2016/0376224, the content of both of which is incorporated herein by reference in its entirety. In some embodiments, the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can be one or more of PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol (1-[8′-(Cholest-5-en-3[beta]-oxy)carboxamido-3′,6′-dioxaoctanyl] carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4-Ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid comprises PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some embodiments, the PEG-lipid comprises a structure selected from:




text missing or illegible when filed


In some embodiments, lipids conjugated with a molecule other than a PEG can also be used in place of PEG-lipid. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic-polymer lipid (GPL) conjugates can be used in place of or in addition to the PEG-lipid.


Exemplary conjugated lipids, i.e., PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates and cationic polymer-lipids are described in the PCT and LIS patent applications listed in Table 2 of WO2019051289A9 and in WO2020106946A1, the contents of all of which are incorporated herein by reference in their entirety.


In some embodiments an LNP comprises a compound of Formula (xix), a compound of Formula (xxi) and a compound of Formula (xxv). In some embodiments a LNP comprising a formulation of Formula (xix), Formula (xxi) and Formula (xxv) is used to deliver a GeneWriter composition described herein to the lung or pulmonary cells.


In some embodiments, the PEG or the conjugated lipid can comprise 0-20% (mol) of the total lipid present in the lipid nanoparticle. In some embodiments, PEG or the conjugated lipid content is 0.5-10% or 2-5% (mol) of the total lipid present in the lipid nanoparticle. Molar ratios of the ionizable lipid, non-cationic-lipid, sterol, and PEG/conjugated lipid can be varied as needed. For example, the lipid particle can comprise 30-70% ionizable lipid by mole or by total weight of the composition, 0-60% cholesterol by mole or by total weight of the composition, 0-30% non-cationic-lipid by mole or by total weight of the composition and 1-10% conjugated lipid by mole or by total weight of the composition. Preferably, the composition comprises 30-40% ionizable lipid by mole or by total weight of the composition, 40-50% cholesterol by mole or by total weight of the composition, and 10-20% non-cationic-lipid by mole or by total weight of the composition. In some other embodiments, the composition is 50-75% ionizable lipid by mole or by total weight of the composition, 20-40% cholesterol by mole or by total weight of the composition, and 5 to 10% non-cationic-lipid, by mole or by total weight of the composition and 1-10% conjugated lipid by mole or by total weight of the composition. The composition may contain 60-70% ionizable lipid by mole or by total weight of the composition, 25-35% cholesterol by mole or by total weight of the composition, and 5-10% non-cationic-lipid by mole or by total weight of the composition. The composition may also contain up to 90% ionizable lipid by mole or by total weight of the composition and 2 to 15% non-cationic lipid by mole or by total weight of the composition. The formulation may also be a lipid nanoparticle formulation, for example comprising 8-30% ionizable lipid by mole or by total weight of the composition, 5-30% non-cationic lipid by mole or by total weight of the composition, and 0-20% cholesterol by mole or by total weight of the composition; 4-25% ionizable lipid by mole or by total weight of the composition, 4-25% non-cationic lipid by mole or by total weight of the composition, 2 to 25% cholesterol by mole or by total weight of the composition, 10 to 35% conjugate lipid by mole or by total weight of the composition, and 5% cholesterol by mole or by total weight of the composition; or 2-30% ionizable lipid by mole or by total weight of the composition, 2-30% non-cationic lipid by mole or by total weight of the composition, 1 to 15% cholesterol by mole or by total weight of the composition, 2 to 35% conjugate lipid by mole or by total weight of the composition, and 1-20% cholesterol by mole or by total weight of the composition; or even up to 90% ionizable lipid by mole or by total weight of the composition and 2-10% non-cationic lipids by mole or by total weight of the composition, or even 100% cationic lipid by mole or by total weight of the composition. In some embodiments, the lipid particle formulation comprises ionizable lipid, phospholipid, cholesterol and a PEG-ylated lipid in a molar ratio of 50:10:38.5:1.5. In some other embodiments, the lipid particle formulation comprises ionizable lipid, cholesterol and a PEG-ylated lipid in a molar ratio of 60:38.5:1.5.


In some embodiments, the lipid particle comprises ionizable lipid, non-cationic lipid (e.g. phospholipid), a sterol (e.g., cholesterol) and a PEG-ylated lipid, where the molar ratio of lipids ranges from 20 to 70 mole percent for the ionizable lipid, with a target of 40-60, the mole percent of non-cationic lipid ranges from 0 to 30, with a target of 0 to 15, the mole percent of sterol ranges from 20 to 70, with a target of 30 to 50, and the mole percent of PEG-ylated lipid ranges from 1 to 6, with a target of 2 to 5.


In some embodiments, the lipid particle comprises ionizable lipid/non-cationic-lipid/sterol/conjugated lipid at a molar ratio of 50:10:38.5:1.5.


In an aspect, the disclosure provides a lipid nanoparticle formulation comprising phospholipids, lecithin, phosphatidylcholine and phosphatidylethanolamine.


In some embodiments, one or more additional compounds can also be included. Those compounds can be administered separately or the additional compounds can be included in the lipid nanoparticles of the invention. In other words, the lipid nanoparticles can contain other compounds in addition to the nucleic acid or at least a second nucleic acid, different than the first. Without limitations, other additional compounds can be selected from the group consisting of small or large organic or inorganic molecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and derivatives thereof, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, an extract made from biological materials, or any combinations thereof.


In some embodiments, a lipid nanoparticle (or a formulation comprising lipid nanoparticles) lacks reactive impurities (e.g., aldehydes or ketones), or comprises less than a preselected level of reactive impurities (e.g., aldehydes or ketones). While not wishing to be bound by theory, in some embodiments, a lipid reagent is used to make a lipid nanoparticle formulation, and the lipid reagent may comprise a contaminating reactive impurity (e.g., an aldehyde or ketone). A lipid regent may be selected for manufacturing based on having less than a preselected level of reactive impurities (e.g., aldehydes or ketones). Without wishing to be bound by theory, in some embodiments, aldehydes can cause modification and damage of RNA, e.g., cross-linking between bases and/or covalently conjugating lipid to RNA (e.g., forming lipid-RNA adducts). This may, in some instances, lead to failure of a reverse transcriptase reaction and/or incorporation of inappropriate bases, e.g., at the site(s) of lesion(s), e.g., a mutation in a newly synthesized target DNA.


In some embodiments, a lipid nanoparticle formulation is produced using a lipid reagent comprising less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content. In some embodiments, a lipid nanoparticle formulation is produced using a lipid reagent comprising less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species. In some embodiments, a lipid nanoparticle formulation is produced using a lipid reagent comprising: (i) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content; and (ii) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species. In some embodiments, the lipid nanoparticle formulation is produced using a plurality of lipid reagents, and each lipid reagent of the plurality independently meets one or more criterion described in this paragraph. In some embodiments, each lipid reagent of the plurality meets the same criterion, e.g., a criterion of this paragraph.


In some embodiments, the lipid nanoparticle formulation comprises less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content. In some embodiments, the lipid nanoparticle formulation comprises less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species. In some embodiments, the lipid nanoparticle formulation comprises: (i) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content; and (ii) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species.


In some embodiments, one or more, or optionally all, of the lipid reagents used for a lipid nanoparticle as described herein or a formulation thereof comprise less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content. In some embodiments, one or more, or optionally all, of the lipid reagents used for a lipid nanoparticle as described herein or a formulation thereof comprise less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species. In some embodiments, one or more, or optionally all, of the lipid reagents used for a lipid nanoparticle as described herein or a formulation thereof comprise: (i) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% total reactive impurity (e.g., aldehyde) content; and (ii) less than 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of any single reactive impurity (e.g., aldehyde) species.


In some embodiments, total aldehyde content and/or quantity of any single reactive impurity (e.g., aldehyde) species is determined by liquid chromatography (LC), e.g., coupled with tandem mass spectrometry (MS/MS), e.g., according to the method described in Example 40. In some embodiments, reactive impurity (e.g., aldehyde) content and/or quantity of reactive impurity (e.g., aldehyde) species is determined by detecting one or more chemical modifications of a nucleic acid molecule (e.g., an RNA molecule, e.g., as described herein) associated with the presence of reactive impurities (e.g., aldehydes), e.g., in the lipid reagents. In some embodiments, reactive impurity (e.g., aldehyde) content and/or quantity of reactive impurity (e.g., aldehyde) species is determined by detecting one or more chemical modifications of a nucleotide or nucleoside (e.g., a ribonucleotide or ribonucleoside, e.g., comprised in or isolated from a template nucleic acid, e.g., as described herein) associated with the presence of reactive impurities (e.g., aldehydes), e.g., in the lipid reagents, e.g., as described in Example 41. In embodiments, chemical modifications of a nucleic acid molecule, nucleotide, or nucleoside are detected by determining the presence of one or more modified nucleotides or nucleosides, e.g., using LC-MS/MS analysis, e.g., as described in Example 41.


In some embodiments, a nucleic acid (e.g., RNA) described herein (e.g., a template nucleic acid or a nucleic acid encoding a GeneWriter) does not comprise an aldehyde modification, or comprises less than a preselected amount of aldehyde modifications. In some embodiments, on average, a nucleic acid has less than 50, 20, 10, 5, 2, or 1 aldehyde modifications per 1000 nucleotides, e.g., wherein a single cross-linking of two nucleotides is a single aldehyde modification. In some embodiments, the aldehyde modification is an RNA adduct (e.g., a lipid-RNA adduct). In some embodiments, the aldehyde-modified nucleotide is cross-linking between bases. In some embodiments, a nucleic acid (e.g., RNA) described herein comprises less than 50, 20, 10, 5, 2, or 1 cross-links between nucleotide.


In some embodiments, LNPs are directed to specific tissues by the addition of targeting domains. For example, biological ligands may be displayed on the surface of LNPs to enhance interaction with cells displaying cognate receptors, thus driving association with and cargo delivery to tissues wherein cells express the receptor. In some embodiments, the biological ligand may be a ligand that drives delivery to the liver, e.g., LNPs that display GalNAc result in delivery of nucleic acid cargo to hepatocytes that display asialoglycoprotein receptor (ASGPR). The work of Akinc et al. Mol Ther 18(7):1357-1364 (2010) teaches the conjugation of a trivalent GalNAc ligand to a PEG-lipid (GalNAc-PEG-DSG) to yield LNPs dependent on ASGPR for observable LNP cargo effect (see, e.g., FIG. 6). Other ligand-displaying LNP formulations, e.g., incorporating folate, transferrin, or antibodies, are discussed in WO2017223135, which is incorporated herein by reference in its entirety, in addition to the references used therein, namely Kolhatkar et al., Curr Drug Discov Technol. 2011 8:197-206; Musacchio and Torchilin, Front Biosci. 2011 16:1388-1412; Yu et al., Mol Membr Biol. 2010 27:286-298; Patil et al., Crit Rev Ther Drug Carrier Syst. 2008 25:1-61; Benoit et al., Biomacromolecules. 2011 12:2708-2714; Zhao et al., Expert Opin Drug Deliv. 2008 5:309-319; Akinc et al., Mol Ther. 2010 18:1357-1364; Srinivasan et al., Methods Mol Biol. 2012 820:105-116; Ben-Arie et al., Methods Mol Biol. 2012 757:497-507; Peer 2010 J Control Release. 20:63-68; Peer et al., Proc Natl Acad Sci USA. 2007 104:4095-4100; Kim et al., Methods Mol Biol. 2011 721:339-353; Subramanya et al., Mol Ther. 2010 18:2028-2037; Song et al., Nat Biotechnol. 2005 23:709-717; Peer et al., Science. 2008 319:627-630; and Peer and Lieberman, Gene Ther. 2011 18:1127-1133.


In some embodiments, LNPs are selected for tissue-specific activity by the addition of a Selective ORgan Targeting (SORT) molecule to a formulation comprising traditional components, such as ionizable cationic lipids, amphipathic phospholipids, cholesterol and poly(ethylene glycol) (PEG) lipids. The teachings of Cheng et al. Nat Nanotechnol 15(4):313-320 (2020) demonstrate that the addition of a supplemental “SORT” component precisely alters the in vivo RNA delivery profile and mediates tissue-specific (e.g., lungs, liver, spleen) gene delivery and editing as a function of the percentage and biophysical property of the SORT molecule.


In some embodiments, the LNPs comprise biodegradable, ionizable lipids. In some embodiments, the LNPs comprise (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate) or another ionizable lipid. See, e.g, lipids of WO2019/067992, WO/2017/173054, WO2015/095340, and WO2014/136086, as well as references provided therein. In some embodiments, the term cationic and ionizable in the context of LNP lipids is interchangeable, e.g., wherein ionizable lipids are cationic depending on the pH.


In some embodiments, multiple components of a Gene Writer system may be prepared as a single LNP formulation, e.g., an LNP formulation comprises mRNA encoding for the Gene Writer polypeptide and an RNA template. Ratios of nucleic acid components may be varied in order to maximize the properties of a therapeutic. In some embodiments, the ratio of RNA template to mRNA encoding a Gene Writer polypeptide is about 1:1 to 100:1, e.g., about 1:1 to 20:1, about 20:1 to 40:1, about 40:1 to 60:1, about 60:1 to 80:1, or about 80:1 to 100:1, by molar ratio. In other embodiments, a system of multiple nucleic acids may be prepared by separate formulations, e.g., one LNP formulation comprising a template RNA and a second LNP formulation comprising an mRNA encoding a Gene Writer polypeptide. In some embodiments, the system may comprise more than two nucleic acid components formulated into LNPs. In some embodiments, the system may comprise a protein, e.g., a Gene Writer polypeptide, and a template RNA formulated into at least one LNP formulation.


In some embodiments, the average LNP diameter of the LNP formulation may be between 10s of nm and 100s of nm, e.g., measured by dynamic light scattering (DLS). In some embodiments, the average LNP diameter of the LNP formulation may be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average LNP diameter of the LNP formulation may be from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In some embodiments, the average LNP diameter of the LNP formulation may be from about 70 nm to about 100 nm. In a particular embodiment, the average LNP diameter of the LNP formulation may be about 80 nm. In some embodiments, the average LNP diameter of the LNP formulation may be about 100 nm. In some embodiments, the average LNP diameter of the LNP formulation ranges from about 1 mm to about 500 mm, from about 5 mm to about 200 mm, from about 10 mm to about 100 mm, from about 20 mm to about 80 mm, from about 25 mm to about 60 mm, from about 30 mm to about 55 mm, from about 35 mm to about 50 mm, or from about 38 mm to about 42 mm.


A LNP may, in some instances, be relatively homogenous. A polydispersity index may be used to indicate the homogeneity of a LNP, e.g., the particle size distribution of the lipid nanoparticles. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A LNP may have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of a LNP may be from about 0.10 to about 0.20.


The zeta potential of a LNP may be used to indicate the electrokinetic potential of the composition. In some embodiments, the zeta potential may describe the surface charge of a LNP. Lipid nanoparticles with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of a LNP may be from about −10 mV to about +20 mV, from about −10 mV to about +15 mV, from about −10 mV to about +10 mV, from about −10 mV to about +5 mV, from about −10 mV to about 0 mV, from about −10 mV to about −5 mV, from about −5 mV to about +20 mV, from about −5 mV to about +15 mV, from about −5 mV to about +10 mV, from about −5 mV to about +5 mV, from about −5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.


The efficiency of encapsulation of a protein and/or nucleic acid, e.g., Gene Writer polypeptide or mRNA encoding the polypeptide, describes the amount of protein and/or nucleic acid that is encapsulated or otherwise associated with a LNP after preparation, relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing the lipid nanoparticle before and after breaking up the lipid nanoparticle with one or more organic solvents or detergents. An anion exchange resin may be used to measure the amount of free protein or nucleic acid (e.g., RNA) in a solution. Fluorescence may be used to measure the amount of free protein and/or nucleic acid (e.g., RNA) in a solution. For the lipid nanoparticles described herein, the encapsulation efficiency of a protein and/or nucleic acid may be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In some embodiments, the encapsulation efficiency may be at least 90%. In some embodiments, the encapsulation efficiency may be at least 95%.


A LNP may optionally comprise one or more coatings. In some embodiments, a LNP may be formulated in a capsule, film, or table having a coating. A capsule, film, or tablet including a composition described herein may have any useful size, tensile strength, hardness or density.


Additional exemplary lipids, formulations, methods, and characterization of LNPs are taught by WO2020061457, which is incorporated herein by reference in its entirety.


In some embodiments, in vitro or ex vivo cell lipofections are performed using Lipofectamine MessengerMax (Thermo Fisher) or TransIT-mRNA Transfection Reagent (Mirus Bio). In certain embodiments, LNPs are formulated using the GenVoy_ILM ionizable lipid mix (Precision NanoSystems). In certain embodiments, LNPs are formulated using 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) or dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA or MC3), the formulation and in vivo use of which are taught in Jayaraman et al. Angew Chem Int Ed Engl 51(34):8529-8533 (2012), incorporated herein by reference in its entirety.


LNP formulations optimized for the delivery of CRISPR-Cas systems, e.g., Cas9-gRNA RNP, gRNA, Cas9 mRNA, are described in WO2019067992 and WO2019067910, both incorporated by reference.


Additional specific LNP formulations useful for delivery of nucleic acids are described in U.S. Pat. Nos. 8,158,601 and 8,168,775, both incorporated by reference, which include formulations used in patisiran, sold under the name ONPATTRO.


Exemplary dosing of Gene Writer LNP may include about 0.1, 0.25, 0.3, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, or 100 mg/kg (RNA). Exemplary dosing of AAV comprising a nucleic acid encoding one or more components of the system may include an MOI of about 1011, 1012, 1013, and 1014 vg/kg.


All publications, patent applications, patents, and other publications and references (e.g., sequence database reference numbers) cited herein are incorporated by reference in their entirety. For example, all GenBank, Unigene, and Entrez sequences referred to herein, e.g., in any Table herein, are incorporated by reference. Unless otherwise specified, the sequence accession numbers specified herein, including in any Table herein, refer to the database entries current as of Mar. 4, 2020. When one gene or protein references a plurality of sequence accession numbers, all of the sequence variants are encompassed.


EXAMPLES

The invention is further illustrated by the following examples. The examples are provided for illustrative purposes only and are not to be construed as limiting the scope or content of the invention in any way.


Example 1: Gene Writer™ Enabling Nucleotide Substitution in Genomic DNA to Correct Alpha-1 Antitrypsin Deficiency Mutation in Human Cells

This example describes the use of a Gene Writer™ gene editing system to alter a genomic sequence at a single nucleotide.


In this example, the Gene Writer™ polypeptide and writing template are provided as DNA transfected into HEK293T cells that possess the PiZ genotype (E342K), a common allele associated with alpha-1 antitrypsin deficiency. The Gene Writer™ polypeptide uses a Cas9 nickase for both DNA-binding and endonuclease functions. The writing template is designed to have homology to the target sequence, while incorporating additional nucleotides at the desired position, such that reverse transcription of the template RNA results in the generation of a new DNA strand containing the substitution.


To create the transversion in the affected human SERPINA1 gene that restores the GAG triplet coding for glutamate in healthy patients, the Gene Writer™ polypeptide is used with a specific template nucleic acid, which encodes a gRNA scaffold for polypeptide binding, a spacer for polypeptide homing, target homology domain to set up TPRT, and a template sequence for reverse transcription that includes the required substitution. An exemplary template RNA carries the sequence (1)TCCCCTCCAGGCCGTGCATA(2)GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG GCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCC(3)TcGTCGATGGTC AGCACAGCCTTAT(4)GCACGGCCTGGA (SEQ ID NO: 1607), where numbers are used to delineate the modules of the template in the order (5′-3′) (1) gRNA spacer, (2) gRNA scaffold, (3) heterologous object sequence, (4) 3′ homology priming domain, and the lowercase “c” indicates the position in the template carrying the nucleotide substitution to be written into the target site to correct the E342K mutation. An exemplary gRNA for providing a second nick as described in embodiments of this system comprises the spacer sequence TTTGTTGAACTTGACCTCGG (SEQ ID NO: 1608) and directs a Cas9 nickase to nick the second strand of the target site within the homologous region. In some embodiments, this second nick improves the efficiency of the edit.


After transfection, cells are incubated for three days to allow for expression of the Gene Writing™ system and conversion of the genomic DNA target, and genomic DNA is extracted from cells. Genomic DNA is then subjected to PCR-based amplification using site-specific primers and amplicons are sequenced on an Illumina MiSeq according to manufacturer's protocols. Sequence analysis is then performed to determine the frequency of reads containing the desired edit.


Example 2: Gene Writer™ Enabling Short Insertion in Genomic DNA to Correct CFTR

This example describes the use of a Gene Writer™ gene editing system to alter a genomic sequence by insertion of a short string of nucleotides.


In this example, the Gene Writer™ polypeptide and writing template are provided as DNA transfected into HEK293T cells that possess the CFTR delta-F508 mutation, a common allele associated with cystic fibrosis. The Gene Writer™ polypeptide uses a Cas9 nickase for both DNA-binding and endonuclease functions. The writing template is designed to have homology to the target sequence, while incorporating additional nucleotides at the desired position, such that reverse transcription of the template RNA results in the generation of a new DNA strand containing the short insertion.


To create a short insertion in the affected human CFTR locus that restores the TTT triplet coding for phenylalanine in healthy patients, the Gene Writer™ polypeptide is used with a specific template, which encodes a spacer for polypeptide homing, target homology domain to set up TPRT, and a template sequence for reverse transcription that includes the 3-nt insertion.


After transfection, cells are incubated for three days to allow for expression of the Gene Writing™ system and conversion of the genomic DNA target. After the incubation period, genomic DNA is extracted from cells. Genomic DNA is then subjected to PCR-based amplification using site-specific primers and amplicons are sequenced on an Illumina MiSeq according to manufacturer's protocols. Sequence analysis is then performed to determine the frequency of reads containing the desired edit.


Example 3: Gene Writer™ Enabling Deletion of Genomic DNA to Correct Duchenne Muscular Dystrophy (DMD)

This example describes the use of a Gene Writer™ gene editing system to alter a genomic sequence by deletion of nucleotides.


One of the most common mutations found in patients with DMD is a deletion that eliminates exon 50 in the rod domain of dystrophin, which places exon 51 out of frame with preceding exons. Such a mutation results in production of truncated dystrophin, leading to the pathological effects of the disease. In order to ameliorate disease, the remainder of the 79 total exons, the splice acceptor site is deleted from exon 51, resulting in restoration of the full-length protein, an approach known as exon skipping.


In this example, the Gene Writer™ polypeptide and writing template are provided as RNA nucleofected into cells containing a deletion in exon 50 that results in a truncated dystrophin product, as described above. Target cells are either patient-derived iPSCs containing the mutation or are synthetically engineered using CRISPR-Cas to generate the deletion. The Gene Writer™ polypeptide uses a Cas9 nickase for both DNA-binding and endonuclease functions. The writing template is designed to have homology to the target sequence, while incorporating a deletion at the desired position, such that reverse transcription of the template RNA results in the generation of a new DNA strand lacking the deleted nucleotides.


To create a short deletion that removes the exon 51 5′ splice acceptor site, the Gene Writer™ polypeptide is used with a specific template that encodes a spacer for polypeptide homing, target homology domain to set up TPRT, and a template sequence for reverse transcription that includes a 5-nt deletion proximal to the Gene Writer™ polypeptide-induced nick, which includes the splice acceptor site.


After transfection, cells are incubated for three days to allow for expression of the Gene Writing™ system and conversion of the genomic DNA target. After the incubation period, genomic DNA is extracted from cells. Genomic DNA is then subjected to PCR-based amplification using site-specific primers and amplicons are sequenced on an Illumina MiSeq according to manufacturer's protocols. Sequence analysis is then performed to determine the frequency of reads containing the desired edit. Protein analysis by Western blot is used to further confirm the expression of the restored dystrophin, as compared to the truncated dystrophin produced in non-edited cells.


Example 4: Gene Writer™ Enabling Large Insertion into Genomic DNA

This example describes the use of a Gene Writer™ gene editing system to alter a genomic sequence by insertion of a large string of nucleotides.


In this example, the Gene Writer™ polypeptide, gRNA, and writing template are provided as DNA transfected into HEK293T cells. The Gene Writer™ polypeptide uses a Cas9 nickase for both DNA-binding and endonuclease functions. The reverse transcriptase function is derived from the highly processive RT domain of an R2 retrotransposase. The writing template is designed to have homology to the target sequence, while incorporating the genetic payload at the desired position, such that reverse transcription of the template RNA results in the generation of a new DNA strand containing the desired insertion.


To create a large insertion in the human HEK293T cell DNA, the Gene Writer™ polypeptide is used in conjunction with a specific gRNA, which targets the Cas9-containing Gene Writer™ to the target locus, and a template RNA for reverse transcription, which contains an RT-binding motif (3′ UTR from an R2 element) for associating with the reverse transcriptase, a region of homology to the target site for priming reverse transcription, and a genetic payload (GFP expression unit). This complex nicks the target site and then performs TPRT on the template, initiating the reaction by using priming regions on the template that are complementary to the sequence immediately adjacent to the site of the nick and copying the GFP payload into the genomic DNA.


After transfection, cells are incubated for three days to allow for expression of the Gene Writing™ system and conversion of the genomic DNA target. After the incubation period, genomic DNA is extracted from cells. Genomic DNA is then subjected to PCR-based amplification using site-specific primers and amplicons are sequenced on an Illumina MiSeq according to manufacturer's protocols. Sequence analysis is then performed to determine the frequency of reads containing the desired edit.


Example 5: Gene Writer™ Edits not Incorporating Binding Sequences from the Template RNA

This example describes the use of a Gene Writer™ gene editing system to alter a genomic sequence by insertion of a genetic payload without causing the insertion of additional sequence from the template molecule.


In this example, the Gene Writer™ polypeptide and writing template are provided as DNA transfected into HEK293T cells. The Gene Writer™ polypeptide uses a Cas9 nickase for both DNA-binding and endonuclease functions. The writing template is designed to have homology to the target sequence, while incorporating the genetic payload (e.g. GFP gene expression unit) at the desired position, such that reverse transcription of the template RNA results in the generation of a new DNA strand containing the desired insertion.


To accomplish specific insertion of a genetic payload without also incorporating extraneous template motifs (e.g. protein binding motif), the layout of the template RNA molecule is such that the protein binding sequences (e.g. UTRs) are terminal to the homology sequences used to write the new payload into the genomic target site.


After transfection, cells are incubated for three days to allow for expression of the Gene Writing™ system and conversion of the genomic DNA target. After the incubation period, genomic DNA is extracted from cells. Genomic DNA is then subjected to PCR-based amplification using site-specific primers and amplicons are sequenced on an Illumina MiSeq according to manufacturer's protocols. Sequence analysis is then performed to determine the frequency of reads containing the desired edit.


Example 6: Gene Writer™ Genome Editing in the Presence of DNA Repair Inhibitors

This example describes the use of a Gene Writer™ gene editing system to alter a genomic sequence by insertion of a genetic payload without causing the insertion of additional sequence from the template molecule.


In this example, experiments will test the effect of different DNA repair pathways on Gene Writing™ via the application of DNA repair pathway inhibitors or DNA repair pathway deficient cell lines. When applying DNA repair pathway inhibitors, PrestoBlue cell viability assay is performed first to determine the toxicity of the inhibitors and whether any normalization should be applied for following assays. SCR7 is an inhibitor for NHEJ, which is applied at a series of dilutions during Gene Writer™ delivery. PARP protein is a nuclear enzyme that binds as homodimers to both single- and double-strand breaks. Thus, its inhibitors are be used in the test of relevant DNA repair pathways, including homologous recombination repair pathway and base excision repair pathway. The experiment procedure is the same with that of SCR7. Cell lines with deficient core proteins of nucleotide excision repair (NER) pathway are used to test the effect of NER on Gene Writing™. After the delivery of the Gene Writer™ system into the cell, ddPCR is used to evaluate the retrotransposition in the context of inhibition of DNA repair pathways. Sequencing analysis is also performed to evaluate whether certain DNA repair pathways play a role in the alteration of the integration junction. In some embodiments, Gene Writing™ into the genome will not be decreased by the knockdown of DNA repair pathways, suggesting that the system does not utilize host cell repair pathways for DNA integration. In some embodiments, Gene Writing™ into the genome will not be decreased by more than 50% by the knockdown of DNA repair pathways, suggesting that the system does not rely on host cell repair pathways for DNA integration.


Example 7: Internal Gene Writer Deletions Demonstrating Protein Domain Modularity

This example describes deletions in a Gene Writer polypeptide that retain functionality and further demonstrate the modularity of the DNA binding domain.


In this example, a series of experiments were performed to test the activity of various mutant retrotransposases, as well as gaining structural knowledge about the protein modularity. This experiment tested removing a polypeptide stretch after the c-myb motif in the DNA binding domain (DBD) and replacing it with a flexible linker (FIG. 8a). The polypeptide stretch removed is referred to as the “natural linker” since it is the intervening region between the DNA binding motifs and the RNA binding domain. The polypeptide region removed spans the following: on the N terminal side at either, location A (predicted random coil following c-myb motif) or location B (end of predicted alpha helix that contains part of the c-myb motif) and the removed region ends at either location v1 (alpha helical region of R2Tg that preceded the predicted −1 RNA binding motif or at location v2 (C-terminal side of an alpha helical region of R2Tg that preceded the predicted −1 RNA binding motif). In place of the polypeptide stretch removed, “natural linker”, is the either of two linkers (Linker A, XTEN: SGSETPGTSESATPES (SEQ ID NO: 1023), and Linker B, 3GS: GGGS (SEQ ID NO: 1024)). For each of these mutant retrotransposases that contain different removed regions (location A-v1, location A-v2, location B-v1, or location B-v2) they were replaced with either linker A or linker B by PCR to a DNA plasmid that expressed R2Tg, thereby yielding these sequences: c-mybA-v1 replaced with 3GS linker (SEQ ID NO: 1024), c-mybA-v2 replaced with 3GS linker (SEQ ID NO: 1024), c-mybA-v1 replaced with XTEN linker, c-mybA-v2 replaced with XTEN linker, c-mybB-v1 replaced with 3GS linker (SEQ ID NO: 1024), c-mybB-v2 replaced with 3GS linker (SEQ ID NO: 1024), c-mybB-v1 replaced with XTEN linker, c-mybB-v2 replaced with XTEN linker, as shown in Table E1 below. The insertion of the linkers was verified by Sanger sequencing and the DNA plasmids were purified for transfection.














R2Tg

SEQ ID


Mutant
Amino Acid Sequence
NO:







R2Tg with

MASCPKPGPPVSAGAMSLESGLTTHSVLAIERGPNSLANSGSDFGGGGL

1646


natural linker

GLPLRLLRVSVGTQTSRSDWVDLVSWSHPGPTSKSQQVDLVSLFPKHRV




deletion c-

DLLSKNDQVDLVAQFLPSKFPPNLAENDLALLVNLEFYRSDLHVYECVH




mybA

FAAHWEGLSGLPEVYEQLAPQPCVGETLHSSLPRDSELFVPEEGSSEKE




location-v1

SEDAPKTSPPTPGKHGLEQTGEEKVMVTVPDKNPPCPCCGTRVNSVLNL




replaced with

IEHLKVSHGKRGVCFRCAKCGKENSNYHSVVCHFPKCRGPETEKAPAGE




3GS linker

WICEVCNRDFTTKIGLGQHKRLAHPAVRNQERIVASQPKETSNRGAHKR




(SEQ ID NO:

CWTKEEEELLIRLEAQFEGNKNINKLIAEHITTKTAKQISDKRRLLSRK




1024)

PAEEPREEPGTCHHTRRAA

GGGS
CFGCLESISQIRTATRDKKDTVTREK




HPKKPFQKWMKDRAIKKGNYLRFQRLFYLDRGKLAKIILDDIECLSCDI




PLSEIYSVFKTRWETTGSFKSLGDFKTYGKADNTAFRELITAKEIEKNV




QEMSKGSAPGPDGITLGDVVKMDPEFSRTMEIFNLWLTTGKIPDMVRGC




RTVLIPKSSKPDRLKDINNWRPITIGSILLRLFSRIVTARLSKACPLNP




RQRGFIRAAGCSENLKLLQTIIWSAKREHRPLGVVFVDIAKAFDTVSHQ




HIIHALQQREVDPHIVGLVSNMYENISTYITTKRNTHTDKIQIRVGVKQ




GDPMSPLLFNLAMDPLLCKLEESGKGYHRGQSSITAMAFADDLVLLSDS




WENMNTNISILETFCNLTGLKTQGQKCHGFYIKPTKDSYTINDCAAWTI




NGTPLNMIDPGESEKYLGLQFDPWIGIARSGLSTKLDFWLQRIDQAPLK




PLQKTDILKTYTIPRLIYIADHSEVKTALLETLDQKIRTAVKEWLHLPP




CTCDAILYSSTRDGGLGITKLAGLIPSVQARRLHRIAQSSDDTMKCFME




KEKMEQLHKKLWIQAGGDRENIPSIWEAPPSSEPPNNVSTNSEWEAPTQ




KDKFPKPCNWRKNEFKKWTKLASQGRGIVNFERDKISNHWIQYYRRIPH




RKLLTALQLRANVYPTREFLARGRQDQYIKACRHCDADIESCAHIIGNC




PVTQDARIKRHNYICELLLEEAKKKDWVVFKEPHIRDSNKELYKPDLIF




VKDARALVVDVTVRYEAAKSSLEEAAAEKVRKYKHLETEVRHLTNAKDV




TFVGFPLGARGKWHQDNFKLLTELGLSKSRQVKMAETFSTVALFSSVDI




VHMFASRARKSMVM






R2Tg with

MASCPKPGPPVSAGAMSLESGLTTHSVLAIERGPNSLANSGSDFGGGGL

1647


natural linker

GLPLRLLRVSVGTQTSRSDWVDLVSWSHPGPTSKSQQVDLVSLFPKHRV




deletion c-

DLLSKNDQVDLVAQFLPSKFPPNLAENDLALLVNLEFYRSDLHVYECVH




mybA

FAAHWEGLSGLPEVYEQLAPQPCVGETLHSSLPRDSELFVPEEGSSEKE




location-v2

SEDAPKTSPPTPGKHGLEQTGEEKVMVTVPDKNPPCPCCGTRVNSVLNL




replaced with

IEHLKVSHGKRGVCFRCAKCGKENSNYHSVVCHFPKCRGPETEKAPAGE




3GS linker

WICEVCNRDFTTKIGLGQHKRLAHPAVRNQERIVASQPKETSNRGAHKR




(SEQ ID NO:

CWTKEEEELLIRLEAQFEGNKNINKLIAEHITTKTAKQISDKRRLLSRK




1024)

PAEEPREEPGTCHHTRRAA

GGGS
TATRDKKDTVTREKHPKKPFQKWMKD




RAIKKGNYLRFQRLFYLDRGKLAKIILDDIECLSCDIPLSEIYSVFKTR




WETTGSFKSLGDFKTYGKADNTAFRELITAKEIEKNVQEMSKGSAPGPD




GITLGDVVKMDPEFSRTMEIFNLWLTTGKIPDMVRGCRTVLIPKSSKPD




RLKDINNWRPITIGSILLRLFSRIVTARLSKACPLNPRQRGFIRAAGCS




ENLKLLQTIIWSAKREHRPLGVVFVDIAKAFDTVSHQHIIHALQQREVD




PHIVGLVSNMYENISTYITTKRNTHTDKIQIRVGVKQGDPMSPLLFNLA




MDPLLCKLEESGKGYHRGQSSITAMAFADDLVLLSDSWENMNTNISILE




TFCNLTGLKTQGQKCHGFYIKPTKDSYTINDCAAWTINGTPLNMIDPGE




SEKYLGLQFDPWIGIARSGLSTKLDFWLQRIDQAPLKPLQKTDILKTYT




IPRLIYIADHSEVKTALLETLDQKIRTAVKEWLHLPPCTCDAILYSSTR




DGGLGITKLAGLIPSVQARRLHRIAQSSDDTMKCFMEKEKMEQLHKKLW




IQAGGDRENIPSIWEAPPSSEPPNNVSTNSEWEAPTQKDKFPKPCNWRK




NEFKKWTKLASQGRGIVNFERDKISNHWIQYYRRIPHRKLLTALQLRAN




VYPTREFLARGRQDQYIKACRHCDADIESCAHIIGNCPVTQDARIKRHN




YICELLLEEAKKKDWVVFKEPHIRDSNKELYKPDLIFVKDARALVVDVT




VRYEAAKSSLEEAAAEKVRKYKHLETEVRHLTNAKDVTFVGFPLGARGK




WHQDNFKLLTELGLSKSRQVKMAETFSTVALFSSVDIVHMFASRARKSM




VM






R2Tg with

MASCPKPGPPVSAGAMSLESGLTTHSVLAIERGPNSLANSGSDFGGGGL

1648


natural linker

GLPLRLLRVSVGTQTSRSDWVDLVSWSHPGPTSKSQQVDLVSLFPKHRV




deletion c-

DLLSKNDQVDLVAQFLPSKFPPNLAENDLALLVNLEFYRSDLHVYECVH




mybA

FAAHWEGLSGLPEVYEQLAPQPCVGETLHSSLPRDSELFVPEEGSSEKE




location-v1

SEDAPKTSPPTPGKHGLEQTGEEKVMVTVPDKNPPCPCCGTRVNSVLNL




replaced with

IEHLKVSHGKRGVCFRCAKCGKENSNYHSVVCHFPKCRGPETEKAPAGE




XTEN linker

WICEVCNRDFTTKIGLGQHKRLAHPAVRNQERIVASQPKETSNRGAHKR






CWTKEEEELLIRLEAQFEGNKNINKLIAEHITTKTAKQISDKRRLLSRK






PAEEPREEPGTCHHTRRAA

SGSETPGTSESATPES
CFGCLESISQIRTA




TRDKKDTVTREKHPKKPFQKWMKDRAIKKGNYLRFQRLFYLDRGKLAKI




ILDDIECLSCDIPLSEIYSVFKTRWETTGSFKSLGDFKTYGKADNTAFR




ELITAKEIEKNVQEMSKGSAPGPDGITLGDVVKMDPEFSRTMEIFNLWL




TTGKIPDMVRGCRTVLIPKSSKPDRLKDINNWRPITIGSILLRLFSRIV




TARLSKACPLNPRQRGFIRAAGCSENLKLLQTIIWSAKREHRPLGVVFV




DIAKAFDTVSHQHIIHALQQREVDPHIVGLVSNMYENISTYITTKRNTH




TDKIQIRVGVKQGDPMSPLLFNLAMDPLLCKLEESGKGYHRGQSSITAM




AFADDLVLLSDSWENMNTNISILETFCNLTGLKTQGQKCHGFYIKPTKD




SYTINDCAAWTINGTPLNMIDPGESEKYLGLQFDPWIGIARSGLSTKLD




FWLQRIDQAPLKPLQKTDILKTYTIPRLIYIADHSEVKTALLETLDQKI




RTAVKEWLHLPPCTCDAILYSSTRDGGLGITKLAGLIPSVQARRLHRIA




QSSDDTMKCFMEKEKMEQLHKKLWIQAGGDRENIPSIWEAPPSSEPPNN




VSTNSEWEAPTQKDKFPKPCNWRKNEFKKWTKLASQGRGIVNFERDKIS




NHWIQYYRRIPHRKLLTALQLRANVYPTREFLARGRQDQYIKACRHCDA




DIESCAHIIGNCPVTQDARIKRHNYICELLLEEAKKKDWVVFKEPHIRD




SNKELYKPDLIFVKDARALVVDVTVRYEAAKSSLEEAAAEKVRKYKHLE




TEVRHLTNAKDVTFVGFPLGARGKWHQDNFKLLTELGLSKSRQVKMAET




FSTVALFSSVDIVHMFASRARKSMVM






R2Tg with

MASCPKPGPPVSAGAMSLESGLTTHSVLAIERGPNSLANSGSDFGGGGL

1649


natural linker

GLPLRLLRVSVGTQTSRSDWVDLVSWSHPGPTSKSQQVDLVSLFPKHRV




deletion c-

DLLSKNDQVDLVAQFLPSKFPPNLAENDLALLVNLEFYRSDLHVYECVH




mybA

FAAHWEGLSGLPEVYEQLAPQPCVGETLHSSLPRDSELFVPEEGSSEKE




location-v2

SEDAPKTSPPTPGKHGLEQTGEEKVMVTVPDKNPPCPCCGTRVNSVLNL




replaced with

IEHLKVSHGKRGVCFRCAKCGKENSNYHSVVCHFPKCRGPETEKAPAGE




XTEN linker

WICEVCNRDFTTKIGLGQHKRLAHPAVRNQERIVASQPKETSNRGAHKR






CWTKEEEELLIRLEAQFEGNKNINKLIAEHITTKTAKQISDKRRLLSRK






PAEEPREEPGTCHHTRRAA

SGSETPGTSESATPES
TATRDKKDTVTREK




HPKKPFQKWMKDRAIKKGNYLRFQRLFYLDRGKLAKIILDDIECLSCDI




PLSEIYSVFKTRWETTGSFKSLGDFKTYGKADNTAFRELITAKEIEKNV




QEMSKGSAPGPDGITLGDVVKMDPEFSRTMEIFNLWLTTGKIPDMVRGC




RTVLIPKSSKPDRLKDINNWRPITIGSILLRLFSRIVTARLSKACPLNP




RQRGFIRAAGCSENLKLLQTIIWSAKREHRPLGVVFVDIAKAFDTVSHQ




HIIHALQQREVDPHIVGLVSNMYENISTYITTKRNTHTDKIQIRVGVKQ




GDPMSPLLFNLAMDPLLCKLEESGKGYHRGQSSITAMAFADDLVLLSDS




WENMNTNISILETFCNLTGLKTQGQKCHGFYIKPTKDSYTINDCAAWTI




NGTPLNMIDPGESEKYLGLQFDPWIGIARSGLSTKLDFWLQRIDQAPLK




PLQKTDILKTYTIPRLIYIADHSEVKTALLETLDQKIRTAVKEWLHLPP




CTCDAILYSSTRDGGLGITKLAGLIPSVQARRLHRIAQSSDDTMKCFME




KEKMEQLHKKLWIQAGGDRENIPSIWEAPPSSEPPNNVSTNSEWEAPTQ




KDKFPKPCNWRKNEFKKWTKLASQGRGIVNFERDKISNHWIQYYRRIPH




RKLLTALQLRANVYPTREFLARGRQDQYIKACRHCDADIESCAHIIGNC




PVTQDARIKRHNYICELLLEEAKKKDWVVFKEPHIRDSNKELYKPDLIF




VKDARALVVDVTVRYEAAKSSLEEAAAEKVRKYKHLETEVRHLTNAKDV




TFVGFPLGARGKWHQDNFKLLTELGLSKSRQVKMAETFSTVALFSSVDI




VHMFASRARKSMVM






R2Tg with

MASCPKPGPPVSAGAMSLESGLTTHSVLAIERGPNSLANSGSDFGGGGL

1650


natural linker

GLPLRLLRVSVGTQTSRSDWVDLVSWSHPGPTSKSQQVDLVSLFPKHRV




deletion c-

DLLSKNDQVDLVAQFLPSKFPPNLAENDLALLVNLEFYRSDLHVYECVH




mybB

FAAHWEGLSGLPEVYEQLAPQPCVGETLHSSLPRDSELFVPEEGSSEKE




location-v1

SEDAPKTSPPTPGKHGLEQTGEEKVMVTVPDKNPPCPCCGTRVNSVLNL




replaced with

IEHLKVSHGKRGVCFRCAKCGKENSNYHSVVCHFPKCRGPETEKAPAGE




3GS linker

WICEVCNRDFTTKIGLGQHKRLAHPAVRNQERIVASQPKETSNRGAHKR




(SEQ ID NO:

CWTKEEEELLIRLEAQFEGNKNINKLIAEHITTKTAKQISDKRRL

GGGS





1024)
CFGCLESISQIRTATRDKKDTVTREKHPKKPFQKWMKDRAIKKGNYLRF




QRLFYLDRGKLAKIILDDIECLSCDIPLSEIYSVFKTRWETTGSFKSLG




DFKTYGKADNTAFRELITAKEIEKNVQEMSKGSAPGPDGITLGDVVKMD




PEFSRTMEIFNLWLTTGKIPDMVRGCRTVLIPKSSKPDRLKDINNWRPI




TIGSILLRLFSRIVTARLSKACPLNPRQRGFIRAAGCSENLKLLQTIIW




SAKREHRPLGVVFVDIAKAFDTVSHQHIIHALQQREVDPHIVGLVSNMY




ENISTYITTKRNTHTDKIQIRVGVKQGDPMSPLLFNLAMDPLLCKLEES




GKGYHRGQSSITAMAFADDLVLLSDSWENMNTNISILETFCNLTGLKTQ




GQKCHGFYIKPTKDSYTINDCAAWTINGTPLNMIDPGESEKYLGLQFDP




WIGIARSGLSTKLDFWLQRIDQAPLKPLQKTDILKTYTIPRLIYIADHS




EVKTALLETLDQKIRTAVKEWLHLPPCTCDAILYSSTRDGGLGITKLAG




LIPSVQARRLHRIAQSSDDTMKCFMEKEKMEQLHKKLWIQAGGDRENIP




SIWEAPPSSEPPNNVSTNSEWEAPTQKDKFPKPCNWRKNEFKKWTKLAS




QGRGIVNFERDKISNHWIQYYRRIPHRKLLTALQLRANVYPTREFLARG




RQDQYIKACRHCDADIESCAHIIGNCPVTQDARIKRHNYICELLLEEAK




KKDWVVFKEPHIRDSNKELYKPDLIFVKDARALVVDVTVRYEAAKSSLE




EAAAEKVRKYKHLETEVRHLTNAKDVTFVGFPLGARGKWHQDNFKLLTE




LGLSKSRQVKMAETFSTVALFSSVDIVHMFASRARKSMVM






R2Tg with

MASCPKPGPPVSAGAMSLESGLTTHSVLAIERGPNSLANSGSDFGGGGL

1651


natural linker

GLPLRLLRVSVGTQTSRSDWVDLVSWSHPGPTSKSQQVDLVSLFPKHRV




deletion c-

DLLSKNDQVDLVAQFLPSKFPPNLAENDLALLVNLEFYRSDLHVYECVH




mybB

FAAHWEGLSGLPEVYEQLAPQPCVGETLHSSLPRDSELFVPEEGSSEKE




location-v2

SEDAPKTSPPTPGKHGLEQTGEEKVMVTVPDKNPPCPCCGTRVNSVLNL




replaced with

IEHLKVSHGKRGVCFRCAKCGKENSNYHSVVCHFPKCRGPETEKAPAGE




3GS linker

WICEVCNRDFTTKIGLGQHKRLAHPAVRNQERIVASQPKETSNRGAHKR




(SEQ ID NO:

CWTKEEEELLIRLEAQFEGNKNINKLIAEHITTKTAKQISDKRRL

GGGS





1024)
TATRDKKDTVTREKHPKKPFQKWMKDRAIKKGNYLRFQRLFYLDRGKLA




KIILDDIECLSCDIPLSEIYSVFKTRWETTGSFKSLGDFKTYGKADNTA




FRELITAKEIEKNVQEMSKGSAPGPDGITLGDVVKMDPEFSRTMEIFNL




WLTTGKIPDMVRGCRTVLIPKSSKPDRLKDINNWRPITIGSILLRLFSR




IVTARLSKACPLNPRQRGFIRAAGCSENLKLLQTIIWSAKREHRPLGVV




FVDIAKAFDTVSHQHIIHALQQREVDPHIVGLVSNMYENISTYITTKRN




THTDKIQIRVGVKQGDPMSPLLFNLAMDPLLCKLEESGKGYHRGQSSIT




AMAFADDLVLLSDSWENMNTNISILETFCNLTGLKTQGQKCHGFYIKPT




KDSYTINDCAAWTINGTPLNMIDPGESEKYLGLQFDPWIGIARSGLSTK




LDFWLQRIDQAPLKPLQKTDILKTYTIPRLIYIADHSEVKTALLETLDQ




KIRTAVKEWLHLPPCTCDAILYSSTRDGGLGITKLAGLIPSVQARRLHR




IAQSSDDTMKCFMEKEKMEQLHKKLWIQAGGDRENIPSIWEAPPSSEPP




NNVSTNSEWEAPTQKDKFPKPCNWRKNEFKKWTKLASQGRGIVNFERDK




ISNHWIQYYRRIPHRKLLTALQLRANVYPTREFLARGRQDQYIKACRHC




DADIESCAHIIGNCPVTQDARIKRHNYICELLLEEAKKKDWVVFKEPHI




RDSNKELYKPDLIFVKDARALVVDVTVRYEAAKSSLEEAAAEKVRKYKH




LETEVRHLTNAKDVTFVGFPLGARGKWHQDNFKLLTELGLSKSRQVKMA




ETFSTVALFSSVDIVHMFASRARKSMVM






R2Tg with

MASCPKPGPPVSAGAMSLESGLTTHSVLAIERGPNSLANSGSDFGGGGL

1652


natural linker

GLPLRLLRVSVGTQTSRSDWVDLVSWSHPGPTSKSQQVDLVSLFPKHRV




deletion c-

DLLSKNDQVDLVAQFLPSKFPPNLAENDLALLVNLEFYRSDLHVYECVH




mybB

FAAHWEGLSGLPEVYEQLAPQPCVGETLHSSLPRDSELFVPEEGSSEKE




location-v1

SEDAPKTSPPTPGKHGLEQTGEEKVMVTVPDKNPPCPCCGTRVNSVLNL




replaced with

IEHLKVSHGKRGVCFRCAKCGKENSNYHSVVCHFPKCRGPETEKAPAGE




XTEN linker

WICEVCNRDFTTKIGLGQHKRLAHPAVRNQERIVASQPKETSNRGAHKR






CWTKEEEELLIRLEAQFEGNKNINKLIAEHITTKTAKQISDKRRL

SGSE








TPGTSESATPES
CFGCLESISQIRTATRDKKDTVTREKHPKKPFQKWMK




DRAIKKGNYLRFQRLFYLDRGKLAKIILDDIECLSCDIPLSEIYSVFKT




RWETTGSFKSLGDFKTYGKADNTAFRELITAKEIEKNVQEMSKGSAPGP




DGITLGDVVKMDPEFSRTMEIFNLWLTTGKIPDMVRGCRTVLIPKSSKP




DRLKDINNWRPITIGSILLRLFSRIVTARLSKACPLNPRQRGFIRAAGC




SENLKLLQTIIWSAKREHRPLGVVFVDIAKAFDTVSHQHIIHALQQREV




DPHIVGLVSNMYENISTYITTKRNTHTDKIQIRVGVKQGDPMSPLLFNL




AMDPLLCKLEESGKGYHRGQSSITAMAFADDLVLLSDSWENMNTNISIL




ETFCNLTGLKTQGQKCHGFYIKPTKDSYTINDCAAWTINGTPLNMIDPG




ESEKYLGLQFDPWIGIARSGLSTKLDFWLQRIDQAPLKPLQKTDILKTY




TIPRLIYIADHSEVKTALLETLDQKIRTAVKEWLHLPPCTCDAILYSST




RDGGLGITKLAGLIPSVQARRLHRIAQSSDDTMKCFMEKEKMEQLHKKL




WIQAGGDRENIPSIWEAPPSSEPPNNVSTNSEWEAPTQKDKFPKPCNWR




KNEFKKWTKLASQGRGIVNFERDKISNHWIQYYRRIPHRKLLTALQLRA




NVYPTREFLARGRQDQYIKACRHCDADIESCAHIIGNCPVTQDARIKRH




NYICELLLEEAKKKDWVVFKEPHIRDSNKELYKPDLIFVKDARALVVDV




TVRYEAAKSSLEEAAAEKVRKYKHLETEVRHLTNAKDVTFVGFPLGARG




KWHQDNFKLLTELGLSKSRQVKMAETFSTVALFSSVDIVHMFASRARKS




MVM






R2Tg with

MASCPKPGPPVSAGAMSLESGLTTHSVLAIERGPNSLANSGSDFGGGGL

1653


natural linker

GLPLRLLRVSVGTQTSRSDWVDLVSWSHPGPTSKSQQVDLVSLFPKHRV




deletion c-

DLLSKNDQVDLVAQFLPSKFPPNLAENDLALLVNLEFYRSDLHVYECVH




mybB

FAAHWEGLSGLPEVYEQLAPQPCVGETLHSSLPRDSELFVPEEGSSEKE




location-v2

SEDAPKTSPPTPGKHGLEQTGEEKVMVTVPDKNPPCPCCGTRVNSVLNL




replaced with

IEHLKVSHGKRGVCFRCAKCGKENSNYHSVVCHFPKCRGPETEKAPAGE




XTEN linker

WICEVCNRDFTTKIGLGQHKRLAHPAVRNQERIVASQPKETSNRGAHKR






CWTKEEEELLIRLEAQFEGNKNINKLIAEHITTKTAKQISDKRRL

SGSE








TPGTSESATPES
TATRDKKDTVTREKHPKKPFQKWMKDRAIKKGNYLRF




QRLFYLDRGKLAKIILDDIECLSCDIPLSEIYSVFKTRWETTGSFKSLG




DFKTYGKADNTAFRELITAKEIEKNVQEMSKGSAPGPDGITLGDVVKMD




PEFSRTMEIFNLWLTTGKIPDMVRGCRTVLIPKSSKPDRLKDINNWRPI




TIGSILLRLFSRIVTARLSKACPLNPRQRGFIRAAGCSENLKLLQTIIW




SAKREHRPLGVVFVDIAKAFDTVSHQHIIHALQQREVDPHIVGLVSNMY




ENISTYITTKRNTHTDKIQIRVGVKQGDPMSPLLFNLAMDPLLCKLEES




GKGYHRGQSSITAMAFADDLVLLSDSWENMNTNISILETFCNLTGLKTQ




GQKCHGFYIKPTKDSYTINDCAAWTINGTPLNMIDPGESEKYLGLQFDP




WIGIARSGLSTKLDFWLQRIDQAPLKPLQKTDILKTYTIPRLIYIADHS




EVKTALLETLDQKIRTAVKEWLHLPPCTCDAILYSSTRDGGLGITKLAG




LIPSVQARRLHRIAQSSDDTMKCFMEKEKMEQLHKKLWIQAGGDRENIP




SIWEAPPSSEPPNNVSTNSEWEAPTQKDKFPKPCNWRKNEFKKWTKLAS




QGRGIVNFERDKISNHWIQYYRRIPHRKLLTALQLRANVYPTREFLARG




RQDQYIKACRHCDADIESCAHIIGNCPVTQDARIKRHNYICELLLEEAK




KKDWVVFKEPHIRDSNKELYKPDLIFVKDARALVVDVTVRYEAAKSSLE




EAAAEKVRKYKHLETEVRHLTNAKDVTFVGFPLGARGKWHQDNFKLLTE




LGLSKSRQVKMAETFSTVALFSSVDIVHMFASRARKSMVM









HEK293T cells were plated in 96-well plates and grown overnight at 37° C., 5% CO2. The HEK293T cells were transfected with plasmids that expressed R2Tg (wild-type), R2 endonuclease mutant, and natural linker mutants. The transfection was carried out using the Fugene HID transfection reagent according to the manufacturer recommendations, where each well received 80 ng of plasmid DNA and 0.5 pL of transfection reagent. All transfections were performed in duplicate and the cells were incubated for 72 h prior to genomic DNA extraction.


Activity of the mutants was measured by a ddPCR assay that quantified the copy number of R2Tg integrations by measuring the number of 3′ junction amplicons (FIG. 8b).


Deletions that begin after the random coil following the c-myb DNA binding motif (location A, c-mybA) are well-tolerated with integration activity near that of wild-type R2Tg. The natural linker region deletion end point is nearly the same for either location vi (N-terminal to the alpha helix preceding the −1 RNA binding motif) or v2 (C-terminal to the alpha helix preceding the −1 RNA binding motif). For the deletion beginning at location A and ending at location v1 or v2, replacement of this polypeptide stretch with the XTEN linker (SEQ ID NO: 1023) seems to retain the most amount of activity whereas replacement with the 3GS linker (SEQ ID NO: 1024) has approximately a 50% reduction in integration activity. For natural linker deletions that begin at location B (c-mybB), these configurations show a more marked reduction in integration activity when compared to wild-type or location A (c-mybA). The difference in activity may be related to the structure of the protein based on the position of the deletion that creates a non-optimal three dimensional structure of the retrotransposase through the location of the linker, length of the linker, or amino acid combination of the linker that is not optimal to connect location B to locations v1 or v2. Even though the N-terminal natural linker deletion start location mybB is a sub-optimal, the C-terminal end of the deletion was most tolerated at v2 with either the 3GS (SEQ ID NO: 1024) or XTEN linker and appears to be the preferential location for having a polypeptide preceding the RBD −1 region.


Example 8: Determination of Target Specificity of a Gene Writer Endonuclease Domain

This example describes using a custom genomic landing pad in human cells to determine whether there is a sequence requirement for target cleavage and subsequent integration by a Gene Writer system.


In this example, cell lines were created to have “landing pads” or stable integrations that mimic a region of rDNA that contain the R2 position to which R2 retrotransposases target for retrotransposition (see FIG. 9). The integrants or landing pads were designed to either have the wild-type region sequence in and around the R2 site found in rDNA, 12-bp of sequence mutation at and around the R2 cleavage site, or 75-bp of sequence mutation at and around the R2 cleavage site (Table E2). The DNA for these different landing pads was chemically synthesized and cloned into the pLenti-N-tGFP vector. The cloned landing pads into the lentiviral expression vector were confirmed and sequence verified by Sanger sequencing of the landing pad. The sequence verified plasmids (9 μg) along with the lentiviral packaging mix (9 μg, obtained from Biosettia) were transfected using Lipofectamine2000™ according the manufacturer instructions into a packaging cell line, LentiX-293T (Takara Bio). The transfected cells were incubated at 37° C., 10% CO2 for 48 hours (including one medium change at 24 hrs) and the viral particle containing medium was collected from the cell culture dish. The collected medium was filtered through a 0.2 μm filter to remove cell debris and prepared for transduction of U2OS cells. The viral containing medium was diluted in DMEM and mixed with polybrene to prepare a dilution series for transduction of U2OS cells where the final concentration of polybrene was 8 μg/ml. The U2OS cells were grown in viral containing medium for 48 hour and then split with fresh medium. The split cells were grown to confluence and transduction efficiency of the different dilutions of virus were measured by GFP expression via flow cytometry and ddPCR detection of the genomic integrated lentivirus that contained GFP and the different rDNA landing pads (WT, 12-bp mutation, or 75-bp mutation). The GFP positive cell line from the 1:10 viral medium dilution (>99% GFP+) was chosen for subsequent experiments and cryopreserved.


To test if mutations in and around the R2 cleavage position can impact the Gene Writer system activity, the R2Tg Gene Writer Driver along with a plasmid that expressed a Gene Writer transgene molecule were electroporated into the different landing pad cell lines. In order to test if the sequence in and around the cleavage site impacted the Gene Writer polypeptide sequence activity to integrate, the homology arms for the Gene Writer template molecule were designed to have 100% homology 100 bp to the left (Gene Writer molecule module A) and 100 bp to the right (Gene Writer molecule module F) of the cleavage position for each of the landing pads. The changes to the homology arms of the Gene Writer template molecule expression plasmid were introduced by PCR and were confirmed by Sanger Sequencing. Either 73 ng of the WT R2Tg Gene Writer Driver or the Endonuclease domain mutant R2Tg Gene Writer Driver expression plasmids were co-nucleofected) using nucleofection program DN100 into each of the different U2OS landing pad cell lines (WT, 12-bp mutant, or 75-bp mutant) with 177 ng of plasmids that expressed the Gene Writer template molecules that had 100% homology to either the WT landing pad, 12-bp mutant landing pad, or 75-bp mutant landing pad. After nucleofection, cells were grown at 37° C., 10% CO2 for 3 days prior to cell lysis and genomic DNA extraction. The extracted gDNA was measured for Gene Writer template molecule integration at the landing pad site by ddPCR. The DNA nicking activity was measured by detection of insertions, deletions, and/or a combination of both insertions and deletions at the landing pad through next-generation sequence analysis of an amplicon that was generated from the landing pad found in the gDNA.


The integration activity of the R2Tg Gene Writer is greatly reduced when the cleavage region is mutated where there is no integration of a Gene Writer template molecule in either of the 12-bp or 75-bp landing pad cell lines (FIG. 10a). Furthermore, integration is not detected with Gene Writer template molecules that have homology arms that correspond to either the 12-bp or 75-bp mutant landing pads. To rule out that the lost integration activity is due to incompatible homology arms, DNA nicking activity was measured by NGS analysis of the landing pad. The nicking activity is independent of the Gene Writer template molecule as the WT R2Tg Gene Writer driver had comparable indels at the WT landing pad with the WT, 12-bp mutant, or 75-bp mutant Gene Writer template molecule (FIG. 10b). The 12-bp and 75-bp landing pads, regardless of Gene Writer template molecule co-nucleofected with the WT R2Tg Gene Writer did not show any reads above background that contained indels. The level of indels was similar to the Gene Writer template driver containing endonuclease mutations.









TABLE E2







Exemplary Landing Pads








Landing



Pad
Sequence 5′-> 3′


Sequence
(rDNA, underline; cleavage region, bold;


Name
mutated sequence, bold-italic





WT
GCTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTGTTGACG




CGATGTGATTTCTGCCCAGTGCTCTGAATGTCAAAGTGAAGAAATTCA





ATGAAGCGCGGGTAAACGGCGGGAGTAACTATGACTCTCTTAAGGTAG






CCAAATGCCTCGTCATCTAATTAGTGACGCGCATGAATGGATGAACGA





GATTCCCACTGTCCCTACCTACTATCCAGCGAAACCACAGCCAAGGGA





AATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGC



GTTACCCAACTTAATCGCCTTGCAGCACATCC (SEQ ID NO:



1654)





12-bp
GCTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTGTTGACG


Mutant

CGATGTGATTTCTGCCCAGTGCTCTGAATGTCAAAGTGAAGAAATTCA





ATGAAGCGCGGGTAAACGGCGGGAGTAACTATGACTCTCTTTCCAATA







TGATT
TGCCTCGTCATCTAATTAGTGACGCGCATGAATGGATGAACGA





GATTCCCACTGTCCCTACCTACTATCCAGCGAAACCACAGCCAAGGGA





AATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGC



GTTACCCAACTTAATCGCCTTGCAGCACATCC (SEQ ID NO:



1655)





75-bp
getcacacaggaaacagctatgaccatgattacgccaagctgttgacg


Mutant

cgatgtgatttctgcccagtgctctgaatgtcaaagtgaagaaattca





atgaagcgcgggtaaacggcgggagtaactatgactctctttccaata







tgattccacccatggcaaattccatggcaccgtcaaggctgagaacgg









gaagcttgtcatcaatggaa
actatccagcgaaaccacagccaaggga





aattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggc



gttacccaacttaatcgccttgcagcacatcc (SEQ ID NO:



1656)









In some embodiments, a Gene Writer is derived from a retrotransposase with some level of target sequence specificity in the endonuclease domain. Thus, it may be desirable to retarget the Gene Writer to a location in the genome that possesses homology to the natural target sequence recognized by an endonuclease domain, referred to as the endonuclease recognition motif (ERM). In some embodiments, this sub-target sequence may be contained in the region surrounding the nick site. In specific embodiments, a 13 nt sequence (TAAGGTAGCCAAA (SEQ TD NO: 1657)) based on the nick site of an R2 element, e.g., R2Tg, is used to search the human genome for suitable locations for retargeting the Gene Writer, wherein a heterologous DNA-binding domain is designed to localize the Gene Writer to an endogenous ERM to direct endonuclease activity and subsequent retrotransposition of a template RNA. In some embodiments, the human genome site possesses 100% identity to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides in the 13 nt motif. In further embodiments, the human genome site containing the ERM is selected from Table E3, and a DNA-binding domain fusion, e.g., ZF, TAL, or dCas9 with a custom gRNA, is designed to localize the polypeptide to the site (e.g., see Example 9). In preferred embodiments, the genome site possesses a safe harbor score of at least 5, 6, 7, 8 as defined in Pellenz et al Hum Gene Ther 30, 814-282 (2019) and shown in Table E3.


In some embodiments, the template RNA (or DNA encoding the template RNA) is designed such that the homology arms match the flanking genomic sequences surrounding the expected nick site at the new target.


Table E3: Human Genome Sites Matching a 13 nt Stretch Around the Nicking Site of R2 Elements.


The human genome was searched for 100% identity to the full 13 nt match or 12 consecutive nucleotides (“Match”). Chromosomal location and start and end coordinates are provided for each match. Score (“Score”) is a metric evaluating each site for eight desirable safe harbor characteristics.

















Chromosome
Start
End
Source
Match
Score




















chr06
123749082
123749094
NC_000006.12
13
8


chr02
5035294
5035305
NC_000002.12
12
8


chr02
145760352
145760341
NC_000002.12
12
8


chr02
147034635
147034624
NC_000002.12
12
8


chr02
181792104
181792115
NC_000002.12
12
8


chr03
34017171
34017182
NC_000003.12
12
8


chr03
74784684
74784695
NC_000003.12
12
8


chr03
110093351
110093362
NC_000003.12
12
8


chr06
14459104
14459093
NC_000006.12
12
8


chr06
119620936
119620947
NC_000006.12
12
8


chr06
145123473
145123462
NC_000006.12
12
8


chr07
12024654
12024665
NC_000007.14
12
8


chr07
52001436
52001447
NC_000007.14
12
8


chr07
115339421
115339410
NC_000007.14
12
8


chr08
126384299
126384310
NC_000008.11
12
8


chr12
84083562
84083573
NC_000012.12
12
8


chrX
117646432
117646421
NC_000023.11
12
8


chr02
106547509
106547521
NC_000002.12
13
7


chr02
226038592
226038604
NC_000002.12
13
7


chr03
102522532
102522520
NC_000003.12
13
7


chr03
110933592
110933604
NC_000003.12
13
7


chr03
119752575
119752563
NC_000003.12
13
7


chr03
172868603
172868615
NC_000003.12
13
7


chr03
191985222
191985210
NC_000003.12
13
7


chr05
6213503
6213515
NC_000005.10
13
7


chr05
58295578
58295566
NC_000005.10
13
7


chr05
129844500
129844512
NC_000005.10
13
7


chr06
1454372
1454360
NC_000006.12
13
7


chr06
48973921
48973909
NC_000006.12
13
7


chr08
18663054
18663066
NC_000008.11
13
7


chr08
93499020
93499032
NC_000008.11
13
7


chr08
119753973
119753985
NC_000008.11
13
7


chr09
86856907
86856919
NC_000009.12
13
7


chr12
29955571
29955583
NC_000012.12
13
7


chr12
118529104
118529092
NC_000012.12
13
7


chr13
65656029
65656041
NC_000013.11
13
7


chr22
34266611
34266623
NC_000022.11
13
7


chrX
26651640
26651628
NC_000023.11
13
7


chrX
119194351
119194363
NC_000023.11
13
7


chrX
139180620
139180608
NC_000023.11
13
7


chr01
106465846
106465857
NC_000001.11
12
7


chr02
964160
964171
NC_000002.12
12
7


chr02
40018947
40018936
NC_000002.12
12
7


chr02
62845403
62845392
NC_000002.12
12
7


chr02
64834920
64834909
NC_000002.12
12
7


chr02
67969608
67969619
NC_000002.12
12
7


chr02
76183118
76183129
NC_000002.12
12
7


chr02
81819286
81819297
NC_000002.12
12
7


chr02
119597238
119597249
NC_000002.12
12
7


chr02
122897376
122897365
NC_000002.12
12
7


chr02
123603423
123603412
NC_000002.12
12
7


chr02
144644206
144644217
NC_000002.12
12
7


chr02
145221757
145221746
NC_000002.12
12
7


chr02
158367531
158367520
NC_000002.12
12
7


chr02
160092083
160092072
NC_000002.12
12
7


chr02
192245037
192245048
NC_000002.12
12
7


chr02
195223552
195223563
NC_000002.12
12
7


chr02
200351999
200351988
NC_000002.12
12
7


chr02
237068525
237068514
NC_000002.12
12
7


chr03
18724351
18724340
NC_000003.12
12
7


chr03
23969399
23969388
NC_000003.12
12
7


chr03
25177339
25177350
NC_000003.12
12
7


chr03
34880863
34880852
NC_000003.12
12
7


chr03
66233879
66233890
NC_000003.12
12
7


chr03
74527939
74527950
NC_000003.12
12
7


chr03
98583025
98583014
NC_000003.12
12
7


chr03
99278452
99278463
NC_000003.12
12
7


chr03
116060228
116060239
NC_000003.12
12
7


chr03
139468578
139468589
NC_000003.12
12
7


chr03
140064054
140064043
NC_000003.12
12
7


chr03
140438138
140438127
NC_000003.12
12
7


chr03
152457330
152457341
NC_000003.12
12
7


chr03
160950736
160950725
NC_000003.12
12
7


chr03
167207758
167207769
NC_000003.12
12
7


chr03
167722472
167722483
NC_000003.12
12
7


chr03
180475661
180475672
NC_000003.12
12
7


chr04
121590786
121590775
NC_000004.12
12
7


chr04
133719599
133719588
NC_000004.12
12
7


chr05
11564132
11564121
NC_000005.10
12
7


chr05
11970221
11970210
NC_000005.10
12
7


chr05
32814431
32814420
NC_000005.10
12
7


chr05
38003029
38003018
NC_000005.10
12
7


chr05
39758118
39758129
NC_000005.10
12
7


chr05
41221615
41221604
NC_000005.10
12
7


chr05
74838717
74838728
NC_000005.10
12
7


chr05
86444529
86444518
NC_000005.10
12
7


chr05
86617117
86617106
NC_000005.10
12
7


chr05
89438360
89438349
NC_000005.10
12
7


chr05
108102395
108102406
NC_000005.10
12
7


chr05
110231750
110231761
NC_000005.10
12
7


chr05
113996496
113996485
NC_000005.10
12
7


chr05
117233050
117233039
NC_000005.10
12
7


chr05
121622921
121622932
NC_000005.10
12
7


chr05
122520704
122520693
NC_000005.10
12
7


chr05
142330490
142330479
NC_000005.10
12
7


chr05
156359105
156359094
NC_000005.10
12
7


chr06
19187842
19187831
NC_000006.12
12
7


chr06
41103469
41103458
NC_000006.12
12
7


chr06
49856872
49856883
NC_000006.12
12
7


chr06
54896309
54896298
NC_000006.12
12
7


chr06
64416107
64416096
NC_000006.12
12
7


chr06
104438997
104439008
NC_000006.12
12
7


chr06
109349688
109349677
NC_000006.12
12
7


chr06
110631149
110631160
NC_000006.12
12
7


chr06
114751383
114751372
NC_000006.12
12
7


chr06
116514339
116514350
NC_000006.12
12
7


chr06
121606126
121606137
NC_000006.12
12
7


chr06
126139788
126139777
NC_000006.12
12
7


chr06
130852449
130852460
NC_000006.12
12
7


chr06
136843057
136843068
NC_000006.12
12
7


chr06
155559692
155559681
NC_000006.12
12
7


chr06
158099232
158099221
NC_000006.12
12
7


chr07
24339208
24339219
NC_000007.14
12
7


chr07
39519736
39519725
NC_000007.14
12
7


chr07
72228733
72228722
NC_000007.14
12
7


chr07
82955239
82955228
NC_000007.14
12
7


chr07
104990180
104990191
NC_000007.14
12
7


chr07
107698186
107698197
NC_000007.14
12
7


chr07
111002449
111002438
NC_000007.14
12
7


chr07
115191852
115191841
NC_000007.14
12
7


chr07
115572755
115572766
NC_000007.14
12
7


chr08
21126162
21126151
NC_000008.11
12
7


chr08
25928055
25928066
NC_000008.11
12
7


chr08
45036535
45036524
NC_000008.11
12
7


chr08
45248484
45248473
NC_000008.11
12
7


chr08
45502096
45502085
NC_000008.11
12
7


chr08
45763984
45763973
NC_000008.11
12
7


chr08
53335054
53335043
NC_000008.11
12
7


chr08
55581238
55581227
NC_000008.11
12
7


chr08
63169546
63169557
NC_000008.11
12
7


chr08
66553887
66553898
NC_000008.11
12
7


chr08
86283378
86283367
NC_000008.11
12
7


chr08
113060704
113060715
NC_000008.11
12
7


chr08
114537195
114537206
NC_000008.11
12
7


chr08
114886082
114886071
NC_000008.11
12
7


chr08
127206415
127206426
NC_000008.11
12
7


chr08
133590421
133590410
NC_000008.11
12
7


chr08
135425161
135425150
NC_000008.11
12
7


chr10
7034863
7034852
NC_000010.11
12
7


chr10
124304797
124304786
NC_000010.11
12
7


chr10
131502495
131502506
NC_000010.11
12
7


chr11
5557203
5557192
NC_000011.10
12
7


chr11
31242576
31242565
NC_000011.10
12
7


chr11
31419537
31419526
NC_000011.10
12
7


chr11
33169689
33169678
NC_000011.10
12
7


chr11
55948947
55948958
NC_000011.10
12
7


chr11
85654901
85654890
NC_000011.10
12
7


chr11
92588724
92588735
NC_000011.10
12
7


chr11
105227927
105227938
NC_000011.10
12
7


chr11
106302916
106302927
NC_000011.10
12
7


chr11
110594096
110594085
NC_000011.10
12
7


chr11
125228337
125228326
NC_000011.10
12
7


chr12
8769205
8769194
NC_000012.12
12
7


chr12
30438984
30438973
NC_000012.12
12
7


chr12
33556205
33556216
NC_000012.12
12
7


chr12
39419629
39419640
NC_000012.12
12
7


chr12
88509246
88509257
NC_000012.12
12
7


chr12
92878719
92878708
NC_000012.12
12
7


chr12
107034133
107034144
NC_000012.12
12
7


chr12
109455187
109455176
NC_000012.12
12
7


chr13
88827671
88827660
NC_000013.11
12
7


chr14
48126959
48126948
NC_000014.9
12
7


chr20
851155
851166
NC_000020.11
12
7


chr22
17367227
17367238
NC_000022.11
12
7


chrX
20420571
20420582
NC_000023.11
12
7


chrX
22545830
22545819
NC_000023.11
12
7


chrX
28899719
28899708
NC_000023.11
12
7


chrX
33013952
33013963
NC_000023.11
12
7


chrX
103880419
103880408
NC_000023.11
12
7


chrX
105612448
105612459
NC_000023.11
12
7


chrX
107929443
107929454
NC_000023.11
12
7


chrX
112571297
112571286
NC_000023.11
12
7


chrX
127584278
127584267
NC_000023.11
12
7


chrX
130836800
130836811
NC_000023.11
12
7


chrX
140554200
140554211
NC_000023.11
12
7


chrX
146697583
146697572
NC_000023.11
12
7


chr01
97461481
97461469
NC_000001.11
13
6


chr01
104600535
104600547
NC_000001.11
13
6


chr02
12589473
12589461
NC_000002.12
13
6


chr02
187173643
187173631
NC_000002.12
13
6


chr03
29181713
29181701
NC_000003.12
13
6


chr04
32171549
32171537
NC_000004.12
13
6


chr04
116646904
116646916
NC_000004.12
13
6


chr04
164205821
164205833
NC_000004.12
13
6


chr04
170244792
170244780
NC_000004.12
13
6


chr05
15818439
15818451
NC_000005.10
13
6


chr05
174059501
174059489
NC_000005.10
13
6


chr06
94936128
94936140
NC_000006.12
13
6


chr06
98142018
98142006
NC_000006.12
13
6


chr06
151814731
151814719
NC_000006.12
13
6


chr07
14490189
14490201
NC_000007.14
13
6


chr07
53075165
53075153
NC_000007.14
13
6


chr07
87815318
87815306
NC_000007.14
13
6


chr07
103485572
103485584
NC_000007.14
13
6


chr08
35951572
35951560
NC_000008.11
13
6


chr08
39327231
39327219
NC_000008.11
13
6


chr08
69690270
69690258
NC_000008.11
13
6


chr08
117816166
117816154
NC_000008.11
13
6


chr08
123134654
123134666
NC_000008.11
13
6


chr09
68817454
68817466
NC_000009.12
13
6


chr09
68894040
68894028
NC_000009.12
13
6


chr09
80470190
80470178
NC_000009.12
13
6


chr10
1642234
1642222
NC_000010.11
13
6


chr10
73077072
73077060
NC_000010.11
13
6


chr10
110134589
110134601
NC_000010.11
13
6


chr11
9150979
9150991
NC_000011.10
13
6


chr11
9153635
9153623
NC_000011.10
13
6


chr11
13413693
13413705
NC_000011.10
13
6


chr11
41773900
41773912
NC_000011.10
13
6


chr11
77886545
77886557
NC_000011.10
13
6


chr11
79988166
79988154
NC_000011.10
13
6


chr11
108008162
108008150
NC_000011.10
13
6


chr12
30847723
30847735
NC_000012.12
13
6


chr12
86693014
86693026
NC_000012.12
13
6


chr12
122128926
122128914
NC_000012.12
13
6


chr13
29622714
29622726
NC_000013.11
13
6


chr14
39336522
39336510
NC_000014.9
13
6


chr15
94819443
94819431
NC_000015.10
13
6


chr17
10951262
10951250
NC_000017.11
13
6


chr19
30854506
30854518
NC_000019.10
13
6


chr20
42688485
42688473
NC_000020.11
13
6


chrX
38138789
38138801
NC_000023.11
13
6


chrX
86361231
86361243
NC_000023.11
13
6


chrX
107051786
107051798
NC_000023.11
13
6


chrX
109054235
109054247
NC_000023.11
13
6


chr01
32830533
32830522
NC_000001.11
12
6


chr01
56714138
56714127
NC_000001.11
12
6


chr01
79950536
79950547
NC_000001.11
12
6


chr01
81600862
81600851
NC_000001.11
12
6


chr01
88351333
88351344
NC_000001.11
12
6


chr01
100720346
100720335
NC_000001.11
12
6


chr01
103153587
103153598
NC_000001.11
12
6


chr01
163679268
163679279
NC_000001.11
12
6


chr01
178138239
178138228
NC_000001.11
12
6


chr01
202443386
202443375
NC_000001.11
12
6


chr01
214381798
214381787
NC_000001.11
12
6


chr01
239483920
239483909
NC_000001.11
12
6


chr02
5995932
5995921
NC_000002.12
12
6


chr02
14869774
14869785
NC_000002.12
12
6


chr02
37466261
37466250
NC_000002.12
12
6


chr02
38845623
38845634
NC_000002.12
12
6


chr02
38849877
38849866
NC_000002.12
12
6


chr02
52660534
52660523
NC_000002.12
12
6


chr02
55372861
55372872
NC_000002.12
12
6


chr02
62005199
62005210
NC_000002.12
12
6


chr02
70287567
70287556
NC_000002.12
12
6


chr02
79359701
79359712
NC_000002.12
12
6


chr02
84655638
84655627
NC_000002.12
12
6


chr02
126324776
126324787
NC_000002.12
12
6


chr02
149537132
149537143
NC_000002.12
12
6


chr02
169529510
169529521
NC_000002.12
12
6


chr02
175817135
175817124
NC_000002.12
12
6


chr02
180079693
180079682
NC_000002.12
12
6


chr02
206324011
206324000
NC_000002.12
12
6


chr02
206814054
206814043
NC_000002.12
12
6


chr02
224807794
224807805
NC_000002.12
12
6


chr02
229238864
229238853
NC_000002.12
12
6


chr02
236280053
236280064
NC_000002.12
12
6


chr03
154343
154354
NC_000003.12
12
6


chr03
8511973
8511984
NC_000003.12
12
6


chr03
16880365
16880376
NC_000003.12
12
6


chr03
18087857
18087846
NC_000003.12
12
6


chr03
47168148
47168137
NC_000003.12
12
6


chr03
47937628
47937617
NC_000003.12
12
6


chr03
48992978
48992989
NC_000003.12
12
6


chr03
82163078
82163067
NC_000003.12
12
6


chr03
103449909
103449898
NC_000003.12
12
6


chr03
120049593
120049604
NC_000003.12
12
6


chr03
143783076
143783065
NC_000003.12
12
6


chr03
149601763
149601752
NC_000003.12
12
6


chr03
167891194
167891183
NC_000003.12
12
6


chr03
181054638
181054627
NC_000003.12
12
6


chr03
191545181
191545170
NC_000003.12
12
6


chr03
197899144
197899155
NC_000003.12
12
6


chr04
668375
668364
NC_000004.12
12
6


chr04
19382020
19382031
NC_000004.12
12
6


chr04
19484541
19484552
NC_000004.12
12
6


chr04
26997338
26997349
NC_000004.12
12
6


chr04
55658608
55658619
NC_000004.12
12
6


chr04
70437852
70437841
NC_000004.12
12
6


chr04
79981798
79981809
NC_000004.12
12
6


chr04
94968197
94968208
NC_000004.12
12
6


chr04
102674459
102674470
NC_000004.12
12
6


chr04
124485434
124485445
NC_000004.12
12
6


chr04
126123159
126123148
NC_000004.12
12
6


chr04
137124764
137124753
NC_000004.12
12
6


chr04
160702860
160702849
NC_000004.12
12
6


chr04
167052375
167052386
NC_000004.12
12
6


chr04
179139043
179139032
NC_000004.12
12
6


chr04
179161408
179161397
NC_000004.12
12
6


chr04
187143772
187143761
NC_000004.12
12
6


chr05
10200709
10200720
NC_000005.10
12
6


chr05
33225853
33225842
NC_000005.10
12
6


chr05
76255175
76255186
NC_000005.10
12
6


chr05
82855245
82855256
NC_000005.10
12
6


chr05
84139572
84139561
NC_000005.10
12
6


chr05
88198462
88198473
NC_000005.10
12
6


chr05
102501084
102501073
NC_000005.10
12
6


chr05
109583817
109583806
NC_000005.10
12
6


chr05
128180682
128180671
NC_000005.10
12
6


chr05
136190403
136190392
NC_000005.10
12
6


chr05
154189555
154189566
NC_000005.10
12
6


chr05
171957271
171957282
NC_000005.10
12
6


chr05
175317578
175317567
NC_000005.10
12
6


chr06
4853151
4853162
NC_000006.12
12
6


chr06
16133021
16133032
NC_000006.12
12
6


chr06
26103447
26103436
NC_000006.12
12
6


chr06
35947570
35947581
NC_000006.12
12
6


chr06
68279419
68279430
NC_000006.12
12
6


chr06
79806546
79806557
NC_000006.12
12
6


chr06
85260407
85260418
NC_000006.12
12
6


chr06
136633874
136633885
NC_000006.12
12
6


chr06
137931054
137931043
NC_000006.12
12
6


chr06
139739984
139739973
NC_000006.12
12
6


chr06
140341418
140341429
NC_000006.12
12
6


chr06
145869806
145869795
NC_000006.12
12
6


chr06
146731539
146731528
NC_000006.12
12
6


chr06
168728425
168728436
NC_000006.12
12
6


chr07
51771646
51771635
NC_000007.14
12
6


chr07
137082304
137082315
NC_000007.14
12
6


chr07
141052267
141052278
NC_000007.14
12
6


chr08
17556548
17556537
NC_000008.11
12
6


chr08
30097319
30097308
NC_000008.11
12
6


chr08
68502659
68502670
NC_000008.11
12
6


chr08
86697209
86697198
NC_000008.11
12
6


chr08
91622182
91622171
NC_000008.11
12
6


chr08
92498179
92498168
NC_000008.11
12
6


chr08
124481608
124481597
NC_000008.11
12
6


chr08
129563081
129563092
NC_000008.11
12
6


chr08
131305462
131305451
NC_000008.11
12
6


chr09
14627274
14627285
NC_000009.12
12
6


chr09
15151836
15151847
NC_000009.12
12
6


chr09
22322306
22322295
NC_000009.12
12
6


chr09
23783142
23783153
NC_000009.12
12
6


chr09
26318093
26318104
NC_000009.12
12
6


chr09
31054959
31054970
NC_000009.12
12
6


chr09
79007585
79007596
NC_000009.12
12
6


chr09
88239264
88239253
NC_000009.12
12
6


chr09
96543680
96543669
NC_000009.12
12
6


chr09
99112802
99112813
NC_000009.12
12
6


chr09
123836553
123836564
NC_000009.12
12
6


chr10
33633573
33633562
NC_000010.11
12
6


chr10
65551995
65551984
NC_000010.11
12
6


chr10
66717930
66717941
NC_000010.11
12
6


chr10
74291798
74291787
NC_000010.11
12
6


chr10
82621770
82621781
NC_000010.11
12
6


chr10
91090519
91090530
NC_000010.11
12
6


chr10
99682921
99682910
NC_000010.11
12
6


chr10
107653284
107653273
NC_000010.11
12
6


chr10
127387876
127387887
NC_000010.11
12
6


chr11
10330421
10330410
NC_000011.10
12
6


chr11
21052051
21052062
NC_000011.10
12
6


chr11
56948810
56948799
NC_000011.10
12
6


chr11
91992913
91992902
NC_000011.10
12
6


chr11
96712150
96712139
NC_000011.10
12
6


chr11
99478699
99478710
NC_000011.10
12
6


chr11
103284503
103284514
NC_000011.10
12
6


chr11
110624774
110624763
NC_000011.10
12
6


chr11
118226686
118226697
NC_000011.10
12
6


chr11
121927186
121927175
NC_000011.10
12
6


chr11
127371998
127372009
NC_000011.10
12
6


chr12
21742376
21742387
NC_000012.12
12
6


chr12
33375091
33375102
NC_000012.12
12
6


chr12
79305333
79305322
NC_000012.12
12
6


chr12
87018030
87018041
NC_000012.12
12
6


chr12
97027085
97027074
NC_000012.12
12
6


chr12
97030674
97030685
NC_000012.12
12
6


chr12
97794786
97794775
NC_000012.12
12
6


chr12
99326334
99326345
NC_000012.12
12
6


chr12
100617295
100617284
NC_000012.12
12
6


chr12
106997614
106997603
NC_000012.12
12
6


chr12
114419769
114419758
NC_000012.12
12
6


chr13
29428703
29428714
NC_000013.11
12
6


chr13
34838980
34838991
NC_000013.11
12
6


chr13
68672648
68672637
NC_000013.11
12
6


chr13
68677576
68677565
NC_000013.11
12
6


chr13
79534292
79534303
NC_000013.11
12
6


chr13
83374368
83374357
NC_000013.11
12
6


chr13
91208120
91208131
NC_000013.11
12
6


chr13
92057240
92057251
NC_000013.11
12
6


chr13
105912154
105912165
NC_000013.11
12
6


chr14
37970959
37970948
NC_000014.9
12
6


chr14
40492006
40491995
NC_000014.9
12
6


chr14
44782915
44782926
NC_000014.9
12
6


chr14
48758306
48758317
NC_000014.9
12
6


chr14
88004548
88004537
NC_000014.9
12
6


chr15
56610753
56610764
NC_000015.10
12
6


chr15
70757589
70757578
NC_000015.10
12
6


chr15
96964230
96964219
NC_000015.10
12
6


chr16
66442829
66442818
NC_000016.10
12
6


chr16
74623964
74623975
NC_000016.10
12
6


chr16
75189302
75189291
NC_000016.10
12
6


chr17
9332911
9332900
NC_000017.11
12
6


chr18
32474384
32474373
NC_000018.10
12
6


chr18
34128952
34128963
NC_000018.10
12
6


chr18
55039826
55039815
NC_000018.10
12
6


chr18
78931519
78931508
NC_000018.10
12
6


chr19
31065225
31065236
NC_000019.10
12
6


chr19
32434028
32434017
NC_000019.10
12
6


chr19
51221292
51221303
NC_000019.10
12
6


chr20
1361969
1361958
NC_000020.11
12
6


chr20
4448895
4448906
NC_000020.11
12
6


chr20
13696489
13696478
NC_000020.11
12
6


chr20
20275384
20275395
NC_000020.11
12
6


chr20
26367536
26367525
NC_000020.11
12
6


chr21
37223237
37223248
NC_000021.9
12
6


chr21
46496495
46496484
NC_000021.9
12
6


chr22
39560335
39560346
NC_000022.11
12
6


chrX
986645
986656
NC_000023.11
12
6


chrX
5921242
5921253
NC_000023.11
12
6


chrX
6765829
6765840
NC_000023.11
12
6


chrX
15504137
15504126
NC_000023.11
12
6


chrX
22546280
22546269
NC_000023.11
12
6


chrX
41199361
41199372
NC_000023.11
12
6


chrX
43885293
43885282
NC_000023.11
12
6


chrX
67874307
67874296
NC_000023.11
12
6


chrX
110216026
110216037
NC_000023.11
12
6


chrX
110566890
110566879
NC_000023.11
12
6


chrX
111357390
111357379
NC_000023.11
12
6


chrX
150589443
150589454
NC_000023.11
12
6


chr01
23207589
23207577
NC_000001.11
13
5


chr01
25897408
25897420
NC_000001.11
13
5


chr01
65491478
65491490
NC_000001.11
13
5


chr01
154831168
154831180
NC_000001.11
13
5


chr02
35254361
35254349
NC_000002.12
13
5


chr02
207969171
207969159
NC_000002.12
13
5


chr03
185371630
185371642
NC_000003.12
13
5


chr04
46469891
46469879
NC_000004.12
13
5


chr04
105058847
105058835
NC_000004.12
13
5


chr04
124730032
124730044
NC_000004.12
13
5


chr04
158619352
158619364
NC_000004.12
13
5


chr06
85949972
85949960
NC_000006.12
13
5


chr06
109604972
109604960
NC_000006.12
13
5


chr10
59089285
59089273
NC_000010.11
13
5


chr10
99263586
99263598
NC_000010.11
13
5


chr11
96315922
96315934
NC_000011.10
13
5


chr15
33186727
33186715
NC_000015.10
13
5


chr15
87091718
87091706
NC_000015.10
13
5


chr16
16972153
16972165
NC_000016.10
13
5


chr16
59986446
59986458
NC_000016.10
13
5


chr18
12587445
12587457
NC_000018.10
13
5


chr18
78691060
78691048
NC_000018.10
13
5


chr19
39627504
39627492
NC_000019.10
13
5


chr19
54674561
54674573
NC_000019.10
13
5


chr20
30512867
30512855
NC_000020.11
13
5


chr20
45173430
45173442
NC_000020.11
13
5


chr21
35062647
35062659
NC_000021.9
13
5


chrX
77412877
77412889
NC_000023.11
13
5


chrX
130349739
130349727
NC_000023.11
13
5


chr01
8663054
8663065
NC_000001.11
12
5


chr01
26335998
26336009
NC_000001.11
12
5


chr01
42582606
42582595
NC_000001.11
12
5


chr01
47032830
47032819
NC_000001.11
12
5


chr01
69196253
69196264
NC_000001.11
12
5


chr01
70300023
70300034
NC_000001.11
12
5


chr01
82771042
82771053
NC_000001.11
12
5


chr01
100102957
100102946
NC_000001.11
12
5


chr01
107996202
107996213
NC_000001.11
12
5


chr01
162211653
162211642
NC_000001.11
12
5


chr01
208646365
208646354
NC_000001.11
12
5


chr01
215734460
215734449
NC_000001.11
12
5


chr01
234143991
234144002
NC_000001.11
12
5


chr01
241045297
241045286
NC_000001.11
12
5


chr02
140780861
140780872
NC_000002.12
12
5


chr02
149162575
149162586
NC_000002.12
12
5


chr02
162692841
162692852
NC_000002.12
12
5


chr02
222738270
222738259
NC_000002.12
12
5


chr03
67248099
67248110
NC_000003.12
12
5


chr03
174292637
174292648
NC_000003.12
12
5


chr04
12331297
12331308
NC_000004.12
12
5


chr04
21504937
21504948
NC_000004.12
12
5


chr04
43962965
43962976
NC_000004.12
12
5


chr04
57433948
57433937
NC_000004.12
12
5


chr04
85682861
85682872
NC_000004.12
12
5


chr04
106114290
106114301
NC_000004.12
12
5


chr04
113028283
113028294
NC_000004.12
12
5


chr04
151151805
151151794
NC_000004.12
12
5


chr04
152051162
152051173
NC_000004.12
12
5


chr04
179052931
179052920
NC_000004.12
12
5


chr05
6661409
6661420
NC_000005.10
12
5


chr05
93549147
93549158
NC_000005.10
12
5


chr05
148916732
148916721
NC_000005.10
12
5


chr05
153193520
153193531
NC_000005.10
12
5


chr05
169165696
169165685
NC_000005.10
12
5


chr06
99056822
99056833
NC_000006.12
12
5


chr07
21203640
21203651
NC_000007.14
12
5


chr07
27364344
27364355
NC_000007.14
12
5


chr07
45331667
45331656
NC_000007.14
12
5


chr08
28102047
28102036
NC_000008.11
12
5


chr08
64148089
64148078
NC_000008.11
12
5


chr08
121058238
121058249
NC_000008.11
12
5


chr08
134902692
134902681
NC_000008.11
12
5


chr09
26814924
26814935
NC_000009.12
12
5


chr09
35739632
35739643
NC_000009.12
12
5


chr09
77017601
77017612
NC_000009.12
12
5


chr09
83041777
83041788
NC_000009.12
12
5


chr09
87072669
87072658
NC_000009.12
12
5


chr09
134613617
134613628
NC_000009.12
12
5


chr10
7938397
7938408
NC_000010.11
12
5


chr10
59688277
59688266
NC_000010.11
12
5


chr10
91834373
91834384
NC_000010.11
12
5


chr10
106036664
106036653
NC_000010.11
12
5


chr11
1648239
1648228
NC_000011.10
12
5


chr11
28286474
28286485
NC_000011.10
12
5


chr11
59609982
59609993
NC_000011.10
12
5


chr11
82154773
82154784
NC_000011.10
12
5


chr12
56884436
56884425
NC_000012.12
12
5


chr12
65309897
65309908
NC_000012.12
12
5


chr12
70312802
70312791
NC_000012.12
12
5


chr12
108169798
108169809
NC_000012.12
12
5


chr13
41643771
41643782
NC_000013.11
12
5


chr13
43730188
43730177
NC_000013.11
12
5


chr13
66772070
66772081
NC_000013.11
12
5


chr13
67266239
67266250
NC_000013.11
12
5


chr13
70438394
70438405
NC_000013.11
12
5


chr13
72462904
72462915
NC_000013.11
12
5


chr13
73589220
73589209
NC_000013.11
12
5


chr13
114256981
114256970
NC_000013.11
12
5


chr14
53548116
53548105
NC_000014.9
12
5


chr14
91128016
91128005
NC_000014.9
12
5


chr15
55623598
55623609
NC_000015.10
12
5


chr15
59650410
59650421
NC_000015.10
12
5


chr15
67895787
67895798
NC_000015.10
12
5


chr15
75030887
75030898
NC_000015.10
12
5


chr15
80376611
80376600
NC_000015.10
12
5


chr17
2259971
2259960
NC_000017.11
12
5


chr17
13599804
13599793
NC_000017.11
12
5


chr17
49970374
49970385
NC_000017.11
12
5


chr17
74411987
74411998
NC_000017.11
12
5


chr18
6692184
6692173
NC_000018.10
12
5


chr18
26936361
26936372
NC_000018.10
12
5


chr18
32164785
32164796
NC_000018.10
12
5


chr18
57372141
57372152
NC_000018.10
12
5


chr18
76028676
76028665
NC_000018.10
12
5


chr18
79860251
79860240
NC_000018.10
12
5


chr20
2767508
2767497
NC_000020.11
12
5


chr20
32334864
32334853
NC_000020.11
12
5


chr20
42969400
42969411
NC_000020.11
12
5


chr21
15405882
15405871
NC_000021.9
12
5


chr21
27128817
27128828
NC_000021.9
12
5


chr21
27724878
27724889
NC_000021.9
12
5


chr21
33775512
33775523
NC_000021.9
12
5


chr22
40201219
40201208
NC_000022.11
12
5


chrX
24583713
24583724
NC_000023.11
12
5


chrX
53003928
53003939
NC_000023.11
12
5


chrX
75537169
75537180
NC_000023.11
12
5


chrX
91187582
91187593
NC_000023.11
12
5


chr01
237603124
237603136
NC_000001.11
13
4


chr02
132279864
132279852
NC_000002.12
13
4


chr02
176672291
176672279
NC_000002.12
13
4


chr04
47096940
47096952
NC_000004.12
13
4


chr05
170123837
170123825
NC_000005.10
13
4


chr10
97944808
97944796
NC_000010.11
13
4


chr10
114226626
114226614
NC_000010.11
13
4


chr13
67884795
67884783
NC_000013.11
13
4


chr14
59591410
59591398
NC_000014.9
13
4


chr16
3659076
3659088
NC_000016.10
13
4


chr18
25418784
25418772
NC_000018.10
13
4


chrX
45634061
45634049
NC_000023.11
13
4


chr01
3217976
3217987
NC_000001.11
12
4


chr01
92837827
92837816
NC_000001.11
12
4


chr01
112701651
112701662
NC_000001.11
12
4


chr01
166000671
166000660
NC_000001.11
12
4


chr01
178801277
178801288
NC_000001.11
12
4


chr02
177290177
177290166
NC_000002.12
12
4


chr02
218084695
218084706
NC_000002.12
12
4


chr02
236494650
236494639
NC_000002.12
12
4


chr04
42894460
42894471
NC_000004.12
12
4


chr04
66200304
66200315
NC_000004.12
12
4


chr06
35644009
35643998
NC_000006.12
12
4


chr06
35671520
35671531
NC_000006.12
12
4


chr09
95179956
95179945
NC_000009.12
12
4


chr09
122078420
122078431
NC_000009.12
12
4


chr09
132891241
132891252
NC_000009.12
12
4


chr09
134244101
134244112
NC_000009.12
12
4


chr10
46934395
46934384
NC_000010.11
12
4


chr10
48117437
48117448
NC_000010.11
12
4


chr10
102716315
102716304
NC_000010.11
12
4


chr12
31614069
31614080
NC_000012.12
12
4


chr13
18693215
18693204
NC_000013.11
12
4


chr14
30845671
30845660
NC_000014.9
12
4


chr14
94062711
94062722
NC_000014.9
12
4


chr17
10363532
10363543
NC_000017.11
12
4


chr17
59667014
59667025
NC_000017.11
12
4


chr17
68278027
68278038
NC_000017.11
12
4


chr18
44686796
44686785
NC_000018.10
12
4


chr18
55570049
55570060
NC_000018.10
12
4


chr20
37099530
37099519
NC_000020.11
12
4


chr21
14473970
14473981
NC_000021.9
12
4


chr21
28191101
28191112
NC_000021.9
12
4


chr01
85362838
85362827
NC_000001.11
12
3


chr14
106817445
106817434
NC_000014.9
12
3


chr17
12074729
12074718
NC_000017.11
12
3


chr21
8217645
8217657
NC_000021.9
13
2


chr21
8400683
8400695
NC_000021.9
13
2


chr21
8444915
8444927
NC_000021.9
13
2


chr01
65227883
65227872
NC_000001.11
12
2


chr17
31347890
31347879
NC_000017.11
12
1









Example 9: Retargeting of a Gene Writer to a Genomic Safe Harbor Site

This example describes a Gene Writer comprising a heterologous DNA binding domain that redirects its activity to a genomic safe harbor site.


In this example, the Gene Writer polypeptide sequence is altered to where its natural DNA binding domain is replaced, mutated/inactivated, and/or joined with another polypeptide sequence that can redirect the Gene Writer system to another genomic location that is not its endogenous or natural binding site. In some instances, the polypeptide sequence that redirects the Gene Writer system to a non-natural genomic location may also be attached and/or inserted to any module of the Gene Writer polypeptide sequence.


In some embodiments, the polypeptide sequence used to redirect the Gene Writer system to a non-natural genomic target encodes for: a zinc finger, a series of adjacent, regularly, or irregularly spaced zinc fingers, a transcription activator-like effector (TALE), a series of adjacent, regularly, or irregularly spaced a transcription activator-like effectors (TALEs), Cas9, Cas9 with mutations to its catalytic residues inactivating the double-stranded DNA endonuclease activity (referred to as catalytically-dead Cas9 (dCas9)), Cas9 with a point mutation or multiple point mutations in a single catalytic domain in order to render Cas9 endonuclease only able to cleave one strand of double-stranded DNA (referred to as Cas9 nickase) (see FIG. 12).


In some embodiments, the polypeptide sequence used to re-direct the Gene Writer system targets a genomic safe-harbor location (e.g., AAVS1 site on human chromosome 19) (Pellenz, S., et. al., Human Gene Therapy, 30(7), 814-828, 2019), see FIGS. 11 and 13. In further embodiments, the polypeptide sequence used to re-direct the Gene Writer polypeptide sequence is used in conjunction with a nucleic acid that targets the genomic safe harbor location (e.g., the polypeptide sequence for catalytic dead Cas9 along with a single-guide RNA targeting the AAVS1 site on chromosome 19).









TABLE E4







Re-targeted Gene Writer constructs. Examples shown are to re-target


R2Tg Gene Writer polypeptide sequence to the AAVS1 site using ZF or


Cas9 domains.








Gene Writer
Polypeptide Sequence (Re-targeting polypeptide


Polypeptide Name
sequence, italic; Linker, bold underline)





AAVS1 Left ZFP

MGIHGVPAAMAERPFQCRICMRNFSYNWHLQRHIRTHTGEKPFACDICGRKFA



attached at v2

RSDHLTTHTKIHTGSQKPFQCRICMRNFSHNYARDCHIRTHTGEKPFACDICG



location of DBD of

RKFAQNSTRIGHTKIHLRGS

GGGS
TATRDKKDTVTREKHPKKPFQKWMKDRAI


R2Tg with 3GS
KKGNYLRFQRLFYLDRGKLAKIILDDIECLSCDIPLSEIYSVFKTRWETTGSF


linker (SEQ ID NO:
KSLGDFKTYGKADNTAFRELITAKEIEKNVQEMSKGSAPGPDGITLGDVVKMD


1024)
PEFSRTMEIFNLWLTTGKIPDMVRGCRTVLIPKSSKPDRLKDINNWRPITIGS



ILLRLFSRIVTARLSKACPLNPRQRGFIRAAGCSENLKLLQTIIWSAKREHRP



LGVVFVDIAKAFDTVSHQHIIHALQQREVDPHIVGLVSNMYENISTYITTKRN



THTDKIQIRVGVKQGDPMSPLLFNLAMDPLLCKLEESGKGYHRGQSSITAMAF



ADDLVLLSDSWENMNTNISILETFCNLTGLKTQGQKCHGFYIKPTKDSYTIND



CAAWTINGTPLNMIDPGESEKYLGLQFDPWIGIARSGLSTKLDFWLQRIDQAP



LKPLQKTDILKTYTIPRLIYIADHSEVKTALLETLDQKIRTAVKEWLHLPPCT



CDAILYSSTRDGGLGITKLAGLIPSVQARRLHRIAQSSDDTMKCFMEKEKMEQ



LHKKLWIQAGGDRENIPSIWEAPPSSEPPNNVSTNSEWEAPTQKDKFPKPCNW



RKNEFKKWTKLASQGRGIVNFERDKISNHWIQYYRRIPHRKLLTALQLRANVY



PTREFLARGRODQYIKACRHCDADIESCAHIIGNCPVTQDARIKRHNYICELL



EEAAAEKVRKYKHLETEVRHLTNAKDVTFVGFPLGARGKWHQDNFKLLTELGL



SKSRQVKMAETFSTVALFSSVDIVHMFASRARKSMVM (SEQ ID NO:



1658)





AAVS1 Left ZFP

MGIHGVPAAMAERPFQCRICMRNFSYNWHLQRHIRTHTGEKPFACDICGRKFA



attached at v2

RSDHLTTHTKIHTGSQKPFQCRICMRNFSHNYARDCHIRTHTGEKPFACDICG



location of DBD of

RKFAQNSTRIGHTKIHLRGS

SGSETPGTSESATPES
TATRDKKDTVTREKHPK


R2Tg with XTEN
KPFQKWMKDRAIKKGNYLRFQRLFYLDRGKLAKIILDDIECLSCDIPLSEIYS


linker
VFKTRWETTGSFKSLGDFKTYGKADNTAFRELITAKEIEKNVQEMSKGSAPGP



DGITLGDVVKMDPEFSRTMEIFNLWLTTGKIPDMVRGCRTVLIPKSSKPDRLK



DINNWRPITIGSILLRLFSRIVTARLSKACPLNPRQRGFIRAAGCSENLKLLQ



TIIWSAKREHRPLGVVFVDIAKAFDTVSHQHIIHALQQREVDPHIVGLVSNMY



ENISTYITTKRNTHTDKIQIRVGVKQGDPMSPLLFNLAMDPLLCKLEESGKGY



HRGQSSITAMAFADDLVLLSDSWENMNTNISILETFCNLTGLKTQGQKCHGFY



IKPTKDSYTINDCAAWTINGTPLNMIDPGESEKYLGLQFDPWIGIARSGLSTK



LDFWLQRIDQAPLKPLQKTDILKTYTIPRLIYIADHSEVKTALLETLDQKIRT



AVKEWLHLPPCTCDAILYSSTRDGGLGITKLAGLIPSVQARRLHRIAQSSDDT



MKCFMEKEKMEQLHKKLWIQAGGDRENIPSIWEAPPSSEPPNNVSTNSEWEAP



TQKDKFPKPCNWRKNEFKKWTKLASQGRGIVNFERDKISNHWIQYYRRIPHRK



LLTALQLRANVYPTREFLARGRODQYIKACRHCDADIESCAHIIGNCPVTQDA



RIKRHNYICELLLEEAKKKDWVVFKEPHIRDSNKELYKPDLIFVKDARALVVD



VTVRYEAAKSSLEEAAAEKVRKYKHLETEVRHLTNAKDVTFVGFPLGARGKWH



QDNFKLLTELGLSKSRQVKMAETFSTVALFSSVDIVHMFASRARKSMVM



(SEQ ID NO: 1659)





AAVS1 Left ZFP

MGIHGVPAAMAERPFQCRICMRNFSYNWHLQRHIRTHTGEKPFACDICGRKFA



attached at v1

RSDHLTTHTKIHTGSQKPFQCRICMRNFSHNYARDCHIRTHTGEKPFACDICG



location of DBD of

RKFAQNSTRIGHTKIHLRGS

GGGS
CFGCLESISQIRTATRDKKDTVTREKHPK


R2Tg with 3GS
KPFQKWMKDRAIKKGNYLRFQRLFYLDRGKLAKIILDDIECLSCDIPLSEIYS


linker (SEQ ID NO:
VFKTRWETTGSFKSLGDFKTYGKADNTAFRELITAKEIEKNVQEMSKGSAPGP


1024)
DGITLGDVVKMDPEFSRTMEIFNLWLTTGKIPDMVRGCRTVLIPKSSKPDRLK



DINNWRPITIGSILLRLFSRIVTARLSKACPLNPRQRGFIRAAGCSENLKLLQ



TIIWSAKREHRPLGVVFVDIAKAFDTVSHQHIIHALQQREVDPHIVGLVSNMY



ENISTYITTKRNTHTDKIQIRVGVKQGDPMSPLLFNLAMDPLLCKLEESGKGY



HRGQSSITAMAFADDLVLLSDSWENMNTNISILETFCNLTGLKTQGQKCHGFY



IKPTKDSYTINDCAAWTINGTPLNMIDPGESEKYLGLQFDPWIGIARSGLSTK



LDFWLQRIDQAPLKPLQKTDILKTYTIPRLIYIADHSEVKTALLETLDQKIRT



AVKEWLHLPPCTCDAILYSSTRDGGLGITKLAGLIPSVQARRLHRIAQSSDDT



MKCFMEKEKMEQLHKKLWIQAGGDRENIPSIWEAPPSSEPPNNVSTNSEWEAP



TQKDKFPKPCNWRKNEFKKWTKLASQGRGIVNFERDKISNHWIQYYRRIPHRK



LLTALQLRANVYPTREFLARGRQDQYIKACRHCDADIESCAHIIGNCPVTQDA



RIKRHNYICELLLEEAKKKDWVVFKEPHIRDSNKELYKPDLIFVKDARALVVD



VTVRYEAAKSSLEEAAAEKVRKYKHLETEVRHLTNAKDVTFVGFPLGARGKWH



QDNFKLLTELGLSKSRQVKMAETFSTVALFSSVDIVHMFASRARKSMVM



(SEQ ID NO: 1660)





AAVS1 Left ZFP

MGIHGVPAAMAERPFQCRICMRNFSYNWHLQRHIRTHTGEKPFACDICGRKFA



attached at v1

RSDHLTTHTKIHTGSQKPFQCRICMRNFSHNYARDCHIRTHTGEKPFACDICG



location of DBD of

RKFAQNSTRIGHTKIHLRGS

SGSETPGTSESATPES
CFGCLESISQIRTATRD


R2Tg with XTEN
KKDTVTREKHPKKPFQKWMKDRAIKKGNYLRFQRLFYLDRGKLAKIILDDIEC


linker
LSCDIPLSEIYSVFKTRWETTGSFKSLGDFKTYGKADNTAFRELITAKEIEKN



VQEMSKGSAPGPDGITLGDVVKMDPEFSRTMEIFNLWLTTGKIPDMVRGCRTV



LIPKSSKPDRLKDINNWRPITIGSILLRLFSRIVTARLSKACPLNPRQRGFIR



AAGCSENLKLLQTIIWSAKREHRPLGVVFVDIAKAFDTVSHQHIIHALQQREV



DPHIVGLVSNMYENISTYITTKRNTHTDKIQIRVGVKQGDPMSPLLFNLAMDP



LLCKLEESGKGYHRGQSSITAMAFADDLVLLSDSWENMNTNISILETFCNLTG



LKTQGQKCHGFYIKPTKDSYTINDCAAWTINGTPLNMIDPGESEKYLGLQFDP



WIGIARSGLSTKLDFWLQRIDQAPLKPLQKTDILKTYTIPRLIYIADHSEVKT



ALLETLDQKIRTAVKEWLHLPPCTCDAILYSSTRDGGLGITKLAGLIPSVQAR



RLHRIAQSSDDTMKCFMEKEKMEQLHKKLWIQAGGDRENIPSIWEAPPSSEPP



NNVSTNSEWEAPTQKDKFPKPCNWRKNEFKKWTKLASQGRGIVNFERDKISNH



WIQYYRRIPHRKLLTALQLRANVYPTREFLARGRQDQYIKACRHCDADIESCA



HIIGNCPVTQDARIKRHNYICELLLEEAKKKDWVVFKEPHIRDSNKELYKPDL



IFVKDARALVVDVTVRYEAAKSSLEEAAAEKVRKYKHLETEVRHLTNAKDVTF



VGFPLGARGKWHQDNFKLLTELGLSKSRQVKMAETFSTVALFSSVDIVHMFAS



RARKSMVM (SEQ ID NO: 1661)





AAVS1 Right ZFP

MGIHGVPAAMAERPFQCRICMRNFSQSSNLARHIRTHTGEKPFACDICGRKFA



attached at v2

RTDYLVDHTKIHTGSQKPFQCRICMRNFSYNTHLTRHIRTHTGEKPFACDICG



location of DBD of

RKFAQGYNLAGHTKIHLRGS

GGGS
TATRDKKDTVTREKHPKKPFQKWMKDRAI


R2Tg with 3GS
KKGNYLRFQRLFYLDRGKLAKIILDDIECLSCDIPLSEIYSVFKTRWETTGSF


linker (SEQ ID NO:
KSLGDFKTYGKADNTAFRELITAKEIEKNVQEMSKGSAPGPDGITLGDVVKMD


1024)
PEFSRTMEIFNLWLTTGKIPDMVRGCRTVLIPKSSKPDRLKDINNWRPITIGS



ILLRLFSRIVTARLSKACPLNPRQRGFIRAAGCSENLKLLQTIIWSAKREHRP



LGVVFVDIAKAFDTVSHQHIIHALQQREVDPHIVGLVSNMYENISTYITTKRN



THTDKIQIRVGVKQGDPMSPLLFNLAMDPLLCKLEESGKGYHRGQSSITAMAF



ADDLVLLSDSWENMNTNISILETFCNLTGLKTQGQKCHGFYIKPTKDSYTIND



CAAWTINGTPLNMIDPGESEKYLGLQFDPWIGIARSGLSTKLDFWLQRIDQAP



LKPLQKTDILKTYTIPRLIYIADHSEVKTALLETLDQKIRTAVKEWLHLPPCT



CDAILYSSTRDGGLGITKLAGLIPSVQARRLHRIAQSSDDTMKCFMEKEKMEQ



LHKKLWIQAGGDRENIPSIWEAPPSSEPPNNVSTNSEWEAPTQKDKFPKPCNW



RKNEFKKWTKLASQGRGIVNFERDKISNHWIQYYRRIPHRKLLTALQLRANVY



PTREFLARGRQDQYIKACRHCDADIESCAHIIGNCPVTQDARIKRHNYICELL



LEEAKKKDWVVFKEPHIRDSNKELYKPDLIFVKDARALVVDVTVRYEAAKSSL



EEAAAEKVRKYKHLETEVRHLTNAKDVTFVGFPLGARGKWHQDNFKLLTELGL



SKSRQVKMAETFSTVALFSSVDIVHMFASRARKSMVM (SEQ ID NO:



1662)





AAVS1 Right ZFP

MGIHGVPAAMAERPFQCRICMRNFSQSSNLARHIRTHTGEKPFACDICGRKFA



attached at v2

RTDYLVDHTKIHTGSQKPFQCRICMRNFSYNTHLTRHIRTHTGEKPFACDICG



location of DBD of

RKFAQGYNLAGHTKIHLRGS

SGSETPGTSESATPES
TATRDKKDTVTREKHPK


R2Tg with XTEN
KPFQKWMKDRAIKKGNYLRFQRLFYLDRGKLAKIILDDIECLSCDIPLSEIYS


linker
VFKTRWETTGSFKSLGDFKTYGKADNTAFRELITAKEIEKNVQEMSKGSAPGP



DGITLGDVVKMDPEFSRTMEIFNLWLTTGKIPDMVRGCRTVLIPKSSKPDRLK



DINNWRPITIGSILLRLFSRIVTARLSKACPLNPRQRGFIRAAGCSENLKLLQ



TIIWSAKREHRPLGVVFVDIAKAFDTVSHQHIIHALQQREVDPHIVGLVSNMY



ENISTYITTKRNTHTDKIQIRVGVKQGDPMSPLLFNLAMDPLLCKLEESGKGY



HRGQSSITAMAFADDLVLLSDSWENMNTNISILETFCNLTGLKTQGQKCHGFY



IKPTKDSYTINDCAAWTINGTPLNMIDPGESEKYLGLQFDPWIGIARSGLSTK



LDFWLQRIDQAPLKPLQKTDILKTYTIPRLIYIADHSEVKTALLETLDQKIRT



AVKEWLHLPPCTCDAILYSSTRDGGLGITKLAGLIPSVQARRLHRIAQSSDDT



MKCFMEKEKMEQLHKKLWIQAGGDRENIPSIWEAPPSSEPPNNVSTNSEWEAP



TQKDKFPKPCNWRKNEFKKWTKLASQGRGIVNFERDKISNHWIQYYRRIPHRK



LLTALQLRANVYPTREFLARGRQDQYIKACRHCDADIESCAHIIGNCPVTQDA



RIKRHNYICELLLEEAKKKDWVVFKEPHIRDSNKELYKPDLIFVKDARALVVD



VTVRYEAAKSSLEEAAAEKVRKYKHLETEVRHLTNAKDVTFVGFPLGARGKWH



QDNFKLLTELGLSKSRQVKMAETFSTVALFSSVDIVHMFASRARKSMVM



(SEQ ID NO: 1663)





AAVS1 Right ZFP

MGIHGVPAAMAERPFQCRICMRNFSQSSNLARHIRTHTGEKPFACDICGRKFA



attached at v1

RTDYLVDHTKIHTGSQKPFQCRICMRNFSYNTHLTRHIRTHTGEKPFACDICG



location of DBD of

RKFAQGYNLAGHTKIHLRGS

GGGS
CFGCLESISQIRTATRDKKDTVTREKHPK


R2Tg with 3GS
KPFQKWMKDRAIKKGNYLRFQRLFYLDRGKLAKIILDDIECLSCDIPLSEIYS


linker (SEQ ID NO:
VFKTRWETTGSFKSLGDFKTYGKADNTAFRELITAKEIEKNVQEMSKGSAPGP


1024)
DGITLGDVVKMDPEFSRTMEIFNLWLTTGKIPDMVRGCRTVLIPKSSKPDRLK



DINNWRPITIGSILLRLFSRIVTARLSKACPLNPRQRGFIRAAGCSENLKLLQ



TIIWSAKREHRPLGVVFVDIAKAFDTVSHQHIIHALQQREVDPHIVGLVSNMY



ENISTYITTKRNTHTDKIQIRVGVKQGDPMSPLLFNLAMDPLLCKLEESGKGY



HRGQSSITAMAFADDLVLLSDSWENMNTNISILETFCNLTGLKTQGQKCHGFY



IKPTKDSYTINDCAAWTINGTPLNMIDPGESEKYLGLQFDPWIGIARSGLSTK



LDFWLQRIDQAPLKPLQKTDILKTYTIPRLIYIADHSEVKTALLETLDQKIRT



AVKEWLHLPPCTCDAILYSSTRDGGLGITKLAGLIPSVQARRLHRIAQSSDDT



MKCFMEKEKMEQLHKKLWIQAGGDRENIPSIWEAPPSSEPPNNVSTNSEWEAP



TQKDKFPKPCNWRKNEFKKWTKLASQGRGIVNFERDKISNHWIQYYRRIPHRK



LLTALQLRANVYPTREFLARGRQDQYIKACRHCDADIESCAHIIGNCPVTQDA



RIKRHNYICELLLEEAKKKDWVVFKEPHIRDSNKELYKPDLIFVKDARALVVD



VTVRYEAAKSSLEEAAAEKVRKYKHLETEVRHLTNAKDVTFVGFPLGARGKWH



QDNFKLLTELGLSKSRQVKMAETFSTVALFSSVDIVHMFASRARKSMVM



(SEQ ID NO: 1664)





AAVS1 Right ZFP

MGIHGVPAAMAERPFQCRICMRNFSQSSNLARHIRTHTGEKPFACDICGRKFA



attached at v1

RTDYLVDHTKIHTGSQKPFQCRICMRNFSYNTHLTRHIRTHTGEKPFACDICG



location of DBD of

RKFAQGYNLAGHTKIHLRGS

SGSETPGTSESATPES
CFGCLESISQIRTATRD


R2Tg with XTEN
KKDTVTREKHPKKPFQKWMKDRAIKKGNYLRFQRLFYLDRGKLAKIILDDIEC


linker
LSCDIPLSEIYSVFKTRWETTGSFKSLGDFKTYGKADNTAFRELITAKEIEKN



VQEMSKGSAPGPDGITLGDVVKMDPEFSRTMEIFNLWLTTGKIPDMVRGCRTV



LIPKSSKPDRLKDINNWRPITIGSILLRLFSRIVTARLSKACPLNPRQRGFIR



AAGCSENLKLLQTIIWSAKREHRPLGVVFVDIAKAFDTVSHQHITHALQQREV



DPHIVGLVSNMYENISTYITTKRNTHTDKIQIRVGVKQGDPMSPLLFNLAMDP



LLCKLEESGKGYHRGQSSITAMAFADDLVLLSDSWENMNTNISILETFCNLTG



LKTQGQKCHGFYIKPTKDSYTINDCAAWTINGTPLNMIDPGESEKYLGLQFDP



WIGIARSGLSTKLDFWLQRIDQAPLKPLQKTDILKTYTIPRLIYIADHSEVKT



ALLETLDQKIRTAVKEWLHLPPCTCDAILYSSTRDGGLGITKLAGLIPSVQAR



RLHRIAQSSDDTMKCFMEKEKMEQLHKKLWIQAGGDRENIPSIWEAPPSSEPP



NNVSTNSEWEAPTQKDKFPKPCNWRKNEFKKWTKLASQGRGIVNFERDKISNH



WIQYYRRIPHRKLLTALQLRANVYPTREFLARGRQDQYIKACRHCDADIESCA



HIIGNCPVTQDARIKRHNYICELLLEEAKKKDWVVFKEPHIRDSNKELYKPDL



IFVKDARALVVDVTVRYEAAKSSLEEAAAEKVRKYKHLETEVRHLTNAKDVTF



VGFPLGARGKWHQDNFKLLTELGLSKSRQVKMAETFSTVALFSSVDIVHMFAS



RARKSMVM (SEQ ID NO: 1665)





AAVS1 Left and

MGIHGVPAAMAERPFQCRICMRNFSYNWHLQRHIRTHTGEKPFACDICGRKFA



Right ZFP

RSDHLTTHTKIHTGSQKPFQCRICMRNFSHNYARDCHIRTHTGEKPFACDICG



(separated by XTEN

RKFAQNSTRIGHTKIHLRGS

SGSETPGTSESATPES

GIHGVPAAMAERPFQCR



linker) attached at

ICMRNFSQSSNLARHIRTHTGEKPFACDICGRKFARTDYLVDHTKIHTGSQKP



v2 location of DBD

FQCRICMRNFSYNTHLTRHIRTHTGEKPFACDICGRKFAQGYNLAGHTKIHLR



of R2Tg with 3GS

GS

GGGS
TATRDKKDTVTREKHPKKPFQKWMKDRAIKKGNYLRFQRLFYLDRGK


linker (SEQ ID NO:
LAKIILDDIECLSCDIPLSEIYSVFKTRWETTGSFKSLGDFKTYGKADNTAFR


1024)
ELITAKEIEKNVQEMSKGSAPGPDGITLGDVVKMDPEFSRTMEIFNLWLTTGK



IPDMVRGCRTVLIPKSSKPDRLKDINNWRPITIGSILLRLFSRIVTARLSKAC



PLNPRQRGFIRAAGCSENLKLLQTIIWSAKREHRPLGVVFVDIAKAFDTVSHQ



HIIHALQQREVDPHIVGLVSNMYENISTYITTKRNTHTDKIQIRVGVKQGDPM



SPLLFNLAMDPLLCKLEESGKGYHRGQSSITAMAFADDLVLLSDSWENMNTNI



SILETFCNLTGLKTQGQKCHGFYIKPTKDSYTINDCAAWTINGTPLNMIDPGE



SEKYLGLQFDPWIGIARSGLSTKLDFWLQRIDQAPLKPLQKTDILKTYTIPRL



IYIADHSEVKTALLETLDQKIRTAVKEWLHLPPCTCDAILYSSTRDGGLGITK



LAGLIPSVQARRLHRIAQSSDDTMKCFMEKEKMEQLHKKLWIQAGGDRENIPS



IWEAPPSSEPPNNVSTNSEWEAPTQKDKFPKPCNWRKNEFKKWTKLASQGRGI



VNFERDKISNHWIQYYRRIPHRKLLTALQLRANVYPTREFLARGRQDQYIKAC



RHCDADIESCAHIIGNCPVTQDARIKRHNYICELLLEEAKKKDWVVFKEPHIR



DSNKELYKPDLIFVKDARALVVDVTVRYEAAKSSLEEAAAEKVRKYKHLETEV



RHLTNAKDVTFVGFPLGARGKWHQDNFKLLTELGLSKSRQVKMAETFSTVALF



SSVDIVHMFASRARKSMVM (SEQ ID NO: 1666)





AAVS1 Left and

MGIHGVPAAMAERPFQCRICMRNFSYNWHLQRHIRTHTGEKPFACDICGRKFA



Right ZFP

RSDHLTTHTKIHTGSQKPFQCRICMRNFSHNYARDCHIRTHTGEKPFACDICG



(separated by XTEN

RKFAQNSTRIGHTKIHLRGS

SGSETPGTSESATPES

GIHGVPAAMAERPFQCR



linker) attached at

ICMRNFSQSSNLARHIRTHTGEKPFACDICGRKFARTDYLVDHTKIHTGSQKP



v2 location of DBD

FQCRICMRNFSYNTHLTRHIRTHTGEKPFACDICGRKFAQGYNLAGHTKIHLR



of R2Tg with XTEN

GS

SGSETPGTSESATPES
TATRDKKDTVTREKHPKKPFQKWMKDRAIKKGNYL


linker
RFQRLFYLDRGKLAKIILDDIECLSCDIPLSEIYSVFKTRWETTGSFKSLGDF



KTYGKADNTAFRELITAKEIEKNVQEMSKGSAPGPDGITLGDVVKMDPEFSRT



MEIFNLWLTTGKIPDMVRGCRTVLIPKSSKPDRLKDINNWRPITIGSILLRLF



SRIVTARLSKACPLNPRQRGFIRAAGCSENLKLLQTIIWSAKREHRPLGVVFV



DIAKAFDTVSHQHIIHALQQREVDPHIVGLVSNMYENISTYITTKRNTHTDKI



QIRVGVKQGDPMSPLLFNLAMDPLLCKLEESGKGYHRGQSSITAMAFADDLVL



LSDSWENMNTNISILETFCNLTGLKTQGQKCHGFYIKPTKDSYTINDCAAWTI



NGTPLNMIDPGESEKYLGLQFDPWIGIARSGLSTKLDFWLQRIDQAPLKPLQK



TDILKTYTIPRLIYIADHSEVKTALLETLDQKIRTAVKEWLHLPPCTCDAILY



SSTRDGGLGITKLAGLIPSVQARRLHRIAQSSDDTMKCFMEKEKMEQLHKKLW



IQAGGDRENIPSIWEAPPSSEPPNNVSTNSEWEAPTQKDKFPKPCNWRKNEFK



KWTKLASQGRGIVNFERDKISNHWIQYYRRIPHRKLLTALQLRANVYPTREFL



ARGRQDQYIKACRHCDADIESCAHIIGNCPVTQDARIKRHNYICELLLEEAKK



KDWVVFKEPHIRDSNKELYKPDLIFVKDARALVVDVTVRYEAAKSSLEEAAAE



KVRKYKHLETEVRHLTNAKDVTFVGFPLGARGKWHQDNFKLLTELGLSKSRQV



KMAETFSTVALFSSVDIVHMFASRARKSMVM (SEQ ID NO: 1667)





AAVS1 Left ZFP

MGIHGVPAAMAERPFQCRICMRNFSYNWHLQRHIRTHTGEKPFACDICGRKFA



attached to N-

RSDHLTTHTKIHTGSQKPFQCRICMRNFSHNYARDCHIRTHTGEKPFACDICG



terminus of R2Tg

RKFAQNSTRIGHTKIHLRGS

SGSETPGTSESATPES
ASCPKPGPPVSAGAMSL


with XTEN linker
ESGLTTHSVLAIERGPNSLANSGSDFGGGGLGLPLRLLRVSVGTQTSRSDWVD



LVSWSHPGPTSKSQQVDLVSLFPKHRVDLLSKNDQVDLVAQFLPSKFPPNLAE



NDLALLVNLEFYRSDLHVYECVHFAAHWEGLSGLPEVYEQLAPQPCVGETLHS



SLPRDSELFVPEEGSSEKESEDAPKTSPPTPGKHGLEQTGEEKVMVTVPDKNP



PCPCCGTRVNSVLNLIEHLKVSHGKRGVCFRCAKCGKENSNYHSVVCHFPKCR



GPETEKAPAGEWICEVCNRDFTTKIGLGQHKRLAHPAVRNQERIVASQPKETS



NRGAHKRCWTKEEEELLIRLEAQFEGNKNINKLIAEHITTKTAKQISDKRRLL



SRKPAEEPREEPGTCHHTRRAAASLRTEPEMSHHAQAEDRDNGPGRRPLPGRA



AAGGRTMDEIRRHPDKGNGQQRPTKQKSEEQLQAYYKKTLEERLSAGALNTFP



RAFKQVMEGRDIKLVINQTAQDCFGCLESISQIRTATRDKKDTVTREKHPKKP



FQKWMKDRAIKKGNYLRFQRLFYLDRGKLAKIILDDIECLSCDIPLSEIYSVF



KTRWETTGSFKSLGDFKTYGKADNTAFRELITAKEIEKNVQEMSKGSAPGPDG



ITLGDVVKMDPEFSRTMEIFNLWLTTGKIPDMVRGCRTVLIPKSSKPDRLKDI



NNWRPITIGSILLRLFSRIVTARLSKACPLNPRQRGFIRAAGCSENLKLLQTI



IWSAKREHRPLGVVFVDIAKAFDTVSHQHITHALQQREVDPHIVGLVSNMYEN



ISTYITTKRNTHTDKIQIRVGVKQGDPMSPLLFNLAMDPLLCKLEESGKGYHR



GQSSITAMAFADDLVLLSDSWENMNTNISILETFCNLTGLKTQGQKCHGFYIK



PTKDSYTINDCAAWTINGTPLNMIDPGESEKYLGLQFDPWIGIARSGLSTKLD



FWLQRIDQAPLKPLQKTDILKTYTIPRLIYIADHSEVKTALLETLDQKIRTAV



KEWLHLPPCTCDAILYSSTRDGGLGITKLAGLIPSVQARRLHRIAQSSDDTMK



CFMEKEKMEQLHKKLWIQAGGDRENIPSIWEAPPSSEPPNNVSTNSEWEAPTQ



KDKFPKPCNWRKNEFKKWTKLASQGRGIVNFERDKISNHWIQYYRRIPHRKLL



TALQLRANVYPTREFLARGRQDQYIKACRHCDADIESCAHIIGNCPVTQDARI



VRYEAAKSSLEEAAAEKVRKYKHLETEVRHLTNAKDVTFVGFPLGARGKWHQD



NFKLLTELGLSKSRQVKMAETFSTVALFSSVDIVHMFASRARKSMVM (SEQ



ID NO: 1668)





AAVS1 Right ZFP

MGIHGVPAAMAERPFQCRICMRNFSQSSNLARHIRTHTGEKPFACDICGRKFA



attached to N-

RTDYLVDHTKIHTGSQKPFQCRICMRNFSYNTHLTRHIRTHTGEKPFACDICG



terminus of R2Tg

RKFAQGYNLAGHTKIHLRGS

SGSETPGTSESATPES
ASCPKPGPPVSAGAMSL


with XTEN linker
ESGLTTHSVLAIERGPNSLANSGSDFGGGGLGLPLRLLRVSVGTQTSRSDWVD



LVSWSHPGPTSKSQQVDLVSLFPKHRVDLLSKNDQVDLVAQFLPSKFPPNLAE



NDLALLVNLEFYRSDLHVYECVHFAAHWEGLSGLPEVYEQLAPQPCVGETLHS



SLPRDSELFVPEEGSSEKESEDAPKTSPPTPGKHGLEQTGEEKVMVTVPDKNP



PCPCCGTRVNSVLNLIEHLKVSHGKRGVCFRCAKCGKENSNYHSVVCHFPKCR



GPETEKAPAGEWICEVCNRDFTTKIGLGQHKRLAHPAVRNQERIVASQPKETS



NRGAHKRCWTKEEEELLIRLEAQFEGNKNINKLIAEHITTKTAKQISDKRRLL



SRKPAEEPREEPGTCHHTRRAAASLRTEPEMSHHAQAEDRDNGPGRRPLPGRA



AAGGRTMDEIRRHPDKGNGQQRPTKQKSEEQLQAYYKKTLEERLSAGALNTFP



RAFKQVMEGRDIKLVINQTAQDCFGCLESISQIRTATRDKKDTVTREKHPKKP



FQKWMKDRAIKKGNYLRFQRLFYLDRGKLAKIILDDIECLSCDIPLSEIYSVF



KTRWETTGSFKSLGDFKTYGKADNTAFRELITAKEIEKNVQEMSKGSAPGPDG



ITLGDVVKMDPEFSRTMEIFNLWLTTGKIPDMVRGCRTVLIPKSSKPDRLKDI



NNWRPITIGSILLRLFSRIVTARLSKACPLNPRQRGFIRAAGCSENLKLLQTI



IWSAKREHRPLGVVFVDIAKAFDTVSHQHIIHALQQREVDPHIVGLVSNMYEN



ISTYITTKRNTHTDKIQIRVGVKQGDPMSPLLFNLAMDPLLCKLEESGKGYHR



GOSSITAMAFADDLVLLSDSWENMNTNISILETFCNLTGLKTQGQKCHGFYIK



PTKDSYTINDCAAWTINGTPLNMIDPGESEKYLGLQFDPWIGIARSGLSTKLD



FWLQRIDQAPLKPLQKTDILKTYTIPRLIYIADHSEVKTALLETLDQKIRTAV



KEWLHLPPCTCDAILYSSTRDGGLGITKLAGLIPSVQARRLHRIAQSSDDTMK



CFMEKEKMEQLHKKLWIQAGGDRENIPSIWEAPPSSEPPNNVSTNSEWEAPTQ



KDKFPKPCNWRKNEFKKWTKLASQGRGIVNFERDKISNHWIQYYRRIPHRKLL



TALQLRANVYPTREFLARGRODQYIKACRHCDADIESCAHIIGNCPVTQDARI



VRYEAAKSSLEEAAAEKVRKYKHLETEVRHLTNAKDVTFVGFPLGARGKWHQD



NFKLLTELGLSKSRQVKMAETFSTVALFSSVDIVHMFASRARKSMVM (SEQ



ID NO: 1669)





AAVS1 Left and

MGIHGVPAAMAERPFQCRICMRNFSYNWHLQRHIRTHTGEKPFACDICGRKFA



Right ZFP attached

RSDHLTTHTKIHTGSQKPFQCRICMRNFSHNYARDCHIRTHTGEKPFACDICG



to N-terminus of

RKFAQNSTRIGHTKIHLRGS

SGSETPGTSESATPES

GIHGVPAAMAERPFQCR



R2Tg with XTEN

ICMRNFSQSSNLARHIRTHTGEKPFACDICGRKFARTDYLVDHTKIHTGSQKP



linker

FQCRICMRNFSYNTHLTRHIRTHTGEKPFACDICGRKFAQGYNLAGHTKIHLR





GS

SGSETPGTSESATPES
ASCPKPGPPVSAGAMSLESGLTTHSVLAIERGPNS



LANSGSDFGGGGLGLPLRLLRVSVGTQTSRSDWVDLVSWSHPGPTSKSQQVDL



VSLFPKHRVDLLSKNDQVDLVAQFLPSKFPPNLAENDLALLVNLEFYRSDLHV



YECVHFAAHWEGLSGLPEVYEQLAPQPCVGETLHSSLPRDSELFVPEEGSSEK



ESEDAPKTSPPTPGKHGLEQTGEEKVMVTVPDKNPPCPCCGTRVNSVLNLIEH



LKVSHGKRGVCFRCAKCGKENSNYHSVVCHFPKCRGPETEKAPAGEWICEVCN



RDFTTKIGLGQHKRLAHPAVRNQERIVASQPKETSNRGAHKRCWTKEEEELLI



RLEAQFEGNKNINKLIAEHITTKTAKQISDKRRLLSRKPAEEPREEPGTCHHT



RRAAASLRTEPEMSHHAQAEDRDNGPGRRPLPGRAAAGGRTMDEIRRHPDKGN



GQQRPTKQKSEEQLQAYYKKTLEERLSAGALNTFPRAFKQVMEGRDIKLVINQ



TAQDCFGCLESISQIRTATRDKKDTVTREKHPKKPFQKWMKDRAIKKGNYLRF



QRLFYLDRGKLAKIILDDIECLSCDIPLSEIYSVFKTRWETTGSFKSLGDFKT



YGKADNTAFRELITAKEIEKNVQEMSKGSAPGPDGITLGDVVKMDPEFSRTME



IFNLWLTTGKIPDMVRGCRTVLIPKSSKPDRLKDINNWRPITIGSILLRLFSR



IVTARLSKACPLNPRQRGFIRAAGCSENLKLLQTIIWSAKREHRPLGVVFVDI



AKAFDTVSHQHIIHALQQREVDPHIVGLVSNMYENISTYITTKRNTHTDKIQI



RVGVKQGDPMSPLLFNLAMDPLLCKLEESGKGYHRGQSSITAMAFADDLVLLS



DSWENMNTNISILETFCNLTGLKTQGQKCHGFYIKPTKDSYTINDCAAWTING



TPLNMIDPGESEKYLGLQFDPWIGIARSGLSTKLDFWLQRIDQAPLKPLQKTD



ILKTYTIPRLIYIADHSEVKTALLETLDQKIRTAVKEWLHLPPCTCDAILYSS



TRDGGLGITKLAGLIPSVQARRLHRIAQSSDDTMKCFMEKEKMEQLHKKLWIQ



AGGDRENIPSIWEAPPSSEPPNNVSTNSEWEAPTQKDKFPKPCNWRKNEFKKW



TKLASQGRGIVNFERDKISNHWIQYYRRIPHRKLLTALQLRANVYPTREFLAR



GRQDQYIKACRHCDADIESCAHIIGNCPVTQDARIKRHNYICELLLEEAKKKD



RKYKHLETEVRHLTNAKDVTFVGFPLGARGKWHQDNFKLLTELGLSKSRQVKM



AETFSTVALFSSVDIVHMFASRARKSMVM (SEQ ID NO: 1670)





AAVS1 Left ZFP

MGIHGVPAAMAERPFQCRICMRNFSYNWHLQRHIRTHTGEKPFACDICGRKFA



attached to N-

RSDHLTTHTKIHTGSQKPFQCRICMRNFSHNYARDCHIRTHTGEKPFACDICG



terminus of R2Tg

RKFAQNSTRIGHTKIHLRGS

SGSETPGTSESATPES
ASCPKPGPPVSAGAMSL


containing DBD
ESGLTTHSVLAIERGPNSLANSGSDFGGGGLGLPLRLLRVSVGTQTSRSDWVD


inactivation
LVSWSHPGPTSKSQQVDLVSLFPKHRVDLLSKNDQVDLVAQFLPSKFPPNLAE


mutations with
NDLALLVNLEFYRSDLHVYECVHFAAHWEGLSGLPEVYEQLAPQPCVGETLHS


XTEN linker
SLPRDSELFVPEEGSSEKESEDAPKTSPPTPGKHGLEQTGEEKVMVTVPDKNP



PSPSSGTRVNSVLNLIEHLKVSHGKRGVCFRCAKCGKENSNYHSVVCHFPKCR



GPETEKAPAGEWISEVSNRDFTTKIGLGQHKRLAHPAVRNQERIVASQPKETS



NRGAHKACATKEEEELLIRLEAQFEGNKNINKLIAEHITTKTAKQISDKRRLL



SRKPAEEPREEPGTCHHTRRAAASLRTEPEMSHHAQAEDRDNGPGRRPLPGRA



AAGGRTMDEIRRHPDKGNGQQRPTKQKSEEQLQAYYKKTLEERLSAGALNTFP



RAFKQVMEGRDIKLVINQTAQDCFGCLESISQIRTATRDKKDTVTREKHPKKP



FQKWMKDRAIKKGNYLRFQRLFYLDRGKLAKIILDDIECLSCDIPLSEIYSVF



KTRWETTGSFKSLGDFKTYGKADNTAFRELITAKEIEKNVQEMSKGSAPGPDG



ITLGDVVKMDPEFSRTMEIFNLWLTTGKIPDMVRGCRTVLIPKSSKPDRLKDI



NNWRPITIGSILLRLFSRIVTARLSKACPLNPRQRGFIRAAGCSENLKLLQTI



IWSAKREHRPLGVVFVDIAKAFDTVSHQHIIHALQQREVDPHIVGLVSNMYEN



ISTYITTKRNTHTDKIQIRVGVKQGDPMSPLLFNLAMDPLLCKLEESGKGYHR



GOSSITAMAFADDLVLLSDSWENMNTNISILETFCNLTGLKTQGQKCHGFYIK



PTKDSYTINDCAAWTINGTPLNMIDPGESEKYLGLQFDPWIGIARSGLSTKLD



FWLQRIDQAPLKPLQKTDILKTYTIPRLIYIADHSEVKTALLETLDQKIRTAV



KEWLHLPPCTCDAILYSSTRDGGLGITKLAGLIPSVQARRLHRIAQSSDDTMK



CFMEKEKMEQLHKKLWIQAGGDRENIPSIWEAPPSSEPPNNVSTNSEWEAPTQ



KDKFPKPCNWRKNEFKKWTKLASQGRGIVNFERDKISNHWIQYYRRIPHRKLL



TALQLRANVYPTREFLARGRODQYIKACRHCDADIESCAHIIGNCPVTQDARI



VRYEAAKSSLEEAAAEKVRKYKHLETEVRHLTNAKDVTFVGFPLGARGKWHQD



NFKLLTELGLSKSRQVKMAETFSTVALFSSVDIVHMFASRARKSMVM (SEQ



ID NO: 1671)





AAVS1 Right ZFP

MGIHGVPAAMAERPFQCRICMRNFSQSSNLARHIRTHTGEKPFACDICGRKFA



attached to N-

RTDYLVDHTKIHTGSQKPFQCRICMRNFSYNTHLTRHIRTHTGEKPFACDICG



terminus of R2Tg

RKFAQGYNLAGHTKIHLRGS

SGSETPGTSESATPES
ASCPKPGPPVSAGAMSL


containing DBD
ESGLTTHSVLAIERGPNSLANSGSDFGGGGLGLPLRLLRVSVGTQTSRSDWVD


inactivation
LVSWSHPGPTSKSQQVDLVSLFPKHRVDLLSKNDQVDLVAQFLPSKFPPNLAE


mutations with
NDLALLVNLEFYRSDLHVYECVHFAAHWEGLSGLPEVYEQLAPQPCVGETLHS


XTEN linker
SLPRDSELFVPEEGSSEKESEDAPKTSPPTPGKHGLEQTGEEKVMVTVPDKNP



PSPSSGTRVNSVLNLIEHLKVSHGKRGVCFRCAKCGKENSNYHSVVCHFPKCR



GPETEKAPAGEWISEVSNRDFTTKIGLGQHKRLAHPAVRNQERIVASQPKETS



NRGAHKACATKEEEELLIRLEAQFEGNKNINKLIAEHITTKTAKQISDKRRLL



SRKPAEEPREEPGTCHHTRRAAASLRTEPEMSHHAQAEDRDNGPGRRPLPGRA



AAGGRTMDEIRRHPDKGNGQQRPTKQKSEEQLQAYYKKTLEERLSAGALNTFP



RAFKQVMEGRDIKLVINQTAQDCFGCLESISQIRTATRDKKDTVTREKHPKKP



FQKWMKDRAIKKGNYLRFQRLFYLDRGKLAKIILDDIECLSCDIPLSEIYSVF



KTRWETTGSFKSLGDFKTYGKADNTAFRELITAKEIEKNVQEMSKGSAPGPDG



ITLGDVVKMDPEFSRTMEIFNLWLTTGKIPDMVRGCRTVLIPKSSKPDRLKDI



NNWRPITIGSILLRLFSRIVTARLSKACPLNPRQRGFIRAAGCSENLKLLQTI



IWSAKREHRPLGVVFVDIAKAFDTVSHQHIIHALQQREVDPHIVGLVSNMYEN



ISTYITTKRNTHTDKIQIRVGVKQGDPMSPLLFNLAMDPLLCKLEESGKGYHR



GQSSITAMAFADDLVLLSDSWENMNTNISILETFCNLTGLKTQGQKCHGFYIK



PTKDSYTINDCAAWTINGTPLNMIDPGESEKYLGLQFDPWIGIARSGLSTKLD



FWLQRIDQAPLKPLQKTDILKTYTIPRLIYIADHSEVKTALLETLDQKIRTAV



KEWLHLPPCTCDAILYSSTRDGGLGITKLAGLIPSVQARRLHRIAQSSDDTMK



CFMEKEKMEQLHKKLWIQAGGDRENIPSIWEAPPSSEPPNNVSTNSEWEAPTQ



KDKFPKPCNWRKNEFKKWTKLASQGRGIVNFERDKISNHWIQYYRRIPHRKLL



TALQLRANVYPTREFLARGRQDQYIKACRHCDADIESCAHIIGNCPVTQDARI



KRHNYICELLLEEAKKKDWVVFKEPHIRDSNKELYKPDLIFVKDARALVVDVT



VRYEAAKSSLEEAAAEKVRKYKHLETEVRHLTNAKDVTFVGFPLGARGKWHQD



NFKLLTELGLSKSRQVKMAETFSTVALFSSVDIVHMFASRARKSMVM (SEQ



ID NO: 1672)





AAVS1 Left and

MGIHGVPAAMAERPFQCRICMRNFSYNWHLQRHIRTHTGEKPFACDICGRKFA



Right ZFP attached

RSDHLTTHTKIHTGSQKPFQCRICMRNFSHNYARDCHIRTHTGEKPFACDICG



to N-terminus of

RKFAQNSTRIGHTKIHLRGS

SGSETPGTSESATPES
GIHGVPAAMAERPFQCR


R2Tg containing

ICMRNFSQSSNLARHIRTHTGEKPFACDICGRKFARTDYLVDHTKIHTGSQKP



DBD inactivation

FQCRICMRNFSYNTHLTRHIRTHTGEKPFACDICGRKFAQGYNLAGHTKIHLR



mutations with

GS

SGSETPGTSESATPES
ASCPKPGPPVSAGAMSLESGLTTHSVLAIERGPNS


XTEN linker
LANSGSDFGGGGLGLPLRLLRVSVGTQTSRSDWVDLVSWSHPGPTSKSQQVDL



VSLFPKHRVDLLSKNDQVDLVAQFLPSKFPPNLAENDLALLVNLEFYRSDLHV



YECVHFAAHWEGLSGLPEVYEQLAPQPCVGETLHSSLPRDSELFVPEEGSSEK



ESEDAPKTSPPTPGKHGLEQTGEEKVMVTVPDKNPPSPSSGTRVNSVLNLIEH



LKVSHGKRGVCFRCAKCGKENSNYHSVVCHFPKCRGPETEKAPAGEWISEVSN



RDFTTKIGLGQHKRLAHPAVRNQERIVASQPKETSNRGAHKACATKEEEELLI



RLEAQFEGNKNINKLIAEHITTKTAKQISDKRRLLSRKPAEEPREEPGTCHHT



RRAAASLRTEPEMSHHAQAEDRDNGPGRRPLPGRAAAGGRTMDEIRRHPDKGN



GQQRPTKOKSEEQLQAYYKKTLEERLSAGALNTFPRAFKQVMEGRDIKLVINQ



TAQDCFGCLESISQIRTATRDKKDTVTREKHPKKPFQKWMKDRAIKKGNYLRF



QRLFYLDRGKLAKIILDDIECLSCDIPLSEIYSVFKTRWETTGSFKSLGDFKT



YGKADNTAFRELITAKEIEKNVQEMSKGSAPGPDGITLGDVVKMDPEFSRTME



IFNLWLTTGKIPDMVRGCRTVLIPKSSKPDRLKDINNWRPITIGSILLRLFSR



IVTARLSKACPLNPRQRGFIRAAGCSENLKLLQTIIWSAKREHRPLGVVFVDI



AKAFDTVSHQHIIHALQQREVDPHIVGLVSNMYENISTYITTKRNTHTDKIQI



RVGVKQGDPMSPLLFNLAMDPLLCKLEESGKGYHRGQSSITAMAFADDLVLLS



DSWENMNTNISILETFCNLTGLKTQGQKCHGFYIKPTKDSYTINDCAAWTING



TPLNMIDPGESEKYLGLQFDPWIGIARSGLSTKLDFWLQRIDQAPLKPLQKTD



ILKTYTIPRLIYIADHSEVKTALLETLDQKIRTAVKEWLHLPPCTCDAILYSS



TRDGGLGITKLAGLIPSVQARRLHRIAQSSDDTMKCFMEKEKMEQLHKKLWIQ



AGGDRENIPSIWEAPPSSEPPNNVSTNSEWEAPTQKDKFPKPCNWRKNEFKKW



TKLASQGRGIVNFERDKISNHWIQYYRRIPHRKLLTALQLRANVYPTREFLAR



GRQDQYIKACRHCDADIESCAHIIGNCPVTQDARIKRHNYICELLLEEAKKKD



WVVFKEPHIRDSNKELYKPDLIFVKDARALVVDVTVRYEAAKSSLEEAAAEKV



RKYKHLETEVRHLTNAKDVTFVGFPLGARGKWHQDNFKLLTELGLSKSRQVKM



AETFSTVALFSSVDIVHMFASRARKSMVM (SEQ ID NO: 1673)






S. pyogenes Cas9

MAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGN



attached at v2

TDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEM



location of DBD of

AKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLV



R2Tg with

DSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLF



XTEN33aa linker

EENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLT





PNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILL





SDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQS





KNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG





SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSR





FAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLL





YEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKED





YFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIV





LTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDK





QSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIAN





LAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRE





RMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR





LSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQ





LLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSR





MNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA





VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMN





FFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKK





TEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE





KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSL





FELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ





LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI





IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDL





SQLGGD

SGGSSGGSSGSETPGTSESATPESSGGSSGGSS
TATRDKKDTVTREK



HPKKPFQKWMKDRAIKKGNYLRFQRLFYLDRGKLAKIILDDIECLSCDIPLSE



IYSVFKTRWETTGSFKSLGDFKTYGKADNTAFRELITAKEIEKNVQEMSKGSA



PGPDGITLGDVVKMDPEFSRTMEIFNLWLTTGKIPDMVRGCRTVLIPKSSKPD



RLKDINNWRPITIGSILLRLFSRIVTARLSKACPLNPRQRGFIRAAGCSENLK



LLQTIIWSAKREHRPLGVVFVDIAKAFDTVSHQHIIHALQQREVDPHIVGLVS



NMYENISTYITTKRNTHTDKIQIRVGVKQGDPMSPLLFNLAMDPLLCKLEESG



KGYHRGQSSITAMAFADDLVLLSDSWENMNTNISILETFCNLTGLKTQGQKCH



GFYIKPTKDSYTINDCAAWTINGTPLNMIDPGESEKYLGLQFDPWIGIARSGL



STKLDFWLQRIDQAPLKPLQKTDILKTYTIPRLIYIADHSEVKTALLETLDQK



IRTAVKEWLHLPPCTCDAILYSSTRDGGLGITKLAGLIPSVQARRLHRIAQSS



DDTMKCFMEKEKMEQLHKKLWIQAGGDRENIPSIWEAPPSSEPPNNVSTNSEW



EAPTQKDKFPKPCNWRKNEFKKWTKLASQGRGIVNFERDKISNHWIQYYRRIP



HRKLLTALQLRANVYPTREFLARGRQDQYIKACRHCDADIESCAHIIGNCPVT



QDARIKRHNYICELLLEEAKKKDWVVFKEPHIRDSNKELYKPDLIFVKDARAL



VVDVTVRYEAAKSSLEEAAAEKVRKYKHLETEVRHLTNAKDVTFVGFPLGARG



KWHQDNFKLLTELGLSKSRQVKMAETFSTVALFSSVDIVHMFASRARKSMVM



(SEQ ID NO: 1674)






S. pyogenes Cas9

MAPKKKRKVGIHGVPAADKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGN



containing catalytic

TDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEM



mutations (dCas9)

AKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLV



attached at v2

DSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLF



location of DBD of

EENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLT



R2Tg with

PNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILL



XTEN33aa linker

SDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQS





KNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG





SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSR





FAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLL





YEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKED





YFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIV





LTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDK





QSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIAN





LAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRE





RMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR





LSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQ





LLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSR





MNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA





VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMN





FFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKK





TEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE





KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSL





FELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ





LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI





IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDL





SQLGGD

SGGSSGGSSGSETPGTSESATPESSGGSSGGSS
TATRDKKDTVTREK



HPKKPFQKWMKDRAIKKGNYLRFQRLFYLDRGKLAKIILDDIECLSCDIPLSE



IYSVFKTRWETTGSFKSLGDFKTYGKADNTAFRELITAKEIEKNVQEMSKGSA



PGPDGITLGDVVKMDPEFSRTMEIFNLWLTTGKIPDMVRGCRTVLIPKSSKPD



LLQTIIWSAKREHRPLGVVFVDIAKAFDTVSHQHIIHALQQREVDPHIVGLVS



NMYENISTYITTKRNTHTDKIQIRVGVKQGDPMSPLLFNLAMDPLLCKLEESG



KGYHRGQSSITAMAFADDLVLLSDSWENMNTNISILETFCNLTGLKTQGQKCH



GFYIKPTKDSYTINDCAAWTINGTPLNMIDPGESEKYLGLQFDPWIGIARSGL



STKLDFWLQRIDQAPLKPLQKTDILKTYTIPRLIYIADHSEVKTALLETLDQK



IRTAVKEWLHLPPCTCDAILYSSTRDGGLGITKLAGLIPSVQARRLHRIAQSS



DDTMKCFMEKEKMEQLHKKLWIQAGGDRENIPSIWEAPPSSEPPNNVSTNSEW



EAPTQKDKFPKPCNWRKNEFKKWTKLASQGRGIVNFERDKISNHWIQYYRRIP



HRKLLTALQLRANVYPTREFLARGRQDQYIKACRHCDADIESCAHIIGNCPVT



VVDVTVRYEAAKSSLEEAAAEKVRKYKHLETEVRHLTNAKDVTFVGFPLGARG



KWHQDNFKLLTELGLSKSRQVKMAETFSTVALFSSVDIVHMFASRARKSMVM



(SEQ ID NO: 1675)






S. pyogenes Cas9

MAPKKKRKVGIHGVPAADKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGN



D10A nickase

TDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEM



mutant attached at

AKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLV



v2 location of DBD

DSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLF



of R2Tg with

EENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLT



XTEN33aa linker

PNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILL





SDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQS





KNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG





SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSR





FAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLL





YEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKED





YFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIV





LTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDK





QSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIAN





LAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRE





RMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR





LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQ





LLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSR





MNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA





VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMN





FFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKK





TEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE





KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSL





FELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ





LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI





IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDL





SQLGGD

SGGSSGGSSGSETPGTSESATPESSGGSSGGSS
TATRDKKDTVTREK



HPKKPFQKWMKDRAIKKGNYLRFQRLFYLDRGKLAKIILDDIECLSCDIPLSE



IYSVFKTRWETTGSFKSLGDFKTYGKADNTAFRELITAKEIEKNVQEMSKGSA



PGPDGITLGDVVKMDPEFSRTMEIFNLWLTTGKIPDMVRGCRTVLIPKSSKPD



RLKDINNWRPITIGSILLRLFSRIVTARLSKACPLNPRQRGFIRAAGCSENLK



LLQTIIWSAKREHRPLGVVFVDIAKAFDTVSHQHIIHALQQREVDPHIVGLVS



NMYENISTYITTKRNTHTDKIQIRVGVKQGDPMSPLLFNLAMDPLLCKLEESG



KGYHRGQSSITAMAFADDLVLLSDSWENMNTNISILETFCNLTGLKTQGQKCH



GFYIKPTKDSYTINDCAAWTINGTPLNMIDPGESEKYLGLQFDPWIGIARSGL



STKLDFWLQRIDQAPLKPLQKTDILKTYTIPRLIYIADHSEVKTALLETLDQK



IRTAVKEWLHLPPCTCDAILYSSTRDGGLGITKLAGLIPSVQARRLHRIAQSS



DDTMKCFMEKEKMEQLHKKLWIQAGGDRENIPSIWEAPPSSEPPNNVSTNSEW



EAPTQKDKFPKPCNWRKNEFKKWTKLASQGRGIVNFERDKISNHWIQYYRRIP



HRKLLTALQLRANVYPTREFLARGRQDQYIKACRHCDADIESCAHIIGNCPVT



QDARIKRHNYICELLLEEAKKKDWVVFKEPHIRDSNKELYKPDLIFVKDARAL



VVDVTVRYEAAKSSLEEAAAEKVRKYKHLETEVRHLTNAKDVTFVGFPLGARG



KWHQDNFKLLTELGLSKSRQVKMAETFSTVALFSSVDIVHMFASRARKSMVM



(SEQ ID NO: 1676)






S. pyogenes Cas9

MAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGN



N863A nickase

TDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEM



mutant attached at

AKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLV



v2 location of DBD

DSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLF



of R2Tg with

EENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLT



XTEN33aa linker

PNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILL





SDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQS





KNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG





SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSR





FAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLL





YEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKED





YFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIV





LTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDK





QSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIAN





LAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRE





RMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR





LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQ





LLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSR





MNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA





VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMN





FFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKK





TEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE





KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSL





FELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ





LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI





IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDL





SQLGGD

SGGSSGGSSGSETPGTSESATPESSGGSSGGSS
TATRDKKDTVTREK



HPKKPFQKWMKDRAIKKGNYLRFQRLFYLDRGKLAKIILDDIECLSCDIPLSE



IYSVFKTRWETTGSFKSLGDFKTYGKADNTAFRELITAKEIEKNVQEMSKGSA



PGPDGITLGDVVKMDPEFSRTMEIFNLWLTTGKIPDMVRGCRTVLIPKSSKPD



LLQTIIWSAKREHRPLGVVFVDIAKAFDTVSHQHIIHALQQREVDPHIVGLVS



NMYENISTYITTKRNTHTDKIQIRVGVKQGDPMSPLLFNLAMDPLLCKLEESG



KGYHRGQSSITAMAFADDLVLLSDSWENMNTNISILETFCNLTGLKTQGQKCH



GFYIKPTKDSYTINDCAAWTINGTPLNMIDPGESEKYLGLQFDPWIGIARSGL



STKLDFWLQRIDQAPLKPLQKTDILKTYTIPRLIYIADHSEVKTALLETLDQK



IRTAVKEWLHLPPCTCDAILYSSTRDGGLGITKLAGLIPSVQARRLHRIAQSS



DDTMKCFMEKEKMEQLHKKLWIQAGGDRENIPSIWEAPPSSEPPNNVSTNSEW



EAPTQKDKFPKPCNWRKNEFKKWTKLASQGRGIVNFERDKISNHWIQYYRRIP



HRKLLTALQLRANVYPTREFLARGRQDQYIKACRHCDADIESCAHIIGNCPVT



QDARIKRHNYICELLLEEAKKKDWVVFKEPHIRDSNKELYKPDLIFVKDARAL



VVDVTVRYEAAKSSLEEAAAEKVRKYKHLETEVRHLTNAKDVTFVGFPLGARG



KWHQDNFKLLTELGLSKSRQVKMAETFSTVALFSSVDIVHMFASRARKSMVM



(SEQ ID NO: 1677)






S. pyogenes Cas9

MAPKKKRKVGIHGVPAADKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGN



D10A nickase

TDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEM



mutant attached at

AKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLV



v2 location of DBD

DSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLF



of R2Tg containing

EENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLT





PNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILL



EN mutation with

SDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQS



XTEN33aa linker

KNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG





SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSR





FAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLL





YEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKED





YFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIV





LTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDK





QSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIAN





LAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRE





LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQ





LLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSR





MNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA





VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMN





FFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKK





TEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE





KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSL





FELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ





LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI





IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDL





SQLGGD

SGGSSGGSSGSETPGTSESATPESSGGSSGGSS
TATRDKKDTVTREK



HPKKPFQKWMKDRAIKKGNYLRFQRLFYLDRGKLAKIILDDIECLSCDIPLSE



IYSVFKTRWETTGSFKSLGDFKTYGKADNTAFRELITAKEIEKNVQEMSKGSA



PGPDGITLGDVVKMDPEFSRTMEIFNLWLTTGKIPDMVRGCRTVLIPKSSKPD



RLKDINNWRPITIGSILLRLFSRIVTARLSKACPLNPRQRGFIRAAGCSENLK



LLQTIIWSAKREHRPLGVVFVDIAKAFDTVSHQHIIHALQQREVDPHIVGLVS



NMYENISTYITTKRNTHTDKIQIRVGVKQGDPMSPLLFNLAMDPLLCKLEESG



KGYHRGQSSITAMAFADDLVLLSDSWENMNTNISILETFCNLTGLKTQGQKCH



GFYIKPTKDSYTINDCAAWTINGTPLNMIDPGESEKYLGLQFDPWIGIARSGL



STKLDFWLQRIDQAPLKPLQKTDILKTYTIPRLIYIADHSEVKTALLETLDQK



IRTAVKEWLHLPPCTCDAILYSSTRDGGLGITKLAGLIPSVQARRLHRIAQSS



DDTMKCFMEKEKMEQLHKKLWIQAGGDRENIPSIWEAPPSSEPPNNVSTNSEW



EAPTQKDKFPKPCNWRKNEFKKWTKLASQGRGIVNFERDKISNHWIQYYRRIP



HRKLLTALQLRANVYPTREFLARGRQDQYIKACRHCDADIESCAHIIGNCPVT



QDARIKRHNYICELLLEEAKKKDWVVFKEPHIRDSNKELYKPALIFVKDARAL



VVDVTVRYEAAKSSLEEAAAEKVRKYKHLETEVRHLTNAKDVTFVGFPLGARG



KWHQDNFKLLTELGLSKSRQVKMAETFSTVALFSSVDIVHMFASRARKSMVM



(SEQ ID NO: 1678)






S. pyogenes Cas9

MAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGN



N863A nickase

TDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEM



mutant attached at

AKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLV



v2 location of DBD

DSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLF



of R2Tg containing

EENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLT



EN mutation with

PNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILL



XTEN33aa linker

SDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQS





KNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG





SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSR





FAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLL





YEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKED





YFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIV





LTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDK





QSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIAN





LAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRE





RMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR





LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKARGKSDNVPSEEVVKKMKNYWRQ





LLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSR





MNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA





VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMN





FFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKK





TEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE





KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSL





FELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ





LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI





IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDL





SQLGGD

SGGSSGGSSGSETPGTSESATPESSGGSSGGSS
TATRDKKDTVTREK



HPKKPFQKWMKDRAIKKGNYLRFQRLFYLDRGKLAKIILDDIECLSCDIPLSE



IYSVFKTRWETTGSFKSLGDFKTYGKADNTAFRELITAKEIEKNVQEMSKGSA



PGPDGITLGDVVKMDPEFSRTMEIFNLWLTTGKIPDMVRGCRTVLIPKSSKPD



RLKDINNWRPITIGSILLRLFSRIVTARLSKACPLNPRQRGFIRAAGCSENLK



LLQTIIWSAKREHRPLGVVFVDIAKAFDTVSHQHIIHALQQREVDPHIVGLVS



NMYENISTYITTKRNTHTDKIQIRVGVKQGDPMSPLLFNLAMDPLLCKLEESG



KGYHRGQSSITAMAFADDLVLLSDSWENMNTNISILETFCNLTGLKTQGQKCH



GFYIKPTKDSYTINDCAAWTINGTPLNMIDPGESEKYLGLQFDPWIGIARSGL



STKLDFWLQRIDQAPLKPLQKTDILKTYTIPRLIYIADHSEVKTALLETLDQK



IRTAVKEWLHLPPCTCDAILYSSTRDGGLGITKLAGLIPSVQARRLHRIAQSS



DDTMKCFMEKEKMEQLHKKLWIQAGGDRENIPSIWEAPPSSEPPNNVSTNSEW



EAPTQKDKFPKPCNWRKNEFKKWTKLASQGRGIVNFERDKISNHWIQYYRRIP



HRKLLTALQLRANVYPTREFLARGRQDQYIKACRHCDADIESCAHIIGNCPVT



QDARIKRHNYICELLLEEAKKKDWVVFKEPHIRDSNKELYKPALIFVKDARAL



VVDVTVRYEAAKSSLEEAAAEKVRKYKHLETEVRHLTNAKDVTFVGFPLGARG



KWHQDNFKLLTELGLSKSRQVKMAETFSTVALFSSVDIVHMFASRARKSMVM



(SEQ ID NO: 1679)






S. pyogenes Cas9

MAPKKKRKVGIHGVPAADKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGN



attached at v2

TDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEM



location of DBD of

AKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLV



R2Tg containing EN

DSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLF



mutation with

EENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLT



XTEN33aa linker

PNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILL





SDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQS





KNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNG





SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSR





FAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLL





YEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKED





YFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIV





LTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDK





QSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIAN





LAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRE





RMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR





LSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQ





LLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSR





MNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA





VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMN





FFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKK





TEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE





KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSL





FELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQ





LFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI





IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDL





SQLGGD

SGGSSGGSSGSETPGTSESATPESSGGSSGGSS
TATRDKKDTVTREK



HPKKPFQKWMKDRAIKKGNYLRFQRLFYLDRGKLAKIILDDIECLSCDIPLSE



IYSVFKTRWETTGSFKSLGDFKTYGKADNTAFRELITAKEIEKNVQEMSKGSA



PGPDGITLGDVVKMDPEFSRTMEIFNLWLTTGKIPDMVRGCRTVLIPKSSKPD



RLKDINNWRPITIGSILLRLFSRIVTARLSKACPLNPRQRGFIRAAGCSENLK



LLQTIIWSAKREHRPLGVVFVDIAKAFDTVSHQHIIHALQQREVDPHIVGLVS



NMYENISTYITTKRNTHTDKIQIRVGVKQGDPMSPLLFNLAMDPLLCKLEESG



KGYHRGQSSITAMAFADDLVLLSDSWENMNTNISILETFCNLTGLKTQGQKCH



GFYIKPTKDSYTINDCAAWTINGTPLNMIDPGESEKYLGLQFDPWIGIARSGL



STKLDFWLQRIDQAPLKPLQKTDILKTYTIPRLIYIADHSEVKTALLETLDQK



IRTAVKEWLHLPPCTCDAILYSSTRDGGLGITKLAGLIPSVQARRLHRIAQSS



DDTMKCFMEKEKMEQLHKKLWIQAGGDRENIPSIWEAPPSSEPPNNVSTNSEW



EAPTQKDKFPKPCNWRKNEFKKWTKLASQGRGIVNFERDKISNHWIQYYRRIP



HRKLLTALQLRANVYPTREFLARGRQDQYIKACRHCDADIESCAHIIGNCPVT



QDARIKRHNYICELLLEEAKKKDWVVFKEPHIRDSNKELYKPALIFVKDARAL



VVDVTVRYEAAKSSLEEAAAEKVRKYKHLETEVRHLTNAKDVTFVGFPLGARG



KWHQDNFKLLTELGLSKSRQVKMAETFSTVALFSSVDIVHMFASRARKSMVM



(SEQ ID NO: 1680)









Example 10: Inactivation of an Endogenous Nucleolar Localization Signal in a Gene Writer

This example describes a Gene Writer in which an endogenous nucleolar localization signal has been inactivated to reduce intracellular targeting of the protein to the nucleolus.


In this example, the nucleolar localization signal (NoLS) of a retrotransposase is computationally predicted using a published algorithm that was trained on validated proteins that localize to the nucleolus (Scott, M. S., et al, Nucleic Acids Research, 38(21), 7388-7399 (2010)). The predicted NoLS sequence is based on both amino acid sequence, amino acid sequence context, and predicted secondary structure of the retrotransposase. The identified sequence is typically rich with basic amino acids (Scott, M. S., et al, Nucleic Acids Research, 38(21), 7388-7399 (2010)) and when these residues are mutated to a simple side-chain, non-basic, amino acids or removed from the retrotransposase polypeptide chain then it can prevent localization to the nucleolus (Yang, C. P., et. al., Journal of Biomedical Science, 22(1), 1-15. (2015), Martin, R. M., et. al., Nucleus, 6(4), 314-325 (2015)). In some embodiments, the NoLS sequence is located in the amino acid region of a retrotransposase that is between the reverse transcriptase polymerase motif and the restriction-like endonuclease motifs. The predicted NoLS region contains lysine, arginine, histidine, and/or glutamine amino acids where nucleolar localization is inactivated by mutation of one or more of these residues to an alanine amino acid residue and/or one or more of these amino acids are removed from the polypeptide chain of the retrotransposase. In some embodiments, the amino acid sequence of the Gene Writer driver of R2Tg found upstream of the RLE is mutated such that lysines (K) are substituted for alanines (A), e.g., the predicted NoLS of R2Tg (amino acids 1,128-1,154 of polypeptide sequence), (APTQKDKFPKPCNWRKNEFKKWTKLAS (SEQ ID NO: 1681)) is mutated at 1, 2, 3, 4, 5, 6, or 7 residues to produce an inactivated NoLS (APTQADAFPAPCNWRANEFAAWTALAS (SEQ ID NO: 1682)).


Example 11: Application of Second-Strand Nicking in a Gene Writer System

This example describes a Gene Writer system in which retrotransposition is paired with targeted second-strand nicking activity in order to increase the efficiency of integration events. The second strand nick can be achieved by (1) a Cas9 nickase fused to a gene writer system, in which the Gene Writer introduces one nick through its endonuclease domain (EN), and the fused nickase Cas9 places another nick on either the top and bottom DNA strands (FIG. 7A), or (2) a GeneWriter system in which the active EN domain introduces a nick, and a Cas9 nickase introduces a second nick on either top or bottom strand of the DNA, upstream or downstream of the Gene Writer induced nick (FIG. 7B).


In the first part of this example, a Cas9 nickase is fused to a Gene Writer protein (FIG. 7A). The Cas9 is targeted to a DNA sequence through a gRNA. The Gene Writer protein introduces a DNA nick through its EN domain, and an additional nick is generated through the nickase Cas9 activity. This additional nick can be targeted to the top or bottom strands of the DNA surrounding the Gene Writer introduced nick (FIG. 8A). Constructs designed and tested include (see schematic FIG. 14A):

    • Cas9-N863A-R2tg (RBD*, RT, EN)
    • Cas9-H840A-R2tg (RBD*, RT, EN)
    • Cas9-D10A-R2tg (RBD*, RT, EN)
    • dCas9-R2tg (RBD*, RT, EN)


The DNA binding domain is the nickase Cas9 that directs the Gene Writer molecule to a DNA target through a gRNA. The RNA binding domain (RBD) in this set of Gene Writer constructs is inactivated with a point mutation (RBD*). As a donor for insertion, constructs in which the R2Tg RNA binding domain is inactive use a gRNA that is extended at its 3′ end to include donor sequence for genome modification (FIG. 14B). These modifications include nucleotide substitutions, nucleotide deletions and nucleotide insertions. In this first set of experiments, the above constructs-R2Tg(RBD*, RT, EN) and dCas9-R2Tg(RBD*, RT, EN) fusions with a 3′ extended gRNA template targeting the AAVS1 locus are delivered to U2OS cells through nucleofection in SE buffer using program DN100. gRNAs used include gRNAs for each construct that target either the bottom or top strand of DNA. After nucleofection, cells are grown in complete medium for 3 days. gDNA is harvested on day 3, and amplicon sequencing followed by computational analysis using CRISPResso (indel analysis tool) are performed. 3′ extended gRNA mediated insertions, deletions or nucleotide substitutions are observed upon delivery of dCas9-R2Tg(RBD*, RT, EN), and increased in frequency when delivering Nickase-Cas9-R2Tg(RBD*, RT, EN) constructs.


In the second part of this example, a Cas9 nickase is fused to a Gene Writer protein (FIG. 7A). The Cas9 is targeted to a DNA sequence through a gRNA. The Gene Writer protein introduces a DNA nick through its EN domain, and an additional nick is generated through the nickase Cas9 activity. This additional nick can be targeted to the top or bottom strands of the DNA surrounding the Gene Writer introduced nick (FIG. 7A). In contrast to the constructs listed above, the RNA binding domain of R2Tg is active (FIG. 15A), and the template used for genome modification is a transgene flanked by UTRs (FIG. 15B). Constructs include (see schematic FIG. 15A):

    • Cas9-N863A-R2tg (RBD, RT, EN)
    • Cas9-H840A-R2tg (RBD, RT, EN)
    • Cas9-D10A-R2tg (RBD, RT, EN)
    • dCas9-R2tg (RBD, RT, EN)


The transgene flanked by UTRs requires homology arms at the site of nicking. To determine the site of nicking for the accurate design of homology arms for the donor transgene DNA, the above listed constructs are nucleofected into 200k U2OS cells with a gRNA targeting the AAVS1 locus using pulse code DN100. After nucleofection, cells are grown in complete medium for 3 days. gDNA is harvested on day 3, and amplicon sequencing followed by computational analysis using CRISPResso as an indel analysis tool are performed. The nicking site of the EN domain is identified from the indels the EN domain produces at the AAVS1 site. Homology arms of 100 bp flanking the EN nicking site are designed and included in the transgene. To achieve genome modification, Cas9-R2Tg fusion constructs listed above are nucleofected into U2OS cells, along with a gRNA targeting either the top or bottom strand of the AAVS1 locus, and the appropriate transgenes harboring homology to the previously determined nicking site. After nucleofection, cells are grown in complete medium for 3 days. gDNA is harvested on day 3, and ddPCR is performed to detect transgene integration at the AAVS1 site. Integrations are observed upon delivery of dCas9-R2Tg(RBD, RT, EN), and increased in frequency when delivering Nickase-Cas9-R2Tg(RBD, RT, EN) constructs.


In another example, a Gene Writer protein is targeted to DNA through its DNA binding domain (FIG. 7B). The Gene Writer protein will introduce a DNA nick at a DNA strand. In addition, a Cas9 nickase is used to generate a second nick either on the top or bottom strands of the DNA, upstream or downstream of the first nick. In this example, a Gene Writer plasmid targeting the AAVS1 site (FIG. 16A) and with a UTR flanked transgene with homology to the AAVS1 site (FIG. 16B) is nucleofected into 200k U2OS cells using pulse code DN100. The following Cas9 constructs are transfected alongside the Gene Writer plasmids (FIG. 16C):

    • Cas9-N863A
    • Cas9-H840A
    • Cas9-D10A
    • dCas9


All Cas9 constructs are co-nucleofected with gRNAs targeting the AAVS1 locus on either the top or bottom strands, upstream or downstream of the Gene Writer introduced nick. After nucleofection, cells are grown in complete medium for 3 days. gDNA is harvested on day 3, and ddPCR is performed to detect transgene integration at the AAVS1 site. Integrations are observed upon delivery of dCas9 and increased in frequency when delivering Nickase-Cas9 constructs.


Example 12: Improved Expression of Gene Writer Polypeptide by Heterologous UTRs

This example describes the use of heterologous UTRs to enhance the intracellular expression of the Gene Writer polypeptide.


In this example, the Gene Writer polypeptide was expressed from mRNA (FIG. 17). In the plasmid template for the mRNA production, the native retrotransposon UTRs were replaced with UTRs optimized for the protein expression (C3 5′UTR and ORM 3′ UTR from Asrani et al., RNA biology 15, 756-762 (2018) or 5′ and 3′ UTRs from Richter et al., Cell 168, 1114-1125 (2017)). The plasmid included the T7 promoter followed by the 5′UTR, the retrotransposon coding sequence, the 3′ UTR, 3GS linker (SEQ ID NO: 1024), SV40 nuclear localization signal (NLS), XTEN linker, HiBit sequence and 96-100 nucleotide long poly(A) tail (SEQ ID NO: 1683). The plasmid was linearized by enzymatic restriction resulting in blunt end or 5′ overhang downstream of poly(A) tail and used for in vitro transcription (IVT) using T7 polymerase (NEB). Following the IVT step the RNA was treated with DNase I (NEB). After the buffer exchange step the enzymatic capping reaction was performed using Vaccinia capping enzyme (NEB) and 2′-O-methyltransferase (NEB) in the presence of GTP and SAM (NEB). The capped RNA was concentrated and buffer exchanged. 50,000 HEK293T cells were transfected with 0.5 μg with the Gene Writer mRNA in the presence or in the absence of the RNA template in 1:1 molar ratio using Neon transfection system (1150 V per pulse, 20 msec per pulse, 2 pulses in 10 μl tips in 96 well format). The RNA template was in vitro transcribed from plasmid as described in Example 14 (Improved Gene Writer components for RNA-based delivery).


After transfection HEK293T cells were grown for 5 hours before assaying the Gene Writer expression by probing its HiBit tag expression using standard protocol www.promega.com/-/media/files/resources/protocols/technical-manuals/500/nano-glo-hibit-lytic-detection-system-technical-manual.pdf?la=en. Protein expression was found to be greatly improved by the use of 5′ and 3′ UTRexp from C3-ORM as compared to using the native UTRs from R2Tg (FIG. 17). The genome integration was assayed 3 days post-transfection using 3′ ddPCR (FIG. 18).


Example 13: Improved Gene Writer Components for Mixed RNA and DNA Delivery

This example describes improvements to the RNA molecule encoding a Gene Writer polypeptide that enhance expression and allow for increased efficiency of retrotransposition when used with a Gene Writer template encoded on plasmid DNA.


In this example, the polypeptide component of the Gene Writer™ system is expressed from mRNA described in Example 12 (Improved expression of Gene Writer polypeptide by heterologous UTRs). The plasmid template was synthesized such that the reporter gene (eGFP) was flanked by R2Tg untranslated regions (UTRs) and 100 bp of homology to its rDNA target. The tempate expression was driven by the mammalian CMV promoter. We introduced the plasmid into HEK393T cells using the FuGENE® HD transfection reagent. HEK293T cells were seeded in 96-well plates at 10,000 cells/well 24 hours before transfection. On the transfection day, 0.5 μl transfection reagent and 80 ng DNA was mixed in 10 μl Opti-MEM and incubated for 15 minutes at room temperature. The transfection mixture was then added to the medium of the seeded cells. Cells were detached and used for the electroporation of 0.5 μg of mRNA per well using Neon transfection system (1150 V per pulse, 20 msec per pulse, 2 pulses in 10 pL tips in 96 well format).


HEK293T cells were transfected with the following test agents:

    • 1. mRNA coding for the polypeptide described above
    • 2. Plasmid encoding template RNA described above
    • 3. Combination of 1 and 2. The plasmid was pre-lipofected 24 hrs before mRNA transfection as described above.


After transfection, HEK293T cells were cultured for 1-3 days and then assayed for site-specific genome editing. Genomic DNA was isolated from each group of HEK293 cells. ddPCR was performed to confirm integration and assess integration efficiency. Taqman probes and primers were designed as described in PCT/US2019/048607 to amplify the expected product across 5′ and 3′ ends of integration junctions. The results of the ddPCR copy number analysis (in comparison to reference gene RPP30) are shown in FIG. 19. The genome integration in the presence of the mRNA and the template plasmid achieved a mean copy number of 0.683 integrants/genome when targeting 3′ junction and of 0.249 integrants/genome when targeting 5′ junction. The mRNA only transfection resulted in a mean copy number of 0.002 integrants/genome, in comparison to 0.0004 integrants/genome for the plasmid only transfection.


Example 14: Improved Gene Writer Components for RNA-Based Delivery

This example describes improvements to the RNA molecule encoding a Gene Writer polypeptide that enhance expression and allow for increased efficiency of retrotransposition when co-delivered with a Gene Writer RNA template.


In this example, the polypeptide component of the Gene Writer™ system is expressed from mRNA described in Example 12 (Improved expression of Gene Writer polypeptide by heterologous UTRs). The plasmid template for the RNA template production included T7 promoter followed by the IRES-expressing reporter gene (eGFP) flanked by R2Tg untranslated regions (UTRs) and 100 bp of homology to its rDNA target. The plasmid template was linearized by enzymatic restriction resulting in blunt end or 5′ overhang downstream of the RNA template sequence and used for in vitro transcription (IVT) using T7 RNA polymerase (NEB). Following the IVT step the RNA was treated with DNase I (NEB) and either enzymatically polyadenylated by poly(A) polymerase (NEB) or not. After the buffer exchange step the enzymatic capping reaction was performed using Vaccinia capping enzyme (NEB) and 2′-O-methyltransferase (NEB) in the presence of GTP and SAM (NEB). The capped RNA was concentrated and buffer exchanged. 50,000 HEK293T cells were co-transfected with 0.5 to 1 μg of the GeneWriter mRNA and the RNA template in 1:4 to 1:12 molar ratios. The Neon transfection system was used for the RNA transfection (1150 V per pulse, 20 msec per pulse, 2 pulses in 10 μL tips in 96 well format).


After transfection, HEK293T cells were cultured for at least 1 day and then assayed for site-specific genome editing. Genomic DNA was isolated from each group of HEK293 cells. ddPCR was performed to confirm integration and assess integration efficiency. Taqman probes and primers were designed as described in PCT/US2019/048607 to amplify the expected product across 5′ and 3′ ends of integration junctions. The mean copy number of 0.498 integrants/genome was achieved in the presence of the 0.5 μg of mRNA and 1:8 molar ratio of Gene Writer mRNA to the RNA template when the RNA template was enzymatically polyadenylated, in comparison to that of 0.031 integrants/genome when the RNA transgene was not polyadenylated.


Example 15: Gene Writers that Deliver Genetic Cargo Containing Introns

This example describes the use of a Gene Writer system to integrate genetic cargo that contains introns by using RNA-based delivery to tune expression of the gene of interest from its newly introduced genomic locus.


In this example, Gene writing technology uses an RNA template encoding a protein of interest including its native or non-native introns. For example, intron 6 of the triose phosphate isomerase (TPI) gene (Nott et al., 2003) will be used as one of the non-native introns in these experiments.


The presence of introns in the genomic copy of a gene and their removal by splicing has been reported to affect nearly every aspect of the gene expression, including its transcription rate, the mRNA processing, export, cell localization, translation and decay (reviewed in Shaul International Journal of Biochemistry and Cell Biology 91B, 145-155 (2017)). The introns can be inserted into different parts of the RNA template (FIG. 21) and depending on the intron location their role in gene expression can differ.


An intron in the 5′ UTRexp, close to the transcription start site, introduces activating chromatin modifications (Bieberstein et al., Cell Reports 2, 62-68 (2012)), improves accuracy of transcription start site recognition and facilitates PolII recruitment (Laxa et al., Plant Physiology 172, 313-327 (2016)), increases rates of transcription initiation (Kwek et al., Nature Structural Biology 9, 800-805 (2002)) and elongation (Lin et al., Nature Structural and Molecular Biology 15, 819-826 (2008)), and improve the productive elongation in the sense relative to the antisense orientation (Almada et al., Nature 499, 360-363 (2013)).


An intron in the 3′ UTRexp limits the mRNA expression to one protein molecule per mRNA: the exon junction complex (EJC) left by spliceosome downstream of stop codon is recognized by the nonsense-mediated decay (NMD) machinery and therefore the mRNA is marked for deletion at the end of the pioneering round of translation (Zhang et al., RNA 4, 801-815 (1998)).


The ability to employ introns in a therapeutic gene may, however, be limited by splicing that occurs prior to integration of the template. For example, an intron in the forward orientation would be spliced out when an RNA template was encoded and delivered on a DNA plasmid, since transcription in the same direction would yield a template RNA that would be spliced prior to integration, thus failing to incorporate the intron in the genome. Additionally, lentivirus constructs designed to deliver a transgene must encode a sequence with an intron in the reverse orientation, since the viral packaging process would result in intron splicing and absence of the intron in packaged viral particles (Miller et al. J Virol 62, 4337-45 (1988)). However, the reverse orientation has also been thought to result in a reduction in viral titer and transduction (Uchida et al., Nat Commun 10, 4479 (2019)). It is worth noting that since the Gene Writer template can be generated through in vitro transcription and delivered directly as RNA, the problem of pre-integration splicing of desired introns can be avoided. In some embodiments, the Gene Writer template may thus contain one or more introns in same-sense orientation with the transcript, which is generated by IVT and delivered to the target cell as RNA.


An intron in any location depicted in FIG. 21 will recruit U1 snRNP that protects mRNA from the premature cleavage and polyadenylation (Kaida et al., Nature 468 664-681 (2010); Berg et al., Cell 150, 53-64 (2012)). In addition, the EJC interacts with components of the TREX (transcription-export) complex and increases the rate of mRNA export from nucleus to cytoplasm 6-10-fold in comparison to the constructs lacking introns (Valencia et al., PNAS 105, 3386-3391 (2008)). It was also demonstrated that the binding of the polypyrimidine tract-binding protein, a splicing regulator protein, mediates a significant increase in the half-life of the spliced transcripts (Lu & Cullen, RNA 9, 618-630 (2003); Millevoi et al., Nucleic Acid Research 37, 4672-4683 (2009)). The efficiency of the mRNA translation was shown to be increased by the presence of the SR proteins (serine-arginine rich proteins, involved in RNA splicing) (Sanford et al., Genes & Development 18, 755-768 (2004); Sato et al., Molecular Cell 29, 255-262 (2008)) and the EJC proteins and its peripheral factors (Nott et al., Genes & Development 18, 210-222 (2004)).


In this example both the template RNAs harboring an intron or introns and Gene Writer polypeptide are delivered to the cells as in vitro transcribed capped RNAs as described in Example 14 (Improved Gene Writer components for RNA-based delivery). One to three days post-transfection the GOI expression and the genomic integration are assayed. In some embodiments, the genome integration and/or protein expression will be higher for the intron-containing RNA template.


Example 16: Engineering of the Retrotransposon 5′ UTR to Improve Efficiency of Integration

This example describes the deletion, replacement, or mutation of the 5′UTR of a retrotransposon to increase integration efficiency.


The 5′UTR region of non-LTR retrotransposons has multiple functions including self-cleaving ribozyme activity, which has been shown in certain elements and is predicted in additional retrotransposons (see modules B and C of FIG. 27-28) (Ruminski et al. J Biol Chem 286, 41286-41295 (2011)). Ribozymal activity is predicted to cleave the RNA within or upstream of the 5′UTR. Either increasing or restricting this activity and structural component of the 5′UTR may benefit retrotransposition efficiency. A prediction of the ribozyme structure of R2Tg is provided in FIG. 29.


In order to evaluate engineering of the 5′UTR, constructs were designed to enhance or diminish these activities (FIG. 20). In case (A), the natural 5′UTR of R2Tg is used to integrate in trans as in previous experiments. Case (B) illustrates deletion of the 5′UTR. (C) and (D) represent cases in which the 5′UTR from the original species (in this case R2Tg from T. guttata) has been replaced by the 5′UTR of a retrotransposon from a distinct species. Case (C) provides an example in which the 5′UTR from A. maritima R2 has replaced that of R2Tg. (D) represents the generic case in which UTRs from additional species may be substituted (“Rx”), such as that from B. mori, D. ananasse, F. auricularia, L. polyphemus, N. giraulti, or O. latipes, or from a retrotransposon selected from a Table herein, or any of Tables 1-3 of PCT/US2019/048607, herein incorporated by reference in its entirety. Case (E) represents the substitution of a ribozyme, such as a hammerhead ribozyme, e.g., RiboJ (Lou et al Nat Biotechnol 30, 1137-1142 (2012)). Case (F) represents the inactivation of the 5′UTR of R2Tg through point mutations, e.g., 75C>T in the 5′ UTR (FIG. 20.B, position indicated by shaded box). 5′UTR sequences are expected to be modular to any insertion sequence mediated by the retrotransposon.


Each case is evaluated as in previous examples by transfection of Gene Writer polypeptide plasmid with template plasmid and evaluation of integration frequency via ddPCR. In some embodiments, substitution or mutation of the 5′ UTR results in increased efficiency of integration.


Example 17: Modifying the 5′ and 3′ Ends of Gene Writer RNA Components to Improve RNA Stability

This example describes the addition of non-coding sequences to the 5′ and 3′ ends of RNA in order to improve stability in a mammalian cell.


The decay of eukaryotic RNAs in cells are mostly carried out by exoribonucleases. In this example, the half-life of RNAs is prolonged by introducing protective sequences and/or modifications at their 5′ and 3′ ends. The most common natural way of protecting the RNA ends is by introduction of 5′ cap structure and 3′ poly(A) tail. In this example, the polypeptide component of the Gene Writer™ system is expressed from mRNA described in Example 12 (Improved expression of Gene Writer polypeptide by heterologous UTRs). The plasmid template for the RNA template production included T7 promoter followed by the IRES-expressing reporter gene (eGFP) flanked by R2Tg untranslated regions (UTRs) and 100 bp of homology to its rDNA target. The plasmid template was linearized by enzymatic restriction resulting in blunt end or 5′ overhang downstream of the RNA template sequence and used for in vitro transcription (IVT) using T7 polymerase (NEB). Following the IVT step the RNA was treated with DNase I (NEB) and either enzymatically polyadenylated by poly(A) polymerase (NEB) or not. After the buffer exchange step the enzymatic capping reaction resulting in cap 1 structure was performed as described in Example 14 (Improved Gene Writer components for RNA-based delivery) or not performed. The template RNA was concentrated and buffer exchanged. 50,000 HEK293T cells were co-transfected with 0.5 μg with the GeneWriter mRNA and the RNA template in 1:1 to 1:8 molar ratios using Neon transfection system (1150 V per pulse, 20 msec per pulse, 2 pulses in 10 μL tips in 96 well format).


After transfection, HEK293T cells were cultured for 1-3 days and then assayed for site-specific genome editing. Genomic DNA was isolated from each group of HEK293 cells. ddPCR was performed to confirm integration and assess integration efficiency. Taqman probes and primers were designed as described in PCT/US2019/048607 to amplify the expected product across 3′ end of integration junctions. The genome integration was improved when the enzymatically capped and poly(A) tailed template was used (FIG. 22).


The mean copy number of 0.498 integrants/genome was achieved in the presence of the 0.5 μg of mRNA and 1:8 molar ratio of mRNA:RNA template when the RNA template was enzymatically polyadenylated, in comparison to that of 0.031 integrants/genome when the RNA transgene was not enzymatically polyadenylated.


3′ End Modifications of RNAs.

It has been reported that the interactions between poly(A) tail shorter than 15-20 nts and the poly(A) binding protein (PABP) are destabilized resulting in the fast degradation of the RNA (Chang et al., Molecular Cell 53, 1044-1052 (2014); Subtelny et al., Nature 508, 66-71 (2014)). To determine the suitable lengths of the poly(A) tail of the template RNA we will test its lengths of 30, 40, 50, 60, 70, 80, 90 and 100 nucleotides. The IVT templates will be produced by PCR using reverse primers encoding the poly(A) tails of the abovementioned length. The IVT, DNase I treatment and capping of Gene Writer and the RNA template will be performed as described in Example 14 (Improved Gene Writer components for RNA-based delivery). After one to three days post-transfection the genomic integration will be assayed. In some embodiments, the genome integration will be higher for the RNA template tailed with a poly(A) tail of a suitable length.


In a cell the RNA degradation is initiated by shortening its poly(A) tail by deadenylases. Since the deadenylases are 3′-5′ exoribonucleases favoring the poly(A) stretches, the terminal uridine, cytidine and most often guanine detected in the natural poly(A) tails of many mRNA were proposed to protect the poly(A) tail from its shortening (Chang et al., Molecular Cell 53, 1044-1052 (2014)). We will assay the Gene Writer and template RNAs with the encoded poly(A) tail with terminal G or C, or intermittent Gs or Cs (similar to that used in Lim et al., Science 361, 701-704 (2018)) according as described before.


Some of the RNAs have been described to evolve alternative ways of protections their 3′ ends. A specific 16-nucleotide long stem-loop structure flanked with unpaired 5 nucleotides on both sides has been reported to protect the 3′ end of mRNA encoding H2a.X histone (Mannironi et al., Nucleic Acid Research 17, 9113-9126 (1989)). It has been shown that the heterologous mRNA ending with the histone stem-loop structure is cell cycle-regulated (Harris et al., Molecular Cellular Biology 11, 2416-2424 (1991); Stauber et al., EMBO Journal 5, 3297-3303 (1986)). The stem-loop structure is recognized and protected by the Stem-Loop Binding Protein (SLBP). The protein accumulates shortly before cells enter S-phase and is rapidly degraded at the end of S-phase (Whifield et al., Molecular Cellular Biology 20, 4188-4198 (2000)). The stem-loop element will be inserted to the 3′ end of the Gene Writer mRNA and the RNA templates and tested as described above to induce cell-cycle specific genome integration events.


Some viral and long non-coding RNAs have evolved to protect their 3′ ends with triple-helical structures (Brown et al., PNAS 109, 19202-19207 (2012)). Additionally, the structural elements of tRNA, Y RNA and vault RNA (reviewed in Labno et al., Biochemica et Biophysica Acta 1863, 3125-3147 (2016)) have been reported to extend half-life of these non-coding RNAs. We will insert the structures to protect the 3′ end of the RNA templates and probe their efficiencies in Gene Writing system as described above.


Finally, we will incorporate dNTP, 2′O-Methylated NTPs or phosphorothioate-NTP at the 3′ of the RNA transgenes to increase the half-life of these molecules by protecting the 3′ end of the RNA from exoribonucleases. We will incorporate single modified nucleotides or their stretches by extending the 3′end of the RNA by the DNA polymerases (for example, Klenow fragment) capable of extending an RNA sequence by adding modified nucleotides (Shcherbakova & Brenowitz, Nature Protocols 3, 288-302 (2008)).


A single nucleotide chemical modification of the 3′ end of the RNA can be done by first oxidation of 3′ terminal end of ribose sugar with sodium periodate to form a reactive aldehyde followed by conjugation of an aldehyde-reactive modified nucleotide. Alternatively, T4 DNA or T4 RNA ligases can be used for the splinted ligation (Moore & Query, Methods in Enzymology 317, 109-123 (2000)) of the stretches of modified nucleotides to the 3′ end of the RNAs.


Chemical ligation of two fragments is also possible. The phosphodiester bond linkage between two RNA substrates can be formed either by activating the phosphomonoester group using a reactive imidazolide or by using a condensing reagent such as cyanogen bromide. A disadvantage of chemical ligation is that it can also result in the creation of a 2′-5′ phosphodiester linkage, together with the desired 3′-5′ phosphodiester linkages.


5′ End Modifications of RNAs

In addition to the cap 1 structure described in Example 14 (Improved Gene Writer components for RNA-based delivery) other 5′ end protection groups will be explored. Particularly, we will use hypermethylated (Wurth et al. Nucleic Acid Res 42, 8663-8677 (2014)), phosphorothioate (Kuhn et al., Gene Therapy 17, 961-971 (2010)), NAD+-derived (Kiledjian, Trends in Cell Biology 28, 454-464 (2018)) and modified (for example, biotinylated: Bednarek et al., Phil Trans R Soc B 373, 20180167 (2018)) cap analogs for co-transcriptional capping.


We will also label the 5′ of the RNA with 5′-[y-thio]triphosphate to create a reactive sulfur group and chemically modify the 5′ end with the protective modifications using a haloacetamide derivative of the modified group.


The proposed modifications to protect 3′ and 5′ end of the RNA will be introduced in RNA templates and/or Gene Writer mRNA (if compatible with translation). The genome integration efficiencies of the RNAs will be tested as described in Example 14 (Improved Gene Writer components for RNA-based delivery).


Example 18: Use of Modified RNA Bases in a Gene Writer System

This example describes Gene Writer systems comprising modified RNA bases to potentially improve features of the system, e.g., increase efficiency of integration, decrease cellular response to foreign nucleic acids. For the Gene Writer polypeptide, the proposed modifications pertaining to the coding region are compatible with translation. For the RNA template, the proposed modifications are compatible with reverse transcription.


In this example, mRNA encoding the Gene Writer polypeptide was in vitro transcribed with a 100% replacement of the corresponding rNTP with one of the modified rNTPs: pseudouridine (Ψ), 1-N-methylpseudouridine (1-Me-Ψ), 5-methoxyuridine (5-MO-U) or 5-methylcytidine (5mC). Otherwise, the RNA preparation, purification and cell transfections were performed as described in the Example 14 (Improved Gene Writer components for RNA-based delivery). The gene integration capacity of the modified mRNAs was compared with that of the non-modified mRNA (GO) using ddPCR, with all polypeptide mRNAs being paired with an unmodified template RNA (FIG. 23). Integration was detected when the polypeptide was encoded using each modified rNTP, with the highest signal coming from 5-MO-U and the lowest from 5mC. This demonstrates that the Gene Writer polypeptide component is functional when expressed from mRNA containing modified bases.


Further, this example describes the modularity of the Gene Writer template molecule where it is composed of all or a subset of exemplary modules listed in FIG. 6 and illustrated in FIG. 5. Individual modules can be produced by chemical or in vitro syntheses as a contiguous nucleic acid molecule or in separate pieces that are later combined together. The individual modules of the Gene Writer template molecule can be chemically modified nucleic acids, be comprised in part or in entirety of non-nucleic acids, re-arranged in order, and/or omitted to form the Gene Writer template molecule.


In some embodiments, the Gene Writer template molecule (all modules, A-F) is synthesized by in vitro transcription where 0-100% replacement of a corresponding rNTP (adenosine, cytidine, guanosine, and/or uridine) is with one or more modified rNTPs (base or ribose modification), e.g., 5′ hydroxyl, 5′ Phosphate, 2′-O-methyl, 2′-O-ethyl, 2′-fluoro, 5 ribothymidine, C-5 propynyl-dC (pdC), C-5 propynyl-dU (pdU), C-5 propynyl-C(pC), C-5 propynyl-U (pU), 5-methyl C, 5-methyl U, 5-methyl dC, 5-methyl dU methoxy, (2,6-diaminopurine), 5′-Dimethoxytrityl-N4-ethyl-2′-deoxyCytidine, C-5 propynyl-fC (pfC), C-5 propynyl-fU (pfU), 5-methyl fC, 5-methyl fU, C-5 propynyl-mC (pmC), C-5 propynyl-fU (pmU), 5-methyl mC, 5-methyl mU, LNA (locked nucleic acid), MGB (minor groove binder) pseudouridine (Ψ), 1-N-methylpseudouridine (1-Me-Ψ), 5-methoxyuridine (5-MO-U). The modified nucleotides in this embodiment rely on incorporation through a transcription reaction which utilizes a natural or mutant polypeptide sequence of a RNA polymerase that readily incorporates modified nucleotides into a RNA transcript that is made in vitro (Padilla, R., Nucleic Acids Research, 30(24), 138e-138, 2002; Ibach, J., et. al., Journal of Biotechnology, 167(3), 287-295, 2013; Meyer, A. J., et. al., Nucleic Acids Research, 43(15), 7480-7488, 2015). The modified Gene Writer template molecule is typically in whole or in part compatible with the reverse transcriptase activity of the Gene Writer polypeptide sequence; for modules or parts of modules of the Gene Writer template molecule used as a template for reverse transcription, preference is given to modifications that are compatible with reverse transcription (Motorin et al., Methods in Enzymology 425 21-53, 2007; Mauger et al., PNAS 116, 24075-24083, 2019). Gene Writer systems with template molecules containing modified rNTPs are tested as described above and in Example 14 (Improved Gene Writer components for RNA-based delivery).


In some embodiments, individual modules are chemically synthesized containing modified nucleotides, e.g., 5′ hydroxyl, 5′ Phosphate, 2′-O-methyl, 2′-O-ethyl, 2′-fluoro, 25 ribothymidine, C-5 propynyl-dC (pdC), C-5 propynyl-dU (pdU), C-5 propynyl-C(pC), C-5 propynyl-U (pU), 5-methyl C, 5-methyl U, 5-methyl dC, 5-methyl dU methoxy, (2,6-diaminopurine), 5′-Dimethoxytrityl-N4-ethyl-2′-deoxyCytidine, C-5 propynyl-fC (pfC), C-5 propynyl-fU (pfU), 5-methyl fC, 5-methyl fU, C-5 propynyl-mC (pmC), C-5 propynyl-fU (pmU), 5-methyl mC, 5-methyl mU, LNA (locked nucleic acid), MGB (minor groove binder) pseudouridine (Ψ), 1-N-methylpseudouridine (1-Me-Ψ), 5-methoxyuridine (5-MO-U), where the individual modules are then ligated together through enzymatic (e.g., splint ligation using T4 DNA ligase, Moore, M. J., & Query, C. C. Methods in Enzymology, 317, 109-123, 2000) or chemical processes (e.g., Fedorova, O. A., et. al., Nucleosides and Nucleotides, 15(6), 1137-1147, 1996) to form a complete Gene Writer template molecule.


An example of a modified Gene Writer template molecule is where modules A and F are each 100 nt of chemically synthesized RNA with cytidine and uridine nucleotides containing 2′-O-methyl ribose modifications and module A contains (3) phosphorothioate linkages between the first 3 nucleotides on the 5′ end and module F contains (3) phosphorothioate linkages between the last 3 nucleotides on the 3′ end of the module. Modules B-E are synthesized by in vitro transcription using an RNA polymerase (RNAP), e.g., T7 RNAP, T3 RNAP, or SP6 RNAP (NEB), or derivatives thereof that possess enhanced properties, e.g., increased fidelity, increased processivity, or increased efficiency of incorporating modified nucleotides. Module A is ligated to the 5′ end of the in vitro transcribed module B-E molecule and module F is ligated on to the 3′ end of the in vitro transcribed module B-E molecule by splint ligation (described by Moore, M. J., & Query, C. C. Methods in Enzymology, 317, 109-123, 2000). This fully assembled template RNA (all modules, A-F) is then used with a Gene Writer polypeptide (or nucleic acid encoding the polypeptide) in a target cell to assess genomic integration as in previous examples. In some embodiments, RNA modifications do not decrease the efficiency of integration greater than 50%, e.g., as measured by ddPCR. In some embodiments, RNA modifications improve the efficiency of integration, e.g., as measured by ddPCR. In some embodiments, RNA modifications improve the reverse transcription reaction, e.g., improve the processivity or fidelity as measured by sequencing of integration events.


Example 19: Gene Writer Templates that do not Incorporate UTRs

This example describes a configuration of the Gene Writer template molecule that results in an exclusion of the UTRs, such that these regions used in retrotransposition are not integrated into the host cell.


In this example, we describe the positioning, omission, and/or substitution of the UTR modules of the Gene Writer template molecule (FIGS. 5 and 6) to result in the Gene Writer driver to not incorporate the UTR modules into the genome as a part of retrotransposition. In some embodiments, the Gene Writer template molecule modules for the 5′ and 3′ UTRs (modules B+C and E of Gene Writer template molecule) are moved to the ends of the molecule so that their function of interacting with the Gene Writer driver does not change but the homology arm is now located adjacent to the heterologous object sequence (module D) where complementarity of the homology arms act as a primer for reverse transcription. In some cases, modules B and/or C are omitted from the Gene Writer template molecule with module E following module F.


Additional examples of not incorporating the UTRs into the genome are removing modules B and C from the Gene Writer template molecule, re-positioning module F (3′ homology arm) to follow module D (heterologous object sequence) and have module E be substituted with a binding ligand such as biotin. This Gene Writer template molecule would now consist of module A (5′ homology arm)-module D (heterologous object sequence)-module F (3′ homology arm)-module E comprised of biotin. The Gene Writer driver polypeptide sequence would be modified to incorporate the amino acid sequence for monomeric streptavidin. This example illustrates how the utility of mediating a non-nucleic acid mediated association of the Gene Writer template molecule with the Gene Writer driver polypeptide sequence.


Example 20: Gene Writers can Integrate Genetic Cargo Independently of the Homology Directed Repair Pathway

This example describes the use of a Gene Writer system in a human cell wherein the homologous recombination repair pathway is inhibited.


In this example, U2OS cells were treated with 30 pmols (1.5˜M) non-targeting control siRNA (Ctrl) or a siRNA against Rad51, a core component of the homologous recombination repair pathway. SiRNAs were co-delivered with R2Tg driver and transgene plasmid in trans (see FIG. 24 for driver and transgene configuration schematic). Specifically, Plasmid expressing R2Tg, control R2Tg with a mutation in the RT domain, or control R2Tg with an endonuclease inactivating mutation were used in conjunction with transgene (FIG. 25 A, B). A total of 250 ng DNA plasmids with a 1:4 molar ration of driver to transgene, along with 30 pmol of siRNAs were nucleofected into 200k U2OS cells resuspended in 20 μL of nucleofection buffer SE using pulse code DN100. Protein lysates collected on day 3 showed the absence of Rad51 in the siRad51 treated condition (FIG. 25C). gDNA was extracted at day 3 and ddPCR assays to detect transgene integration at the rDNA locus was performed. The results of the ddPCR copy number analysis (in comparison to reference gene RPP30) are shown in FIG. 26. The absence of Rad51 leads to a ˜20% reduction in R2Tg mediated transgene integration at the rDNA locus both at the 3′ and 5′ junctions (FIG. 26), indicating that R2TG mediated transgene insertion is not wholly dependent on the presence of the homologous recombination pathway, and can occur in the absence of the endogenous HR pathway. In some embodiments, HR independence enables Gene Writing to work in cells and tissues with endogenously low levels of HR, e.g., liver, brain, retina, muscle, bone, nerve, cells in G0 or G1 phase, non-dividing cells, senescent cells, terminally differentiated cells. In some embodiments, HR independence enables Gene Writing to work in cells or in patients or tissues containing cells with mutations in genes involved in the HR pathway, e.g., BRCA1, BRCA2, P53, RAD51.


Example 21: Gene Writers can Integrate Genetic Cargo Independently of the Single-Stranded Template Repair Pathway

This example describes the use of a Gene Writer system in a human cell wherein the single-stranded template repair (SSTR) pathway is inhibited.


In this example, the SSTR pathway will be inhibited using siRNAs against the core components of the pathway: FANCA, FANCD2, FANCE, USP1. Control siRNAs of a non-target control will also be included. 200k U2OS cells will be nucleofected with 30 pmols (1.5 μM) siRNAs, as well as R2Tg driver and transgene plasmids (trans configuration). Specifically, 250 ng of Plasmids expressing R2Tg, control R2Tg with a mutation in the RT domain, or control R2Tg with an endonuclease inactivating mutation) are used in conjunction with transgene at a 1:4 molar ratio (driver to transgene). Transfections of U2OS cells is performed in SE buffer using program DN100. After nucleofection, cells are grown in complete medium for 3 days. gDNA is harvested on day 3 and ddPCR is performed to assess integration at the rDNA site. Transgene integration at rDNA is detected in the absence of core SSTR pathway components.


Example 22: Gene Writer Systems with Enhanced Activity for Target Vs Non-Target Cells

This example describes the incorporation of regulatory sequences into Gene Writer systems in order to decrease integration activity in non-target cells.


In this example, genetic regulation is accomplished through (i) using tissue-specific promoters to upregulate component expression and integration in target cells and (ii) using miRNA binding sites to decrease integration in non-target cells that have increased endogenous levels of the corresponding miRNA. Target cells used are human hepatocytes and non-target cells are hematopoetic stem cells (HSCs). The driver of integration here is a plasmid encoding the Gene Writer polypeptide (e.g., R2Tg retrotransposase) driven by different promoters and with scrambled or specific miRNA binding sites after the coding sequence. The template for integration is encoded on plasmid DNA, such that transcription results in a homology- and UTR-flanked heterologous object sequence. The heterologous object sequence may comprise a reporter gene that is driven by different promoters and with scrambled or specific miRNA binding sites after the coding sequence. The control promoter used here is CMV and the control for miRNA binding site is a randomly scrambled version of the binding site for miR-142. The target tissue-specific promoter used here is ApoE.HCR.hAAT, which is expressed in liver cells, and the off-target tissue-specific miRNA binding site is complementary to miR-142 (uguaguguuuccuacuuuaugga (SEQ ID NO: 1684)), which is expressed in HSCs.


Target cells and non-target cells are nucleofected with a combination of Gene Writer polypeptide (1) and template (2) selected from:

    • Gene Writer polypeptide constructs (1):
      • a. Non-specific driver: CMV-R2Tg
      • b. Non-specific inactivated driver: CMV-R2Tg(EN*)
      • c. Tissue-specific driver: ApoE.HCR.hAAT-R2Tg-miR142
      • d. Tissue-specific inactivated driver: ApoE.HCR.hAAT-R2Tg(EN*)-miR142
    • Gene Writer template constructs (2):
      • a. Non-specific transgene: CMV-gfp
      • b. Tissue-specific transgene: ApoE.HCR.hAAT-gfp-miR142


Cells are incubated for at least three days and subsequently evaluated for integration efficiency and reporter expression. For integration efficiency, ddPCR is performed to quantify the average number of integrations per genome for each sample. In some embodiments, the ratio between the integration efficiency in target cells and non-target cells is higher when using a template paired with the tissue-specific driver (la) vs a non-specific driver (1c). To assess reporter expression, cells are analyzed by flow cytometry to detect GFP fluorescence and RT-qPCR to detect transcription. In some embodiments, the ratio between fluorescence in target cells and non-target cells is higher when using a driver paired with a tissue-specific transgene cassette (2b) vs a non-specific transgene cassette (2a). In some embodiments, the ratio between transcript levels in target cells and non-target cells is higher when using a driver paired with a tissue-specific transgene cassette (2b) vs a non-specific transgene cassette (2a). In some embodiments, the combination of a tissue-specific driver (la) with a tissue-specific transgene cassette (2b) results in the highest ratio of transcription or expression between target and non-target cells. Alternatively, a screening assay can be performed in the same cell line artificially expressing or not expressing a given miRNA, e.g., the on-target screening cell is a HEK293T cell and the non-target cell is mimicked by introducing overexpression of miR-142 in HEK293T cells.


Example 23: Correction of Alpha-1 Antitrypsin Deficiency Using Lipid Nanoparticles Comprising Gene Writers

This example describes the use of a Gene Writer™ gene editing system to alter a genomic sequence at a single nucleotide in vivo. More specifically, the Gene Writer™ polypeptide and writing template are delivered to mouse liver cells via lipid nanoparticles to correct the SERPINA1 PiZ mutation causing alpha-1 antitrypsin deficiency.


Formulation and treatment of murine models with LNPs (LNP-INT01 system) carrying Cas9 and gRNA are taught by Finn et al. Cell Reports 22:2227-2235 (2018), the methods of which are incorporated herein by reference.


Capped and polyadenylated Gene Writer polypeptide mRNA containing N1-methyl pseudo-U is generated by in vitro transcription using a linearized plasmid DNA template and T7 RNA polymerase. The polypeptide mRNA is purified from enzyme and nucleotides using a MegaClear Transcription Clean-up Kit, in accordance with the manufacturer's protocol (ThermoFisher). The transcript concentration is determined by measuring the light absorbance at 260 nm (Nanodrop), and the transcript is analyzed by capillary electrophoresis by TapeStation (Agilent). Template RNA comprising the mutation correcting sequence is also prepared by in vitro transcription and translation using similar methods. In this example, the template RNA comprises the sequence as exemplified in Example 1.


LNPs are formulated with an amine-to-RNA-phosphate (N:P) ratio of 4.5. The lipid nanoparticle components are dissolved in 100% ethanol with the following molar ratios: 45 mol % LP01 lipid, 44 mol % cholesterol, 9 mol % DSPC, and 2 mol % PEG2k-DMG. The RNA cargo (1:40 molar ratio of polypeptide mRNA:template RNA) is dissolved in 50 mM acetate buffer (pH 4.5), resulting in a concentration of RNA cargo of approximately 0.45 mg/mL. LNPs are formed by microfluidic mixing of the lipid and RNA solutions using a Precision Nanosystems NanoAssemblr Benchtop Instrument, in accordance with the manufacturer's protocol. After mixing, the LNPs are collected and diluted in PBS (approximately 1:1), and then the remaining buffer is exchanged into PBS (100-fold excess of sample volume) overnight at 4 C under gentle stirring using a 10 kDa Slide-a-Lyzer G2 Dialysis Cassette (ThermoFisher Scientific). The resultant mixture is then filtered using a 0.2-mm sterile filter. The filtrate is stored at 2 C-8 C. Multi-dose formulations may be formulated using 25 mM citrate, 100 mM NaCl cargo buffer (pH 5), and buffer exchanged by TFF into tris-saline sucrose buffer (TSS) buffer (5% sucrose, 45 mM NaCl, and 50 mM Tris). Formulated LNPs have an average size of 105 nm. Encapsulation efficiencies are determined by ribogreen assay (Leung et al., 2012). Particle size and polydispersity are measured by dynamic light scattering (DLS) using a Malvern Zetasizer DLS instrument.


NSG-PiZ mice carrying the human SERPINA1 PiZ allele (E342K) are acquired from The Jackson Laboratory. To assess the ability of Gene Writing to edit the mutant allele in vivo, LNPs are dosed via the lateral tail vein at 3 mg/kg in a volume of 0.2 mL per animal. Excipient-treated animals are used as negative controls for all studies. Animals are euthanized at various time points by exsanguination via cardiac puncture under isoflurane anesthesia. In some embodiments, animals are euthanized at one week post-treatment to be analyzed for Gene Writing. Liver tissue is collected from the median or left lateral lobe from each animal for DNA extraction and analysis.


For NGS analysis of editing efficiency, PCR primers are designed around the target site, and the region of interest is amplified from extracted genomic DNA. Additional PCR is performed in accordance with the manufacturer's protocols (Illumina) to add the appropriate chemistry for sequencing, and amplicons are then sequenced on an Illumina MiSeq. Sequencing reads are aligned to the mouse reference genome after eliminating those having low quality scores. The resultant files containing the reads are mapped to the reference genome (BAM files), where reads that overlap the target region of interest are selected, and the number of wild-type reads versus the number of reads that contain the SERPINA1 reversion mutation encoded in the template RNA are calculated. The editing percentage (e.g., the “editing efficiency” or “percent editing”) is defined as the total number of reversion sequence reads over the total number of sequence reads.


In some embodiments, this example is repeated with additional groups of mice and a redosing regimen is used to analyze dose-to-effect properties of the system. In these experiments, mice are assigned to groups for weekly dosing up to 4 weeks, with euthanasia and tissue analysis as described herein being performed each week. In some embodiments, mice that receive more doses of the LNP formulation demonstrate higher Gene Writing efficiency by sequencing, e.g., mice receiving 2 doses one week apart that are analyzed at week three show a higher fraction of gene corrected reads by NGS of liver tissue samples as compared to mice receiving a single dose and analyzed at week three. In application, dosing in this manner may allow tuning of therapeutic intervention after evaluating patient response to one or more doses.


Example 24: Using Gene Writing to Address Repeat Expansion Diseases

This example describes the use of a Gene Writer™ gene editing system to treat a repeat expansion disease by rewriting a normal number of repeats into the locus. More specifically, the Gene Writer™ polypeptide and writing template are delivered to mouse CNS via AAV to reset the CAG repeats in HTT as per the custom template RNA to cure Huntington Disease. Healthy humans tend to carry between 10 and 35 CAG repeats within the huntingtin gene (HTT), while those with Huntington Disease may possess between 36 to greater than 120 repeats.


In this example, the template RNA is designed to correct the CAG repeat region of the HTT gene by encoding a sequence with 10 such repeats and homology to the flanking target sequence to fully write across the target locus. Multiple examples of such template RNAs could be designed, with an exemplary template RNA, as encoded in DNA, comprising the sequence (1)GGCGGCTGAGGAAGCTGAGG(2)GTTTTAGAGCTAGAAATAGCAAGTTAAAATAA GGCTAGTCCGTTATCAACTTGAAAAAGTGGGACCGAGTCGGTCC(3)AGTCCCTCAAG TCCTTCcagcagcagcagcagcagcagcagcagcagccgccaccgccgccgccgccgccgccgcctcct(4)CAGCTTCC TCAG (SEQ ID NO: 1685), where numbers are used to delineate the modules of the template in the order (5′-3′) (1) gRNA spacer, (2) gRNA scaffold, (3) heterologous object sequence, (4) 3′ homology priming domain, with the repeat correction being encoded in (3). The CAG repeat region is followed by a short repeat region encoding for 11 proline residues (8 residues being encoded by CCG triplets). Without wishing to be bound by theory, this region is included in (3) to place (4) in a more unique region to prevent mispriming. An exemplary gRNA for providing a second nick as described in embodiments of this system comprises the spacer sequence CGCTGCACCGACCGTGAGTT (SEQ ID NO: 1630) and directs a Cas9 nickase to nick the second strand of the target site within the homologous region. In some embodiments, this second nick improves the efficiency of the edit.


In order to deliver a complete Gene Writing system to the CNS, in this example, the Gene Writer is split across two AAV genomes, with the first encoding the nickase Cas9 domain fused to intein-N of a split intein pair (DnaE Intein-N: CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCL EDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN (SEQ ID NO: 1638)) and the second encoding the RT domain fused to an intein-C of a split intein pair (DnaE Intein-C, MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN (SEQ ID NO: 1640)) and the template RNA. The two polypeptide components are expressed from a polymerase II promoter, e.g. a neuronal cell-specific promoter described herein, and the template RNA and gRNA for providing a second nick are expressed from a polymerase III promoter, e.g. a U6 promoter. When co-infecting a cell, the two polypeptide components reconstitute a complete Gene Writer polypeptide with N-terminal Cas9 and C-terminal RT and the template RNA is expressed and reverse transcribed into the target locus. To achieve delivery for cells of the CNS (specifically the claudate nucleus and the putamen of the basal ganglia), the pseudotyped system rAAV2/1 is used here, where the AAV2 ITRs are used to package the described nucleic acids into particles with AAV1 capsid. AAV preparation and mouse injection and harvesting protocols used here follow the teachings of Monteys et al. Mol Ther 25(1):12-23 (2017).


FVB-Tg(YAC128)53Hay/J mice are acquired from The Jackson Laboratory. These transgenic mice express the full-length human huntingtin protein with ˜118 glutamine repeats (CAG trinucleotide repeats) and develop hyperkinesis at three months of age. At 8 weeks of age, mice are treated with a combination 1:1 of rAAV2/1-Cas9 virus and rAAV-MMLV_RT/hU6templateRNA virus. For rAAV injections, mice are anesthetized with isoflurane and 5 μL of rAAV mixture injected unilaterally into the right striata at 0.2 μL/min. After three weeks, mice are sacrificed and brain tissue taken for genomic DNA extraction and NGS analysis.


For NGS analysis of editing efficiency, PCR primers are designed flanking the target site, and the region of interest is amplified from extracted genomic DNA. Additional PCR is performed in accordance with the manufacturer's protocols (Illumina) to add the necessary chemistry for sequencing, and amplicons are then sequenced on an Illumina MiSeq. Sequencing reads are aligned to the mouse reference genome after eliminating those having low quality scores. The resultant files containing the reads are mapped to the reference genome (BAM files), where reads that overlap the target region of interest are selected, and the number of diseased allele (>35 CAG repeats) reads versus the number of repaired allele (10-35 CAG repeats) reads are calculated. The editing percentage (e.g., the “editing efficiency” or “percent editing”) is defined as the total number of repaired reads, as defined above, over the total number of sequence reads.


Example 25: Delivery of a Gene Writing System by LNP and AAV Vehicles

This example describes the use of a Gene Writer™ gene editing system to alter a genomic sequence at a single nucleotide in vivo. More specifically, the Gene Writer™ polypeptide and writing template are delivered to mouse liver cells via a combination of lipid nanoparticles (mRNA encoding polypeptide) and AAV (DNA encoding the RNA template) to correct the SERPINA1 PiZ mutation causing alpha-1 antitrypsin deficiency.


Capped and tailed mRNA encoding the Gene Writer polypeptide are prepared by in vitro transcription and formulated into LNP-INT01 as described in Example 23, but without template RNA co-formulation.


In this example, the template RNA is encoded as DNA and delivered via AAV. The teachings of Cunningham et al. Mol Ther 16(6):1081-1088 (2008) describe the use of rAAV2/8 with the human alpha-1 antitrypsin (hAAT) promoter and two copies of the hepatic control region of the apolipoprotein E enhancer (ApoE) to effectively transduce and drive expression of cargo in juvenile mouse liver. Accordingly, rAAV2/8.ApoE-hAAT.PiZ (rAAV2/8.PiZ) as described here comprises the above described AAV and promoter system driving expression of an RNA template for correcting the PiZ mutation, in addition to a second nick-directing gRNA being driven by a U6 promoter (RNA sequences previously described in Example 1).


NGS-PiZ mice carrying the human SERPINA1 PiZ allele (E342K) are acquired from The Jackson Laboratory. To assess the activity of Gene Writing to edit the mutant allele in vivo, 8-week-old mice are dosed i.p. with ˜1011 vg of rAAV2/8.PiZ to express the template RNA and via the lateral tail vein with formulated LNPs at 3 mg/kg in a volume of 0.2 mL per animal to express the Gene Writer polypeptide. Animals are euthanized at various time points by exsanguination via cardiac puncture under isoflurane anesthesia. In some embodiments, animals are euthanized at one week post-treatment to be analyzed for Gene Writing. Liver tissue is collected from the median or left lateral lobe from each animal for DNA extraction and analysis.


For NGS analysis of editing efficiency, PCR primers are designed around the target site, and the region of interest is amplified from extracted genomic DNA. Additional PCR is performed in accordance with the manufacturer's protocols (Illumina) to add the necessary chemistry for sequencing, and amplicons are then sequenced on an Illumina MiSeq. Sequencing reads are aligned to the mouse reference genome after eliminating those having low quality scores. The resultant files containing the reads are mapped to the reference genome (BAM files), where reads that overlap the target region of interest are selected, and the number of wild-type reads versus the number of reads that contain the SERPINA1 reversion mutation encoded in the template RNA are calculated. The editing percentage is defined as the total number of reversion sequence reads over the total number of sequence reads.


Example 26: Application of a Gene Writer™ System for Delivering Therapeutic Gene to Liver in a Human Chimeric Liver Mouse Model

This example describes a Gene Writer™ genome editing system delivered to the liver in vivo for integration and stable expression of a genetic payload. Specifically, LNPs are used to deliver a Gene Writing system capable of integrating a complete OTC expression cassette to treat a humanized mouse model of OTC-deficiency.


In this example, a Gene Writing system is used to treat a humanized mouse model of OTC deficiency, in which human hepatocytes derived from patients with OTC deficiency are engrafted into a mouse model (Ginn et al JHEP Reports 2019). An exemplary Gene Writing system for large payload integration comprises a Cas9-directed reverse transcriptase system utilizing a highly processive reverse transcriptase, e.g., MarathonRT. An exemplary template RNA component comprises, from 5′ to 3′, (1) a gRNA spacer with homology to the AAVS1 safe harbor site, (2) a gRNA scaffold, (3) a heterologous object sequence, and (4) a 3′ target homology region for annealing to the genomic DNA immediately upstream of the first strand nick to prime TPRT of the heterologous object sequence. An exemplary sequence for (1) is GGGGCCACTAGGGACAGGAT (SEQ ID NO: 1689). Region (2) carries the gRNA scaffold as described in this application, generally comprising the sequence GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAA AGTGGGACCGAGTCGGTCC (SEQ ID NO: 1591). In this example, (3) comprises a complete OTC expression cassette, where a liver-codon-optimized sequence encoding human OTC (UniProt P00480) is in operable association with the ApoE.hAAT promoter system as described in Example 25. An exemplary sequence for (4) is CTGTCCCTAGTG (SEQ ID NO: 1690). An exemplary sequence of an additional gRNA spacer for generating a second strand nick to improve the efficiency of integration is AGAGAGATGGCTCCAGGAAA (SEQ ID NO: 1691).


Eight to 12-week-old female Fah−/−Rag2−/−Il2rg−/− (FRG) mice are engrafted with human hepatocytes, isolated from pediatric donors or purchased from Lonza (Basel, Switzerland), as described previously (Azuma et al Nat Biotechnol 2007). Engrafted mice are cycled on and off 2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3-cyclohexanedione (NTBC) in drinking water to promote liver repopulation. Blood is collected every two weeks and at the end of the experiment to measure the levels of human albumin, used as a marker to estimate the level of engraftment, in serum by enzyme-linked immunosorbent assay (ELISA; Bethyl Laboratories, Inc., Montgomery, TX). Eleven weeks after engraftment, mice are treated with the Gene WriterTMs formulated as in Example 23. For treatment, LNPs are delivered via the lateral tail vein at 3 mg/kg in a volume of 0.2 mL per animal.


After vector injection, mice are cycled on NTBC for another 5 weeks before being euthanized. DNA and RNA are subsequently extracted from liver lysates by standard methods. OTC expression is subsequently assayed by performing RT-qPCR on isolated RNA samples using sequence-specific primers. Levels of human OTC are also measured throughout the experiment by using a human OTC ELISA kit (e.g., Aviva Systems Biology OTC ELISA Kit (Human) (OKCD07437)) on serum at Days −7, 0, 2, 4, 7, 14, 21, 28, and 35 post-injection, following the manufacturer's recommended protocol.


For analysis of editing efficiency, a ddPCR assay is performed using a pair of primers that anneal across either the 5′ junction or the 3′ junction of integration, with one primer in each set annealing to the heterologous object sequence, and the other to an appropriate region of the AAVS1 site on the genome. The assay is normalized to a reference gene to quantify the number of target site integrations per genome.


To analyze integrations at the target site, long-read sequencing across the integration site is performed. PCR primers are designed flanking the target site, and the region of interest is amplified from extracted genomic DNA. Additional PCR is performed in accordance with the manufacturer's protocols (PacBio) to add the necessary chemistry for sequencing, and amplicons are then sequenced via PacBio. Sequencing reads are aligned to the mouse reference genome after eliminating those having low quality scores. The resultant files containing the reads are mapped to the reference genome (BAM files), where reads that contain an insertion sequence relative to the reference genome are selected for further analysis to determine completeness of integration, defined in this example as containing the complete promoter and coding sequence of OTC.


Example 27: Gene Writers for Integration of a CAR in T-Cells Ex Vivo

This example describes delivery of a Gene Writer™ genome editing system to T-cells ex vivo for integration and stable expression of a genetic payload. Specifically, LNPs are used to deliver a Gene Writing system capable of integrating a chimeric antigen receptor (CAR) into the TRAC locus to generate CAR-T cells for treating B-cell lymphoma.


In this example, a Gene Writing system comprises a Gene Writing polypeptide, e.g., a nickase Cas9 and R2Tg reverse transcriptase domain, as described herein, a gRNA for directing nickase activity to the target locus, and a template RNA comprising, from 5′ to 3′:

    • (1) 100 nt homology to target site 3′ of first strand nick
    • (2) 5′ UTR from R2Tg
    • (3) Heterologous object sequence
    • (4) 3′ UTR from R2Tg
    • (5) 100 nt homology to target site 5′ of first strand nick


Wherein (3) comprises the coding sequence for the CD19-specific Hul9-CD828Z (Genbank MN698642; Brudno et al. Nat Med 26:270-280 (2020)) CAR molecule. The Gene Writer in this example is guided to the 5′ end of the first exon of TRAC by using a targeted gRNA, e.g., TCAGGGTTCTGGATATCTGT (SEQ ID NO: 1692), in order to place the cargo under endogenous expression control from that locus while disrupting the endogenous TCR, as taught by Eyquem et al. Nature 543:113-117 (2017). These three components (polypeptide, gRNA, and template) all comprise RNA, which is synthesized by in vitro transcription (e.g., polypeptide mRNA, template RNA) or chemical synthesis (gRNA).


The LNP formulation used in this example has been screened and validated for delivery to T-cells ex vivo, being taught in Billingsley et al. Nano Lett 20(3):1578-1589 (2020), which is incorporated herein by reference in its entirety. Specifically, the LNP formulation C14-4, comprising cholesterol, phospholipid, lipid-anchored PEG, and the ionizable lipid C14-4 (FIG. 2C of Billingsley et al. Nano Lett 20(3):1578-1589 (2020)) was used to encapsulate all three RNA components in a molar ratio of polypeptide mRNA:gRNA:template RNA of about 1:40:40.


Additional edits can be performed on T-cells in order to improve activity of the CAR-T cells against their cognate target. In some embodiments, a second LNP formulation of C14-4 as described comprises a Cas9/gRNA preformed RNP complex, wherein the gRNA targets the Pdcd1 exon 1 for PD-1 inactivation, which can enhance anti-tumor activity of CAR-T cells by disruption of this inhibitory checkpoint that can otherwise trigger suppression of the cells (see Rupp et al. Sci Rep 7:737 (2017)). The application of both nanoparticle formulation thus enables lymphoma targeting by providing the anti-CD19 cargo, while simultaneously boosting efficacy by knocking out the PD-1 checkpoint inhibitor. In some embodiments, cells may be treated with the nanoparticles simultaneously. In some embodiments, the cells may be treated with the nanoparticles in separate steps, e.g., first deliver the RNP for generating the PD-1 knockout, and subsequently treat cells with the nanoparticles carrying the anti-CD19 CAR. In some embodiments, the second component of the system that improves T cell efficacy may result in the knockout of PD-1, TCR, CTLA-4, HLA-I, HLA-II, CS1, CD52, B2M, MHC-I, MHC-II, CD3, FAS, PDC1, CISH, TRAC, or a combination thereof. In some embodiments, knockdown of PD-1, TCR, CTLA-4, HLA-I, HLA-II, CS1, CD52, B2M, MHC-I, MHC-II, CD3, FAS, PDC1, CISH, or TRAC may be preferred, e.g., using siRNA targeting PD-1. In some embodiments, siRNA targeting PD-1 may be achieved using self-delivering RNAi as described by Ligtenberg et al. Mol Ther 26(6):1482-1493 (2018) and in WO2010033247, incorporated herein by reference in its entirety, in which extensive chemical modifications of siRNAs, conferring the resulting hydrophobically modified siRNA molecules the ability to penetrate all cell types ex vivo and in vivo and achieve long-lasting specific target gene knockdown without any additional delivery formulations or techniques. In some embodiments, one or more components of the system may be delivered by other methods, e.g., electroporation. In some embodiments, additional regulators are knocked in to the cells for overexpression to control T cell- and NK cell-mediated immune responses and macrophage engulfment, e.g., PD-L1, HLA-G, CD47 (Han et al. PNAS 116(21):10441-10446 (2019)). Knock-in may be accomplished through application of an additional Gene Writing system with a template carrying an expression cassette for one or more such factors (3) with targeting to a safe harbor locus, e.g., AAVS1, e.g., using gRNA GGGGCCACTAGGGACAGGAT (SEQ ID NO: 1689) to target the Gene Writer polypeptide to AAVS1.


LNPs are used to treat primary T cells activated by Dynabeads at a 1:1 CD4+:CD8+ ratio at 450 ng/μL total mRNA concentrations. The resulting T cell populations are analyzed for integration, expression, and effect. For assessing integration, ddPCR is used with primers producing an amplicon extending from within the integrated CAR to the flanking genomic TRAC sequence. Comparing signal to a reference gene (e.g., RPP30), allows quantification of the average copy number per genome and integration efficiency. To analyze expression, flow cytometry with immunological probes is used to assess the level and percent of cells displaying surface CAR expression. To analyze activity of the CAR-T cells, treated cells are assessed via a co-plated cancer cell killing assay. By engineering Nalm6 ALL cells to express luciferase, cancer cell killing can be assessed by change in luminescence after co-culture with CAR-T cells as compared to signal from Nalm6 cells alone Billingsley et al. Nano Lett 20(3):1578-1589 (2020). Thus, a Gene Writing system can be used to generate CAR-T cells ex vivo with the desired cytotoxic activity.


Example 28: Gene Writers for integration of a CAR in T-cells in vivo

This example describes a Gene Writer™ genome editing system delivered to T-cells in vivo for integration and stable expression of a genetic payload. Specifically, targeted nanoparticles are used to deliver a Gene Writing system capable of integrating a chimeric antigen receptor (CAR) expression cassette into the murine Rosa26 locus to generate CAR-T cells in a murine model.


In this example, a Gene Writing system comprises a Gene Writing polypeptide, e.g., a nickase Cas9 and R2Tg reverse transcriptase domain, as described herein, a gRNA for directing nickase activity to the target locus, and a template RNA comprising, from 5′ to 3′:

    • (1) 100 nt homology to target site 3′ of first strand nick
    • (2) 5′ UTR from R2Tg
    • (3) Heterologous object sequence
    • (4) 3′ UTR from R2Tg
    • (5) 100 nt homology to target site 5′ of first strand nick


Wherein (3) comprises the coding sequence for the CD19-specific m194-1BBz CAR driven by the EF1a promoter (Smith et al. Nat Nanotechnol 12(8):813-820 (2017)). The Gene Writer in this example is guided to the murine Rosa26 locus using a gRNA, e.g., ACTCCAGTCTTTCTAGAAGA (SEQ ID NO: 1693) (Chu et al. Nat Biotechnol 33(5):543-548 (2015)). Production of RNA molecules is as according to examples provided herein, e.g., by in vitro transcription (e.g., Gene Writer polypeptide mRNA, template RNA) and by chemical synthesis (e.g., gRNA). Modifications to the RNA components of the system are as described elsewhere. For Gene Writer mRNA, the sequence additionally comprises a 5′ UTR (e.g., GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC (SEQ ID NO: 1603)) and a 3′ UTR (e.g., UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGA (SEQ ID NO: 1604)) flanking the coding sequence. This combination of 5′ UTR and 3′ UTR has been shown to result in good expression of an operably linked ORF (Richner et al. Cell 168(6): P1114-1125 (2017)).


In order to achieve delivery specifically to T-cells, targeted LNPs (tLNPs) are generated that carry a conjugated mAb against CD4. See, e.g., Ramishetti et al. ACS Nano 9(7):6706-6716 (2015). Alternatively, conjugating a mAb against CD3 can be used to target both CD4+ and CD8+ T-cells (Smith et al. Nat Nanotechnol 12(8):813-820 (2017)). In other embodiments, the nanoparticle used to deliver to T-cells in vivo is a constrained nanoparticle that lacks a targeting ligand, as taught by Lokugamage et al. Adv Mater 31(41):e1902251 (2019).


The tLNP can be made by first preparing the nucleic acid mix (e.g., polypeptide mRNA:gRNA:template RNA molar ratio of 1:40:40) with a mixture of lipids (cholesterol, DSPC, PEG-DMG, Dlin-MC3-DMA, and DSPE-PEG-maleimide) and then chemically conjugating the desired DTT-reduced mAb (e.g., anti-CD4, e.g., clone YTS.177) to the maleimide functional group on the LNPs. See Ramishetti et al. ACS Nano 9(7):6706-6716 (2015).


Six to 8 week old C57BL6/J mice are injected intravenously with formulated LNP at a dose of 1 mg RNA/kg body weight. Blood is collected at one day and three days post-administration in heparin-coated collection tubes, and the leukocytes are isolated by density centrifugation using Ficoll-Paque PLUS (GE Healthcare). Five days post-administration, animals are euthanized and blood and organs (spleen, lymph nodes, bone marrow cells) are harvested for T-cell analysis. Expression of the anti-CD19 CAR is detected by FACS using specific immunological sorting. Positive cells are confirmed for integration by ddPCR on the sorted population, where primers are used that flank an integration junction, e.g., one primer of the pair annealing to the integrated cargo and the other to genomic DNA from the Rosa26 target site.


Example 29: Assessment of Distance and PAM Orientation Between the First and Second Nicks to Reduce Non-Templated Indel Formation During Gene Writing

This examples describes how the placement of a second nick used in a Gene Writing system can be optimized to (1) increase the frequency of installation of a desired edit using a Gene Writer polypeptide with a template RNA, while (2) decreasing undesired insertions and/or deletions that may arise as a byproduct of the second nick.


An exemplary Gene Writing system can install a desired genomic modification (e.g., an insertion, deletion, or point mutation) using 1) a template RNA that comprises a gRNA and a heterologous object sequence comprising the desired genomic modification, and 2) a Gene Writing polypeptide comprising a nickase Cas9 (e.g., Cas9 N863A) fused to a reverse transcriptase (RT) (e.g., an RT domain from MMLV). In said exemplary Gene Writing system, the Cas9-RT fusion introduces a first nick, which exposes an available 3′OH to initiate the reverse transcriptase reaction using the template RNA as a template for target primed reverse transcription. The placement of a second nick adjacent to, but on the opposite strand as the first nick, enhances the installation of the desired genome modification.


In this experiment, a 3 nt insertion (CTT) is directed to the HEK3 locus. The template RNA for the insertion comprises (1) a gRNA spacer with homology to the HEK3 site, (2) a gRNA scaffold, (3) a heterologous object sequence including the CTT insertion, and (4) a 3′ target homology region for annealing to the genomic DNA immediately upstream of the first strand nick to set up target-primed reverse transcription of the heterologous object sequence. The sequence of the template RNA used is (5′-3′) GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCT AGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTCTGCCATCA<AAG>CG TGCTCAGTCTG (SEQ ID NO: 1694), where “< >” is used to denote the insertion sequence.


In addition, a set of second nick gRNAs, targeting a nick to the opposite DNA strand as the first nick, were designed that place a second nick either upstream or downstream of the location of the desired CTT insertion at various distances ranging from 26 to 257 bp. The upstream second nick creates a set of nicks with an inward orientation, with the PAM sites out (PAM-out), while the downstream second nick creates a set of nicks with an outward orientation, with the PAM sites inside the nicks (PAM-in), as described herein. Second nick gRNAs were designed using a web-based tool and are listed in Tables E5 and E6. The distance between dual nicks indicates the distance between the first nick directed by the template RNA and the second nick directed by the second nick gRNA, and the PAM orientation (e.g., “PAM-in” and thus outward orientation, or “PAM-out” and thus inward orientation) is provided with respect to the first nick as depicted in FIG. 31.









TABLE E5







gRNA targeting the second nick upstream of the


first nick in “PAM-out” orientation













Distance




PAM

between




orientation

dual




to first

nicks

PAM


nick
Orientation
 (nts)
sgRNA Sequence
Sequence














out
antisense
28
TGGGCCCCAAGGATTGACCC
AGG





(SEQ ID NO: 1695)






out
antisense
33
CCCAAGGATTGACCCAGGCC
AGG





(SEQ ID NO: 1696)






out
antisense
34
CCAAGGATTGACCCAGGCCA
GGG





(SEQ ID NO: 1697)






out
antisense
38
GGATTGACCCAGGCCAGGGC
TGG





(SEQ ID NO: 1698)






out
antisense
108
GCAGAAATAGACTAATTGCA
TGG





(SEQ ID NO: 1699)






out
antisense
109
CAGAAATAGACTAATTGCAT
GGG





(SEQ ID NO: 1700)






out
antisense
120
TAATTGCATGGGCGTTTCCC
TGG





(SEQ ID NO: 1701)






out
antisense
121
AATTGCATGGGCGTTTCCCT
GGG





(SEQ ID NO: 1702)






out
antisense
136
TCCCTGGGATCCCTGTCTCC
AGG





(SEQ ID NO: 1703)






out
antisense
161
TCTCTCATCCATGCCTTTCT
AGG





(SEQ ID NO: 1704)






out
antisense
197
CCCTTGCTTAAAACTCTCCA
AGG





(SEQ ID NO: 1705)






out
antisense
222
TCTCATGCCAAGCTCCCTGC
AGG





(SEQ ID NO: 1706)






out
antisense
232
AGCTCCCTGCAGGACATCCC
AGG





(SEQ ID NO: 1707)






out
antisense
240
GCAGGACATCCCAGGCCCTC
TGG





(SEQ ID NO: 1708)






out
antisense
241
CAGGACATCCCAGGCCCTCT
GGG





(SEQ ID NO: 1709)






out
antisense
255
CCCTCTGGGACAGCAGCTCA
CGG





(SEQ ID NO: 1710)






out
antisense
256
CCTCTGGGACAGCAGCTCAC
GGG





(SEQ ID NO: 1711)
















TABLE E6







gRNA targeting the second nick downstream of the


first nick in “PAM-in” orientation













Distance




PAM

between




orientation

dual




to first

nicks

PAM


nick
Orientation
(nts)
sgRNA Sequence
Sequence














In
antisense
26
GACGCCCTCTGGAGGAAGCA
GGG





(SEQ ID NO: 1712)






In
antisense
27
CGACGCCCTCTGGAGGAAGC
AGG





(SEQ ID NO: 1713)






In
antisense
34
TGTCCTGCGACGCCCTCTGG
AGG





(SEQ ID NO: 1714)






Ir
antisense
37
AGCTGTCCTGCGACGCCCTC
TGG





(SEQ ID NO: 1715)






In
antisense
63
GCACATACTAGCCCCTGTCT
AGG





(SEQ ID NO: 1716)






In
antisense
90
GTCAACCAGTATCCCGGTGC
AGG





(SEQ ID NO: 1717)






In
antisense
96
AAACTTGTCAACCAGTATCC
CGG





(SEQ ID NO: 1718)






In
antisense
133
CCAGGGACCTCCCTAGGTGC
TGG





(SEQ ID NO: 1719)






In
antisense
139
CCCCTTCCAGGGACCTCCCT
AGG





(SEQ ID NO: 1720)






In
antisense
150
GGTGAGGCTGGCCCCTTCCA
GGG





(SEQ ID NO: 1721)






In
antisense
151
TGGTGAGGCTGGCCCCTTCC
AGG





(SEQ ID NO: 1722)






In
antisense
162
CCCTCCTCTCCTGGTGAGGC
TGG





(SEQ ID NO: 1723)






In
antisense
166
AGGTCCCTCCTCTCCTGGTG
AGG





(SEQ ID NO: 1724)






In
antisense
171
GGGCCAGGTCCCTCCTCTCC
TGG





(SEQ ID NO: 1725)






In
antisense
186
GAGCTCGACCCTGAAGGGCC
AGG





(SEQ ID NO: 1726)






I1
antisense
191
CTGTTGAGCTCGACCCTGAA
GGG





(SEQ ID NO: 1727)






In
antisense
192
TCTGTTGAGCTCGACCCTGA
AGG





(SEQ ID NO: 1728)






In
antisense
233
GCTGAAAGCCACTGGGCTCT
GGG





(SEQ ID NO: 1729)






In
antisense
234
TGCTGAAAGCCACTGGGCTC
TGG





(SEQ ID NO: 1730)






In
antisense
240
TGCAGGTGCTGAAAGCCACT
GGG





(SEQ ID NO: 1731)






In
antisense
241
ATGCAGGTGCTGAAAGCCAC
TGG





(SEQ ID NO: 1732)






In
antisense
257
TTGATCTCTGATTTTCATGC
AGG





(SEQ ID NO: 1733)









To conduct the experiment, 200,000 U2OS cells in 20 μL SE buffer are nucleofected with 800 ng of plasmid encoding the Gene Writer polypeptide (N863ACas9-RT), 200 ng of template RNA, and 83 ng of a second nick gRNA listed in Tables E5 and E6. The Lonza Amaxa nucleofection system is used with the nucleofection code DN100. After nucleofection, 80 μL of DMEM+10% FBS medium are added to the cell suspension and the cells are plate in a 24 well plate with 500 μL of DMEM+10% FBS. Genomic DNA is extracted at day 3 post-nucleofection.


To analyze extracted DNA for the desired CTT insertion, amplicon sequencing is performed as described herein by amplifying the BEK locus using primers surrounding the first nick. The anticipated 300-350 bp amplicon is then sequenced on an Illumina MiSeq. The frequency of the desired CCT insertions is determined using the CRISPResso computational analysis pipeline (Clement et al. Nat Biotechnol 37(3):224-226 (2019)).


To measure undesired insertions and/or deletions arising as byproducts of the reaction, long-range amplification is performed with primers located >1.5 kb upstream and downstream of the first nick site, producing an amplicon >3 kb. This amplicon is sequenced using long-read sequencing (e.g., PacBio) and analyzed for the presence of insertions and deletions resulting from the dual nicking.


In some embodiments, a reaction using a second nick gRNA that cuts downstream of the first nick and provides a “PAM in” or outward orientation results in fewer unintended mutations (e.g., mutations in the target site other than the targeted CTT insertion) as compared to gRNAs placed upstream of the first nick at a similar distance but providing a “PAM-out” or inward orientation, as measured by the methods described herein. In other embodiments, a second nick gRNA that cuts upstream of the first nick and provides a “PAM-out” or inward orientation results in fewer undesired mutations (e.g., mutations in the target site other than the targeted CTT insertion) when the distance between the first and second nick is at least 100 nt as compared to a second nick gRNA providing a distance between the first and second nick of less than 100 nt, as measured by the methods described herein.


Thus, in some embodiments, a preferred design for a second nick gRNA is one resulting in 1) a “PAM-in” or outward orientation, or 2) a “PAM-out” or inward orientation with at least 100 nt separation between the first and second nicks (FIG. 32).


Example 30: Design and Human Cell Expression of Gene Writing Systems Utilizing Various Cas-RT Fusions

This example describes the construction and expression of Gene Writing polypeptides comprising fusions of Cas and reverse transcriptase domains in mammalian cells. Gene Writing polypeptides with these domains have been shown herein to enable the precise, site-specific modification of a DNA target from an RNA template molecule. Here, we describe the expression of a library of domains to create novel systems that may have diverse functional characteristics. More specifically, described here are fusion proteins comprising 1) a Cas-nuclease containing a mutation inactivating one endonuclease active site, e.g., the Cas9 nickase Cas9(N863A); 2) a peptide-linker to connect the functional protein domains, e.g., a sequence from Table 38 or 42, e.g., SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 1589); and 3) a reverse transcriptase (RT), e.g., an RT domain described in this application, e.g., an RT domain comprising a sequence from Table 1, Table 3, Table 30, Table 31, or Table 41, or a derivative thereof may be used in such an assay, collectively referred to in this Example as Cas-RT. Accordingly, Cas-RT fusion proteins are assembled on a plasmid and co-delivered with a single guide RNA (sgRNA) expression plasmid to validate system expression in human cells.


Gene Writer polypeptides generated by Cas-RT domain fusions assayed here comprised: (1) a Cas9 wild-type or Cas9(N863A) nickase domain; (2) a peptide linker (SGGSSGGSSGSETPGTSESATPESSGGSSGGSS (SEQ ID NO: 1589)); (3) a selection of RT domains from Table 1 and Table 30 taken from diverse sources; and (4) at least one nuclear localization signal. U2OS or HEK293T cells were transfected by Lonza Amaxa nucleofection of 250,000 cells/well with ˜800 ng of Cas9(N863A)-RT fusion plasmid with 200 ng of a sgRNA plasmid. To assess the expression level of Cas9-RT fusions, cell lysates were collected on day 2 post-transfection and analyzed by Western blot using a primary antibody against Cas9. Several Cas9-RT fusions showed appreciable protein expression (FIG. 33), suggestive of expression levels sufficient for Gene Writing activity. Notably, a wide range of expression levels is observed for the different Cas9-RT constructs, demonstrating the impact of the fusion design and RT selection on expression level of Cas-RT in cells.


Example 31: Improvement of Expression of Cas-RT Fusions Through Linker Selection

This example demonstrates the optimization of Cas-RT fusions to improve protein expression in mammalian cells. As described in Example 30, construction of novel Cas-RT fusions by the simple substitution of new functional domains may result in low or moderate expression of the Gene Writer polypeptide. Thus, it is contemplated here that modified configurations of the fusion may be advantageous in the context of different domains. Without wishing to be limited by the example, one such approach for improving the expression and stability of new fusions is through the use of a linker library. Here, the peptide linker sequence between the Cas and RT domains of the Cas-RT fusion is varied using a library of linker sequences. More specifically, linkers from Table 42 were used to generate new variants of a Cas9-RT fusion construct previously demonstrating low protein expression (see Example 30 and FIG. 33) and delivered to human cells to screen for improved Cas-RT protein expression.


A set of 22 peptide linkers (Table 42) with varying degrees of length, flexibility, hydrophobicity, and secondary structure was first used to generate variants of a Cas-RT fusion protein by substitution of the original linker (see Example 30). HEK293T cells were transfected by electroporation of 250,000 cells/well with ˜800 ng of each Cas9-RT fusion plasmid along with 200 ng of a single-guide RNA plasmid. To assess the expression level of Cas9-RT fusions, cell lysates were collected on day 2 post-transfection and analyzed by Western blot using a primary antibody against Cas9. Linker 10 listed in Table 42 significantly improved Cas-RT fusion expression (FIG. 34), demonstrating the potentially profound impact of the peptide linker sequence on Cas-RT expression.









TABLE 42







Peptide sequences used as linkers between


the Cas and RT domains in Gene Writer


polypeptides comprising Cas-RT fusions












SEQ ID



#
Linker sequence
NO:
Notes













1
GGS

Short





2
GGGGS
1535
Flexible, short





3
(GGGGS)2
3303
Flexible





4
(GGGGS)3
3304
Flexible, long





5
(GGGGS)4
3305
Flexible, very





long





6
(G)6
3310
Flexible





7
(G)8
3312
Flexible





8
GSAGSAAG
3410
Flexible



SGEF







9
(GSSGSS)
1736
Mid





10
(GSSGSS)2
3314
Mid, Flexible





11
(GSSGSS)3
3316
Mid





12
SGSETPGTS
1023
XTEN



ESATPES







13
(EAAAK)
1534
Rigid helix,





short





14
(EAAAK)2
3317
Rigid helix,





mid





15
(EAAAK)3
3318
Rigid helix,





long





16
PAP

Rigid, short





17
PAPAP
3322
Rigid, short





18
PAPAPAPAP
3324
Rigid, mid





19
A(EAAAK)4AL
3407
Rigid, very



EA(EAAAK)4A

long with





helices





20
GGGGS(EAAAK)
3408
Flexible-



GGGGS

helix-flex





21
(EAAAK)GGGGS
3409
Helix-flex-



(EAAAK)

helix





22
SGGSSGGSSGS
1589
Flexible-



ETPGTSESATP

XTEN-



ESSGGSSGGSS

flexible









Example 32: Cas-Mediated Cleavage Activity of Gene Writers Comprising Cas-RT Fusions

This example demonstrates the ability of Cas-RT fusions to retain functionality of the protein domains. Specifically, by assaying cells treated with Gene Writer polypeptides comprising a cleavage-competent Cas domain (cleavase), DNA binding can be read by target site analysis to demonstrate activity of Cas in the context of the fusions. Here, such Cas-RT cleavase fusions in which both nuclease active sites are functional, e.g., Cas9 (wild-type)-RT, were co-delivered on plasmid vectors along with a sgRNA-expression plasmid to target the Cas to the AAVS 1 site in human cells. Analysis of indel formation at the predicted cleavage site in AAVS 1 by Cas-RT cleavase fusions functioned as a readout of both DNA binding activity and endonuclease activity, thereby confirming effective DNA targeting by the Cas-RT fusions.


Cas-RT fusions with fully functional endonuclease domains, e.g., comprising wild-type Cas9 with both nuclease active sites intact, e.g., Cas9(N863), were generated from Cas-RT fusion proteins described herein, e.g., comprising a Cas9 nickase, e.g., Cas9(N863A), in order to increase the sensitivity of detection of DNA binding and cleavage. Since the intact Cas9 nuclease can cut both strands to generate a double-stranded cleavage event in the genome, repair of these sites generates a higher mutation (indel) signal than repair of a single-stranded DNA nick. Thus, the frequency of indel formation of the fusions was compared to that of unfused, wild-type Cas9 in order to assess the maintenance of Cas functionality when placed in the context of the novel Cas-RT fusions.


U2OS or HEK293T cells were transfected by Lonza Amaxa nucleofection of 250,000 cells/well with ˜800 ng of Cas9(WT)-RT fusion plasmid along with 200 ng of a sgRNA plasmid to produce the gRNA targeting Cas9 to AAVS1 (Table 43 gRNA P7). To assess the DNA binding and cleavage activity of Cas9-RT cleavase fusions, genomic DNA (gDNA) was collected on day 3 post-transfection. Indel patterns in the gDNA were analyzed by amplicon sequencing at loci targeted by the sgRNA. Sequencing results were analyzed by the CRISPResso2 pipeline (Clement et al Nat Biotechnol 37(3):224-226 (2019)). All tested Cas-RT cleavase fusions showed indel formation commensurate to their respective protein expression levels (FIG. 33), indicating that Cas-mediated DNA binding activity is retained in Cas-RT fusions (FIG. 35).


Example 33: Gene Writers Comprising Cas-RT Fusions with Various RT Domains Enable Precise Editing in Human Cells

This example demonstrates the ability of multiple tested Cas-RT fusions to programmably install mutations in genomic DNA in human cells. More specifically, the reverse transcriptase domain of Cas-RT fusions, e.g., an RT domain described in this application, was varied to determine the genome editing capacity of Cas-RT fusions employing novel RT combinations. Template RNAs were co-delivered on plasmid vectors along with Cas-RT expression plasmids in human cells to determine the Rewriting activity of Cas-RT fusions.


In order to generate domain libraries for Gene Writer polypeptides, Cas effector proteins were selected; see in Table 37 and Table 40A. Additional Cas9 domains were further selected for use in the Gene Writer polypeptides described herein, as features including PAM requirements of a target sequence, predicted mutations for conferring nickase activity (e.g., D10A, H840A, or N863A for SpCas9), and gRNA features including single-guide composition, e.g., specific spacer parameters and gRNA scaffold sequence for conferring polypeptide binding for the cognate Cas enzyme, were able to be determined (Table 40A). Linker sequences to connect Cas and RT domains were collected based on a search for diversity of length, flexibility, and composition in order to optimize fusion proteins (Tables 38 and 42). Optimization of fusion expression by linker screening is further described in Example 31. Reverse transcriptase domains were mined from a variety of sources using literature and RT protein domain signatures as described in this application, including from non-LTR retrotransposons, LTR retrotransposons, group II introns, diversity-generating elements, retrons, telomerases, retroplasmids, retroviruses, and polymerases with evolved RNA-dependent DNA polymerase activity (e.g., an RT domain comprising a sequence from Table 1, Table 3, Table 30, Table 31, Table 44, or Table N41, or a derivative thereof may be used in such an assay).


Specifically, to assess the use of novel RT domains in the context of a Gene Writer polypeptide to successfully edit the genome, a subset of exemplary RT domains from retroviruses was selected for fusion to a Cas9(N863A) nickase. Briefly, a database of POL proteins from Retroviridae was first generated and then prioritized (see The UniProt Consortium Nucleic Acids Res 47(D1):D506-D515 (2019); Mitchell et al. Nucleic Acids Res 47(D1):D351-D360 (2019)). Though not wishing to be limited by such example, retroviral RTs from the genera Betaretrovirus, Deltaretrovirus, Gammaretrovirus, and Spumavirus may function as monomeric proteins (see, for example, Table 1 from Herschhorn et al Cell Mol Life Sci 67(16):2717-2747 (2010)) and thus may be advantageous for use in a fusion protein, as described herein. A selection of retroviral monomeric RT sequences emerging from the analysis with these criteria is shown in Table 44. Further, mutations that have been shown to stabilize RT domains, as described in this application and in the literature (Table 45) (Anzalone et al Nat Biotechnol 38(7):824-844 (2020); Baranauskas et al Protein Eng Des Sel 25(10):657-668 (2012); Arezi and Hogrefe Nucleic Acids Res 37(2):473-481 (2009); Yasukawa et al J Biotechnol 150(3):299-306 (2010); the findings of which as they relate to improving RT stability and function are incorporated herein in their entirety), were analyzed for application to candidate RT domains (positions provided here based on the MMLV RT amino acid sequence as reference). As examples, MMLV RT with the mutational profile L139P/D200N/T330P/L603W/E607K showed an approximately 65-fold increase in processivity and 48-fold increase in template affinity (Baranauskas et al Protein Eng Des Sel 25(10):657-668 (2012)) and increased efficiency of prime editing of genomic DNA by a range of 1.6-5.1-fold with mutational profile D200N/T306K/W313F/T330P/L603W (Anzalone et al Nat Biotechnol 38(7):824-844 (2020)). From these studies, the core set of D200N/T330P/L603W was identified and an alignment of RT domains from the retroviral genera described here was used to predict the relevant amino acid positions where conserved (FIG. 36 A). The additional mutations T306K and/or W313F were also applied where relevant and L139P and/or E607K was used when neither mutation of the T306K/W313F set was able to be applied (FIG. 36 B). Cas9 nickase fusions with these wild-type RT domains or mutational variants with potentially improved activity were generated and exemplary fusions are described in Table 46.


To generate precise edits using Gene Writer Cas-RT fusions, Template RNAs were constructed to template reverse transcription of an edit into the genomic target site by the RT domain. Template RNAs were designed to comprise (i) a gRNA spacer sequence for guiding the Cas-RT to the target region, e.g., a sequence complementary to a 20-nucleotide sequence in the HEK3 locus; (ii) a primer-binding sequence capable of complementary base pairing with a single strand of the nicked DNA for target-primed reverse transcription; (iii) a heterologous object sequence providing a template for reverse transcription that further comprises the intended final target sequence; and (iv) a gRNA scaffold sequence to associate with the Cas9 domain of the Cas9-RT polypeptide fusion. The constructs employed here specifically followed the 5′ to 3′ orientation (i), (iv), (iii), (ii). Template RNAs encoded on plasmids were cloned such that expression was driven by the U6 promoter and transcription termination controlled by a 7 nt polyT stretch following the primer-binding sequence at the 3′ end of the Template RNA cassette. Template compositions are described in Table 43 (Templates P1, P2, P3).


U2OS or HEK293T cells were transfected by electroporation of 250,000 cells/well with ˜800 ng of Cas9-RT(MMLV) fusion expression plasmid, 200 ng of a Template RNA expression plasmid, and 83 ng of an additional second-nick gRNA (2gRNA P5) expression plasmid (Table 43). To assess the genome editing capacity of Cas-RT fusions, genomic DNA (gDNA) was collected on day 3 post-transfection. The frequency of intended (exact and scarless edit as designed) versus unintended (any non-intended changes to the target sequence) edits (“Activity ratio”) at target loci were analyzed by amplicon sequencing. As used herein, amplicon sequencing of a target site comprises the use of site-specific primers in PCR amplification of the target site, sequencing of amplicons on an Illumina MiSeq, and detection and characterization of editing events using the CRISPResso2 pipeline (Clement et al Nat Biotechnol 37(3):224-226 (2019)). Several Cas-RT fusions showed appreciable genome editing activity, with multiple Cas-RT fusions having Activity Ratios of ˜3 (FIG. 37), demonstrating that various Cas-RT fusions drawing from reverse transcriptase domains described herein can efficiently and precisely encode edits into the human genome.









TABLE 43







List of Template RNA and gRNA used in select examples.













Name
Description
spacer
scaffold
RT+ins
PBS
Template RNA





Template
HEK3_8PBS
GGCCCAGACTGA
GTTTTAGA
TCTGCCATCAA
CGTG
GGCCCAGACTGAG


P1
_10RT
GCACGTGA (SEQ
GCTAGAAA
AG (SEQ ID NO:
CTCA
CACGTGAGTTTTAG



(CTTat1)
ID NO: 3574)
TAGCAAGT
3576)

AGCTAGAAATAGC





TAAAATAA


AAGTTAAAATAAG





GGCTAGTC


GCTAGTCCGTTATC





CGTTATCA


AACTTGAAAAAGT





ACTTGAAA


GGCACCGAGTCGGT





AAGTGGCA


GCTCTGCCATCAAA





CCGAGTCG


GCGTGCTCA (SEQ





GTGC (SEQ


ID NO: 3577)





ID NO: 3575)








Template
HEK3_13PB
GGCCCAGACTGA
GTTTTAGA
TCTGCCATCAA
CGTG
GGCCCAGACTGAG


P2
S_10RT
GCACGTGA (SEQ
GCTAGAAA
AG (SEQ ID NO:
CTCA
CACGTGAGTTTTAG



(CTTat1)
ID NO: 3574)
TAGCAAGT
3576)
GTCT
AGCTAGAAATAGC





TAAAATAA

G
AAGTTAAAATAAG





GGCTAGTC

(SEQ
GCTAGTCCGTTATC





CGTTATCA

ID
AACTTGAAAAAGT





ACTTGAAA

NO:
GGCACCGAGTCGGT





AAGTGGCA

3578)
GCTCTGCCATCAAA





CCGAGTCG


GCGTGCTCAGTCTG





GTGC (SEQ


(SEQ ID NO: 3579)





ID NO: 3575)








Template
HEK3_17PB
GGCCCAGACTGA
GTTTTAGA
TCTGCCATCAA
CGTG
GGCCCAGACTGAG


P3
S_10RT
GCACGTGA (SEQ
GCTAGAAA
AG (SEQ ID NO:
CTCA
CACGTGAGTTTTAG



(CTTat1)
ID NO: 3574)
TAGCAAGT
3576)
GTCT
AGCTAGAAATAGC





TAAAATAA

GGGC
AAGTTAAAATAAG





GGCTAGTC

C
GCTAGTCCGTTATC





CGTTATCA

(SEQ
AACTTGAAAAAGT





ACTTGAAA

ID
GGCACCGAGTCGGT





AAGTGGCA

NO:
GCTCTGCCATCAAA





CCGAGTCG

3580)
GCGTGCTCAGTCTG





GTGC (SEQ


GGCC (SEQ ID NO:





ID NO: 3575)


3581)





Template
HBB_13PBS_
GCATGGTGCACC
GTTTTAGA
AGACTTCTCCA
GAGT
GCATGGTGCACCTG


P4
10RT
TGACTCCTG
GCTAGAAA
CAG (SEQ ID
CAGG
ACTCCTGGTTTTAG



(TtoAat4)
(SEQ ID NO: 3582)
TAGCAAGT
NO: 3583)
TGCA
AGCTAGAAATAGC





TAAAATAA

C
AAGTTAAAATAAG





GGCTAGTC

(SEQ
GCTAGTCCGTTATC





CGTTATCA

ID
AACTTGAAAAAGT





ACTTGAAA

NO:
GGCACCGAGTCGGT





AAGTGGCA

3584)
GCAGACTTCTCCAC





CCGAGTCG


AGGAGTCAGGTGC





GTGC (SEQ


AC (SEQ ID NO:





ID NO: 3575)


3585)





2gRNA
HEK3_+90
GTCAACCAGTAT
GTTTTAGA
NA
NA
NA


P5

CCCGGTGC (SEQ
GCTAGAAA







ID NO: 1717)
TAGCAAGT








TAAAATAA








GGCTAGTC








CGTTATCA








ACTTGAAA








AAGTGGCA








CCGAGTCG








GTGC (SEQ








ID NO: 3575)








2gRNA
HBB_+72
GCCTTGATACCA
GTTTTAGA
NA
NA
NA


P6

ACCTGCCCA
GCTAGAAA







(SEQ ID NO: 3586)
TAGCAAGT








TAAAATAA








GGCTAGTC








CGTTATCA








ACTTGAAA








AAGTGGCA








CCGAGTCG








GTGC (SEQ








ID NO: 3575)








gRNA
g19_AAVS1
GTCCCCTCCACC
GTTTTAGA
NA
NA
NA


P7

CCACAGTG (SEQ
GCTAGAAA







ID NO: 3587)
TAGCAAGT








TAAAATAA








GGCTAGTC








CGTTATCA








ACTTGAAA








AAGTGGCA








CCGAGTCG








GTGC (SEQ








ID NO: 3575)









Example 34: Multiplexing of a Gene Writer System to Simultaneously Edit Multiple Loci in a Human Cell

This example demonstrates the use of a Gene Writer system to edit multiple sites in the genome. In some applications, it may be of high value to be able to engineer multiple locations in the genome, e.g., to correct multiple genetic mutations or to optimize an engineered cell for cell therapy by performing multiple simultaneous modifications ex vivo or in vivo. In this example, a 3-plasmid system is utilized comprising: 1) a Gene Writer polypeptide expression plasmid, e.g., a plasmid encoding a Cas9 nickase fused to a reverse transcriptase (Cas-RT); 2) a Template plasmid, e.g., a plasmid encoding an expression cassette for a Template RNA that determines the genome site and the edit to instill at that site; and 3) a second-nick gRNA expression plasmid, e.g., a plasmid encoding an additional gRNA sequence to direct a second-strand nick for Cas9 at a location proximal to the target site.


In this example, two genome loci, the HBB gene and the human HEK3 locus, were targeted using gRNA comprising spacer sequences with identity to these sites to determine the ability to target multiple loci in parallel. To assess targeting of either locus separately or both simultaneously, cells were treated with different compositions of the Template plasmids to enable targeting of: 1) HEK3 alone, 2) HBB alone, or 3) both HBB and the HEK3 locus. Specifically, 800 ng of plasmid encoding the Cas9-RT(MMLV) fusion (Table 46), 200 ng of plasmid encoding the HEK3-modifying Template (Template P2, Table 43) and/or plasmid encoding the HBB-modifying Template (Template P4, Table 43), and 83 ng of plasmid encoding the HEK3 second-nick gRNA (2gRNA P5, Table 43) and/or plasmid encoding the HBB second-nick gRNA (2gRNA P6, Table 43) were nucleofected using nucleofection program DS_150 into HEK293T cells. After nucleofection, cells were grown at 37° C., 5% C02 for 3 days prior to cell lysis and genomic DNA extraction. Primers specific to each locus were used to amplify the region and amplicons were sequenced using an Illumina MiSeq. Perfect correction and indel rates were analyzed using the CRISPResso2 pipeline (Clement et al Nat Biotechnol 37(3):224-226 (2019)) to determine Gene Writing efficacy. Table 51 lists the components of the Gene Writer System used in this example.















TABLE 51





Name
Description
spacer
scaffold
RT+ins
PBS
Template RNA







Template
HEK
GGCCCA
GTTTTAGAGCT
TCTGC
CGTGCTC
GGCCCAGACTGAGCACGT


P1
3_8P
GACTGA
AGAAATAGCA
CATCA
A
GAGTTTTAGAGCTAGAAA



BS_1
GCACGT
AGTTAAAATA
AAG

TAGCAAGTTAAAATAAGG



ORT
GA (SEQ
AGGCTAGTCC
(SEQ

CTAGTCCGTTATCAACTTG



(CTTa
ID NO:
GTTATCAACTT
ID NO:

AAAAAGTGGCACCGAGTC



t1)
3574)
GAAAAAGTGG
3576)

GGTGCTCTGCCATCAAAG





CACCGAGTCG


CGTGCTCA (SEQ ID NO:





GTGC (SEQ ID


3577)





NO: 3575)








Template
HEK
GGCCCA
GTTTTAGAGCT
TCTGC
CGTGCTC
GGCCCAGACTGAGCACGT


P2
3_13
GACTGA
AGAAATAGCA
CATCA
AGTCTG
GAGTTTTAGAGCTAGAAA



PBS_
GCACGT
AGTTAAAATA
AAG
(SEQ ID
TAGCAAGTTAAAATAAGG



10RT
GA (SEQ
AGGCTAGTCC
(SEQ
NO: 3578)
CTAGTCCGTTATCAACTTG



(CTT
ID NO:
GTTATCAACTT
ID NO:

AAAAAGTGGCACCGAGTC



at1)
3574)
GAAAAAGTGG
3576)

GGTGCTCTGCCATCAAAG





CACCGAGTCG


CGTGCTCAGTCTG (SEQ ID





GTGC (SEQ ID


NO: 3579)





NO: 3575)








Template
HEK
GGCCCA
GTTTTAGAGCT
TCTGC
CGTGCTC
GGCCCAGACTGAGCACGT


P3
3_17
GACTGA
AGAAATAGCA
CATCA
AGTCTG
GAGTTTTAGAGCTAGAAA



PBS_
GCACGT
AGTTAAAATA
AAG
GGCC
TAGCAAGTTAAAATAAGG



10RT
GA (SEQ
AGGCTAGTCC
(SEQ
(SEQ ID
CTAGTCCGTTATCAACTTG



(CTT
ID NO:
GTTATCAACTT
ID NO:
NO: 3580)
AAAAAGTGGCACCGAGTC



at1)
3574)
GAAAAAGTGG
3576)

GGTGCTCTGCCATCAAAG





CACCGAGTCG


CGTGCTCAGTCTGGGCC





GTGC (SEQ ID


(SEQ ID NO: 3581)





NO: 3575)








Template
HBB_
GCATGG
GTTTTAGAGCT
AGAC
GAGTCA
GCATGGTGCACCTGACTC


P4
13P
TGCACC
AGAAATAGCA
TTCTC
GGTGCA
CTGGTTTTAGAGCTAGAA



BS_1
TGACTC
AGTTAAAATA
CACA
C (SEQ ID
ATAGCAAGTTAAAATAAG



ORT
CTG
AGGCTAGTCC
G (SEQ
NO: 3584)
GCTAGTCCGTTATCAACTT



(TtoA
(SEQ ID
GTTATCAACTT
ID NO:

GAAAAAGTGGCACCGAGT



at4)
NO: 3582)
GAAAAAGTGG
3583)

CGGTGCAGACTTCTCCAC





CACCGAGTCG


AGGAGTCAGGTGCAC





GTGC (SEQ ID


(SEQ ID NO: 3585)





NO: 3575)








2gRNA
HEK3_+90
GTCAAC
GTTTTAGAGCT
NA
NA
NA


P5

CAGTAT
AGAAATAGCA







CCCGGT
AGTTAAAATA







GC (SEQ
AGGCTAGTCC







ID NO:
GTTATCAACTT







1717)
GAAAAAGTGG








CACCGAGTCG








GTGC (SEQ ID








NO: 3575)








2gRNA
HBB_+72
GCCTTG
GTTTTAGAGCT
NA
NA
NA


P6

ATACCA
AGAAATAGCA







ACCTGC
AGTTAAAATA







CCA
AGGCTAGTCC







(SEQ ID
GTTATCAACTT







NO: 3586)
GAAAAAGTGG








CACCGAGTCG








GTGC (SEQ ID








NO: 3575)








gRNA
g19_
GTCCCC
GTTTTAGAGCT
NA
NA
NA


P7
AAV
TCCACC
AGAAATAGCA






S1
CCACAG
AGTTAAAATA







TG (SEQ
AGGCTAGTCC







ID NO:
GTTATCAACTT







3587)
GAAAAAGTGG








CACCGAGTCG








GTGC (SEQ ID








NO: 3575)









When tested independently, both targets saw a high degree of precise correction, with approximately 36% editing in HEK3 and 23% editing in HBB (FIG. 38). Further, when targeted at the same time, approximately 34% editing of HEK3 and 14% editing of HBB target sites was achieved with precise correction conferred by the respective Template RNAs. Additionally, insertions and deletions were observed with low frequency in all conditions, with indels for each locus reaching similar levels when tested alone or in combination. Though not the express intent of this example, the lack of increase in indels during simultaneous editing is a positive indicator for the potential to increase the number of loci targetable in parallel without compromising the precision of each individual edit.


Example 35: Delivery of DNA-Free Gene Writer Systems Through Nucleofection of Human Cells

This example describes the application of a Gene Writer system to edit the genome in human cells via delivery of RNA components, e.g., mRNA encoding the Gene Writer polypeptide and an RNA template. Without wishing to be bound by theory, the ability to deliver only RNA components in the absence of DNA is expected to confer major advantages to this system, including a reduction in immunogenicity and cellular toxicity linked to the detection of DNA in the cytoplasm and the availability of lipid nanoparticles systems described herein, the majority of which are optimized for RNA delivery, that can circumvent issues associated with viral delivery of nucleic acid therapeutics (e.g., manufacturing challenges, pre-existing immunity, immunogenic response to viral proteins). The reduction in cellular toxicity through use of an RNA system may be especially important for the modification of more sensitive cell types, such as primary cells. Further, nucleofection may be an effective method of delivering these systems to a patient's cells, e.g., for ex vivo cell engineering. Thus, it is of significant value to demonstrate the capacity of a Gene Writing system to function appropriately when delivered as all RNA and in the absence of DNA. Specifically, this example demonstrates delivery of an all-RNA Gene Writing system to modify the genome of HEK293T cells. To demonstrate RNA-based Gene Writing is not limited to a single composition, two versions of a Cas-RT fusion polypeptide are employed that comprise an RT domain derived from either Moloney murine leukemia virus (Cas9-RT(MMILV)) or porcine endogenous retrovirus (Cas9-RT(PERV)) (Table 46).


Gene Writer polypeptide-encoding mRNAs (1) were generated using T7 polymerase-driven in vitro transcription. In general, plasmids encoding the mRNA constructs comprised a transcriptional cassette comprising the following components: T7 promoter, 5′UTR, Gene Writer coding sequence (Cas9 nickase fused with a reverse transcriptase by a peptide linker and further comprising a nuclear localization signal), 3′UTR, and an 80 nt polyA tail (SEQ ID NO: 3666). In this example, RNA molecules were prepared using unmodified nucleotides from linearized plasmid template. The mRNAs encoding Cas9-RT(MMLV) or Cas9-RT(PERV) (Table47) were co-transcriptionally capped with CleanCap AG (TriLink BioTechnologies).


Gene Writer Template RNAs (2) encoding genomic edits were generated by chemical synthesis and purified by standard desalting. The first and last three bases of each Template RNA comprised 2′-O-methyl phosphorothioate modifications. Template RNAs of varying length were designed to introduce different mutations into the human HEK3 locus (Table 48).


Where indicated, second nick gRNAs (3) were generated by chemical synthesis and comprised the following sequence modifications:











(SEQ ID NO: 3588)



mG*mC*mA*rGrArArArUrArGrArCrUrArArUrUr






GrCrArGrUrUrUrUrArGrArGrCrUrArGrArArAr






UrArGrCrArArGrUrUrArArArArUrArArGrGrCr






UrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrAr






ArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGr






UrGrCmU*mU*mU*rU.






To assay the RNA Gene Writing systems described herein, HEK293T cells were plated 2 days before nucleofection to obtain 70-80% confluency on the day of nucleofection. RNAs were mixed according to the following combinations: i) Cas9-RT mRNA (1) only; ii) Cas9-RT mRNA (1), template RNA (2), and second nick gRNA (3); or iii) Cas9-RT mRNA (1) and template RNA (2). RNA mixes comprised 4.5 μg of the Cas9-RT mRNA (1), 5 μM final concentration of template RNA (2), and 1.3 μM final concentration of second nick gRNA (3). Mixes were nucleofected into approximately 200,000 cells using the Lonza Amaxa Nucleofector 96 Well Shuttle System, as according to manufacturer's protocols. Cells were then lysed and genomic DNA was collected 72 hours after nucleofection. Amplicon sequencing libraries were prepared using primers to amplify across the target site and Illumina sequencing was performed. Precise correction and indel rates were analyzed using the CRISPResso2 pipeline (Clement et al Nat Biotechnol 37(3):224-226 (2019)).


In these experiments, approximately 20% precise Writing activity was achieved with Cas9-RT(MMLV) using Template 1 (Table 48). A drop in activity was observed for templates that were longer than 120 nt in length; Template 4, which encoded the same edit as Template 1, but with an addition of 20 nt at the 3′ end of the RT template, showed an approximately 3.1-fold drop in precise Writing activity and an approximately 2.4-fold drop in the ratio of precise corrections to indels (FIG. 39). The use of Cas9-RT-encoding mRNAs with different UTRs and capping approaches produced similar levels of activity, though there was a slight increase with mRNA-5 (Table 49; FIG. 40). The all-RNA nucleofection of the Gene Writer Cas9-RT(PERV) with Template 1 and the second-nick gRNA further resulted in a precise Writing efficiency of approximately 7% (FIG. 41). Across the experiments of this example, the addition of the second-nick gRNA resulted in an increase in Writing activity.


Table 48 provides sequences of Template RNA molecules used in all-RNA Gene Writing Examples. The spacer sequence of each Template RNA described here was kept constant and comprised 20 nt (5′-GGCCCAGACTGAGCACGTGA-3′ (SEQ ID NO: 3574)) of 100% identity to a target site in the human HEK3 locus (also known as LINC01509) (sequence maps to NC_000009.12:107422339.107422358, assembly GRCh38.p13). A Template RNA will typically comprise the components shown in the table, such that spacer+scaffold+RT+edit+PBS+Tail can yield the complete molecule.









TABLE 48







Template RNAs used in various Examples disclosed herein

















Descrip-





TemplateRNA
RNA



Name
tion
spacer
scaffold
RT+edit
PBS
Tail
Combined
Sequence
Length





Template
HEK3_13P
GGCC
GTTT
TCTG
CGT
TTTT
GGCC
mG*mG*mC*r
126


1
BS_10RT(C
CAGA
TAGA
CCAT
GCT

CAGA
CrCrArGrAr




TTat1)
CTGA
GCTA
CAAA
CA

CTGA
CrUrGrArGr





GCAC
GAAA
G
GTC

GCAC
CrArCrGrUr





GTGA
TAGC
(SEQ ID
TG

GTGA
GrArGrUrUr





(SEQ
AAGT
NO: 3576)
(SEQ

GTTT
UrUrArGrAr





ID
TAAA

ID

TAGA
GrCrUrArGr





NO:
ATAA

NO: 

GCTA
ArArArUrAr





3574)
GGCT

3578)

GAAA
GrCrArArGr






AGTC



TAGC
UrUrArArAr






CGTT



AAGT
ÅrUrArArGr






ATCA



TAAA
GrCrUrArGr






ACTT



ATAA
UrCrCrGrUr






GAAA



GGCT
UrArUrCrAr






AAGT



AGTC
ArCrUrUrGr






GGCA



CGTT
ArArArArAr






CCGA



ATCA
GrUrGrGrCr






GTCG



ACTT
ArCrCrGrAr






GTGC



GAAA
GrUrCrGrGr






(SEQ ID



AAGT
UrGrCrUrCr






NO: 3575)



GGCA
UrGrCrCrAr










CCGA
UrCrArArAr










GTCG
GrCrGrUrGr










GTGC
CrUrCrArGr










TCTG
UrCrUrGrU*










CCAT
mU*mU*mU










CAAA
(SEQ ID 










GCGT
NO: 3590)










GCTC











AGTC











TGTT











TT 











(SEQ











ID











NO:











3589)







Template
HEK3_13P
GGCC
GTTT
TCTG
CGT
TTTT
GGCC
mG*mG*mC*r
133


2
BS_10RT
CAGA
TAGA
CCAT
GCT

CAGA
CrCrArGrAr




(10
CTGA
GCTA
CACA
CA

CTGA
CrUrGrArGr




nts at1)
GCAC
GAAA
TGTA
GTC

GCAC
CrArCrGrUr





GTGA
TAGC
GTTG
TG

GTGA
GrArGrUrUr





(SEQ
AAGT
(SEQ ID
(SEQ

GTTT
UrUrArGrAr





ID
TAAA
NO: 3591)
ID

TAGA
GrCrUrArGr





NO:
ATAA

NO: 

GCTA
ArArArUrAr





3574)
GGCT

3578)

GAAA
GrCrArArGr






AGTC



TAGC
UrUrArArAr






CGTT



AAGT
ArUrArArGr






ATCA



TAAA
GrCrUrArGr






ACTT



ATAA
UrCrCrGrUr






GAAA



GGCT
UrArUrCrAr






AAGT



AGTC
ArCrUrUrGr






GGCA



CGTT
ArArArArAr






CCGA



ATCA
GrUrGrGrCr






GTCG



ACTT
ArCrCrGrAr






GTGC



GAAA
GrUrCrGrGr






(SEQ ID



AAGT
UrGrCrUrCr






NO: 3575)



GGCA
UrGrCrCrAr










CCGA
UrCrArCrAr










GTCG
UrGrUrArGr










GTGC
UrUrGrCrGr










TCTG
UrGrCrUrCr










CCAT
ArGrUrCrUr










CACA
GrU*mU*mU*










TGTA
mU (SEQ ID










GTTG
NO: 3593)










CGTG











CTCA











GTCT











GTTT











T 











(SEQ











ID











NO:











3592)







Template
HEK3_13P
GGCC
GTTT
TCTG
CGT
TTTT
GGCC
mG*mG*mC*
143


3
BS_10RT
CAGA
TAGA
CCAT
GCT

CAGA
rCrC




(20
CTGA
GCTA
CACA
CA

CTGA
rArGrArCrU




nts at1)
GCAC
GAAA
TGTA
GTC

GCAC
rGrArGrCrA





GTGA
TAGC
GTTG
TG

GTGA
rCrGrUrGrA





(SEQ
AAGT
AGGT
(SEQ

GTTT
rGrUrUrUrU





NO:
TAAA
CAAT
ID

TAGA
rArGrArGrC





3574)
ATAA
GA 
NO: 

GCTA
rUrArGrArA






GGCT
(SEQ ID
3578)

GAAA
rArUrArGrC






AGTC
NO:3594)


TAGC
rArArGrUrU






CGTT



AAGT
rArArArArU






ATCA



TAAA
rArArGrGrC






ACTT



ATAA
rUrArGrUrC






GAAA



GGCT
rCrGrUrUrA






AAGT



AGTC
rUrCrArArC






GGCA



CGTT
rUrUrGrArA






CCGA



ATCA
rArArArGrU






GTCG



ACTT
rGrGrCrArC






GTGC



GAAA
rCrGrArGrU






(SEQ ID



AAGT
rCrGrGrUrG






NO: 3575)



GGCA
rCrUrCrUrG










CCGA
rCrCrArUrC










GTCG
rArCrArUrG










GTGC
rUrArGrUrU










TCTG
rGrArGrGrU










CCAT
rCrArArUrG










CACA
rArCrGrUrG










TGTA
rCrUrCrArG










GTTG
rUrCrUrGrU










AGGT
*mU*mU*mU 










CAAT
(SEQ










GACG
ID NO:










TGCT
3596)










CAGT











CTGT











TTT 











(SEQ 











ID











NO:











3595)










Template
HEK3 13P
GGCC
GTTT
GGAA
CGT
TTTT
GGCC
mG*mG*mC*r
146


4
BS_30RT
CAGA
TAGA
GCAG
GCT

CAGA
CrCrArGrAr




(CTTat1)
CTGA
GCTA
GGCT
CA

CTGA
CrUrGrArGr





GCAC
GAAA
TCCT
GTC

GCAC
CrArCrGrUr





GTGA
TAGC
TTCC
TG

GTGA
GrArGrUrUr





(SEQ
AAGT
TCTG
(SEQ

GTTT
UrUrArGrAr





ID
TAAA
CCAT
ID

TAGA
GrCrUrArGr





NO:
ATAA
CAAA
NO: 

GCTA
ArArArUrAr





3574)
GGCT
G 
3578)

GAAA
GrCrArArGr






AGTC
(SEQ ID


TAGC
UrUrArArAr






CGTT
NO: 3597)


AAGT
ÅrUrArArGr






ATCA



TAAA
GrCrUrArGr






ACTT



ATAA
UrCrCrGrUr






GAAA



GGCT
UrArUrCrAr






AAGT



AGTC
ArCrUrUrGr






GGCA



CGTT
ArArArArAr






CCGA



ATCA
GrUrGrGrCr






GTCG



ACTT
ArCrCrGrAr






GTGC



GAAA
GrUrCrGrGr






(SEQ ID



AAGT
UrGrCrGrGr






NO: 3575)



GGCA
ArArGrCrAr










CCGA
GrGrGrCrUr










GTCG
UrCrCrUrUr










GTGC
UrCrCrUrCr










GGAA
UrGrCrCrAr










GCAG
UrCrArArAr










GGCT
GrCrGrUrGr










TCCT
CrUrCrArGr










TTCC
UrCrUtGrU*










TCTG
mU*mU*mU










CCAT
(SEQ ID NO:










CAAA
3599)










GCGT











GCTC











AGTC











TGTT











TT 











(SEQ











ID











NO:











3598)







Template
HEK3 13P
GGCC
GTTT
GGAA
CGT
TTTT
GGCC
mG*mG*mC*r
153


5
BS_30RT
CAGA
TAGA
GCAG
GCT

CAGA
CrCrArGrAr




(10
CTGA
GCTA
GGCT
CA

CTGA
CrUrGrArGr




nts at1)
GCAC
GAAA
TCCT
GTC

GCAC
CrArCrGrUr





GTGA
TAGC
TTCC
TG

GTGA
GrArGrUrUr





(SEQ
AAGT
TCTG
(SEQ

GTTT
UrUrArGrAr





ID
TAAA
CCAT
ID

TAGA
GrCrUrArGr





NO:
ATAA
CACA
NO: 

GCTA
ArArArUrAr





3574)
GGCT
TGTA
3578)

GAAA
GrCrArArGr






AGTC
GTTG


TAGC
UrUrArArAr






CGTT
(SEQ ID


AAGT
ArUrArArGr






ATCA
NO: 3600)


TAAA
GrCrUrArGr






ACTT



ATAA
UrCrCrGrUr






GAAA



GGCT
UrArUrCrAr






AAGT



AGTC
ArCrUrUrGr






GGCA



CGTT
ArArArArAr






CCGA



ATCA
GrUrGrGrCr






GTCG



ACTT
ArCrCrGrAr






GTGC



GAAA
GrUrCrGrGr






(SEQ ID



AAGT
UrGrCrGrGr






NO: 3575)



GGCA
ArArGrCrAr










CCGA
GrGrGrCrUr










GTCG
UrCrCrUrUr










GTGC
UrCrCrUrCr










GGAA
UrGrCrCrAr










GCAG
UrCrArCrAr










GGCT
UrGrUrArGr










TCCT
UrUrGrCrGr










TTCC
UrGrCrUrCr










TCTG
ArGrUrCrUr










CCAT
GrU*mU*mU*










CACA
mU (SEQ ID










TGTA
NO: 3602)










GTTG











CGTG











CTCA











GTCT











GTTT











T 











(SEQ











ID











NO:











3601)







Template
HEK3 13P
GGCC
GTTT
GGAA
CGT
TTTT
GGCC
mG*mG*mC*r
163


6
BS_30RT
CAGA
TAGA
GCAG
GCT

CAGA
CrCrArGrAr




(20
CTGA
GCTA
GGCT
CA

CTGA
CrUrGrArGr




nts at1)
GCAC
GAAA
TCCT
GTC

GCAC
CrArCrGrUr





GTGA
TAGC
TTCC
TG

GTGA
GrArGrUrUr





(SEQ
AAGT
TCTG
(SEQ

GTTT
UrUrArGrAr





ID
TAAA
CCAT
ID

TAGA
GrCrUrArGr





NO:
ATAA
CACA
NO: 

GCTA
ArArArUrAr





3574)
GGCT
TGTA
3578)

GAAA
GrCrArArGr






AGTC
GTTG


TAGC
UrUrArArAr






CGTT
AGGT


AAGT
ArUrArArGr






ATCA
CAAT


TAAA
GrCrUrArGr






ACTT
GA


ATAA
UrCrCrGrUr






GAAA
(SEQ ID


GGCT
UrArUrCrAr






AAGT
NO: 3603)


AGTC
ArCrUrUrGr






GGCA



CGTT
ArArArArAr






CCGA



ATCA
GrUrGrGrCr






GTCG



ACTT
ArCrCrGrAr






GTGC



GAAA
GrUrCrGrGr






(SEQ ID



AAGT
UrGrCrGrGr






NO: 3575)



GGCA
ArArGrCrAr










CCGA
GrGrGrCrUr










GTCG
UrCrCrUrUr










GTGC
UrCrCrUrCr










GGAA
UrGrCrCrAr










GCAG
UrCrArCrAr










GGCT
UrGrUrArGr










TCCT
UrUrGrArGr










TTCC
GrUrCrArAr










TCTG
UrGrArCrGr










CCAT
UrGrCrUrCr










CACA
ArGrUrCrUr










TGTA
GrU*mU*mU*










GTTG
mU(SEQ ID 










AGGT
NO: 3605)










CAAT











GACG











TGCT











CAGT











CTGT











TTT











(SEQ











ID











NO:











3604)
















TABLE 49







Different production and compositions of Gene Writer


polypeptide mRNAs used in various Examples














Transcription


Modified




Name
template
Capping
Poly A
NTPs
5′ UTR
3′ UTR





mRNA-1
PCR
CleanCap
Added
None
AGGAAA
GCTGGAGCCTCG



amplicon
(AG); co-
during

TAAGAG
GTGGCCATGCTTC




transcrip-
amplifi-

AGAAAA
TTGCCCCTTGGGC




tional
cation

GAAGAG
CTCCCCCCAGCCC







TAAGAA
CTCCTCCCCTTCC







GAAATA
TGCACCCGTACCC







TAAGAG
CCGTGGTCTTTGA







CCACC
ATAAAGTCTGA







(SEQ ID
(SEQ ID







NO: 3606)
NO: 3607)





mRNA-2
PCR
CleanCap
Added
5moU
AGGAAA
GCTGGAGCCTCG



amplicon
(AG); co-
during

TAAGAG
GTGGCCATGCTTC




transcrip-
amplifi-

AGAAAA
TTGCCCCTTGGGC




tional
cation

GAAGAG
CTCCCCCCAGCCC







TAAGAA
CTCCTCCCCTTCC







GAAATA
TGCACCCGTACCC







TAAGAG
CCGTGGTCTTTGA







CCACC
ATAAAGTCTGA







(SEQ ID
(SEQ ID







NO: 3606)
NO: 3607)





mRNA-3
PCR
Enzymatic,
Added
None
GGGAAA
GCTGGAGCCTCG



amplicon
2′O
during

TAAGAG
GTGGCCATGCTTC




Methylated
amplifi-

AGAAAA
TTGCCCCTTGGGC




(Cap1);
cation

GAAGAG
CTCCCCCCAGCCC




post-


TAAGAA
CTCCTCCCCTTCC




transcrip-


GAAATA
TGCACCCGTACCC




tional


TAAGAG
CCGTGGTCTTTGA







CCACC
ATAAAGTCTGA







(SEQ ID
(SEQ ID







NO: 3608)
NO: 3607)





mRNA-4
PCR
Enzymatic,
Added
5moU
GGGAAA
GCTGGAGCCTCG



amplicon
2′O
during

TAAGAG
GTGGCCATGCTTC




Methylated
amplifi-

AGAAAA
TTGCCCCTTGGGC




(Cap1);
cation

GAAGAG
CTCCCCCCAGCCC




post-


TAAGAA
CTCCTCCCCTTCC




transcrip-


GAAATA
TGCACCCGTACCC




tional


TAAGAG
CCGTGGTCTTTGA







CCACC
ATAAAGTCTGA







(SEQ ID
(SEQ ID







NO: 3608)
NO: 3607)





mRNA-5
Linearized
CleanCap
Plasmid
None
AGGAAA
GCTGCCTTCTGCG



plasmid
(AG); co-
-

TAAGAG
GGGCTTGCCTTCT




transcrip-
encoded

AGAAAA
GGCCATGCCCTTC




tional


GAAGAG
TTCTCTCCCTTGC







TAAGAA
ACCTGTACCTCTT







GAAATA
GGTCTTTGAATAA







TAAGAG
AGCCTGAGTAGG







CCACC
AAGTCTA







(SEQ ID
(SEQ ID







NO: 3606)
NO: 3609)









Example 36: Use of Modified Nucleotides in an all-RNA Gene Writer System

This example describes the application of a Gene Writer system to edit the genome in human cells via delivery of RNA components, e.g., mRNA encoding the Gene Writer polypeptide and an RNA template. Further to the demonstration of the DNA-free system in Example 35, this example describes the incorporation of modified nucleotides, e.g., 5-methoxyuridine, into the mRNA encoding the Gene Writer polypeptide, and the incorporation of modified nucleotides, e.g. 2′-O-methyl phosphorothioate, into the Gene Writer template RNA.


Gene Writer polypeptide-encoding mRNAs (1) were generated using T7 polymerase-driven in vitro transcription of an amplicon generated from a plasmid by PCR. The plasmid encoding the mRNA construct comprised a transcriptional cassette comprising the following components: T7 promoter, 5′UTR, Gene Writer coding sequence (Cas9 nickase fused with a reverse transcriptase by a peptide linker and further comprising a bipartite SV40 NLS), and a 3′UTR. A poly A tail component was added such that it was encoded in the amplicon serving as the template for RNA transcription. In this example, mRNA molecules were prepared by incorporating one modified nucleotide, 5-methoxyuridine (5moU), into the transcription reaction. The mRNA encoding Cas9-RT(MMLV) (Table 47) was capped either co-transcriptionally with CleanCap AG (TriLink BioTechnologies) or post-transcriptionally via enzymatic capping (2′O methylated, Cap1) (Table 49).


Gene Writer Template RNAs (2) encoding genomic edits were generated by chemical synthesis and purified by standard desalting. The first and last three bases of each Template RNA comprised 2′-O-methyl phosphorothioate modifications. Here, Template 1 was used to introduce a CTT insertion into the human HEK3 locus (Table 48).


Where indicated, second nick gRNAs (3) were generated by chemical synthesis and comprised the following sequence modifications:











(SEQ ID NO: 3588)



mG*mC*mA*rGrArArArUrArGrArCrUrArArUrUrGr






CrArGrUrUrUrUrArGrArGrCrUrArGrArArArUrAr






GrCrArArGrUrUrArArArArUrArArGrGrCrUrArGr






UrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArAr






GrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCmU*mU






*mU*rU.






To assay the RNA Gene Writing systems described herein, HEK293T cells were plated 2 days before nucleofection to obtain 70-80% confluency on the day of nucleofection. RNAs were mixed according to the following combinations: i) Cas9-RT mRNA (1) only; ii) Cas9-RT mRNA (1), template RNA (2), and second nick gRNA (3); or iii) Cas9-RT mRNA (1) and template RNA (2). RNA mixes comprised 4.5 μg of the Cas9-RT mRNA (1), 5 μM final concentration of template RNA (2), and 1.3 μM final concentration of second nick gRNA (3). Mixes were nucleofected into approximately 200,000 cells using the Lonza Amaxa Nucleofector 96 Well Shuttle System, as according to manufacturer's protocols. Cells were then lysed and genomic DNA was collected 72 hours after nucleofection. Amplicon sequencing libraries were prepared using primers to amplify across the target site and Illumina sequencing was performed. Precise correction and indel rates were analyzed using the CRISPResso2 pipeline (Clement et al Nat Biotechnol 37(3):224-226 (2019)).


In these experiments, approximately 20% precise Writing activity was achieved using an all RNA Gene Writing system that incorporated modified nucleotides (5moU) in the mRNA encoding the Gene Writer polypeptide (FIG. 42). Notably, the incorporation of the modified nucleotide 5moU did not result in an observable inhibitory effect on Writing efficiency. Similar efficiencies resulted from the mRNA capping methods assayed here (see Table 49). A slight decrease in efficiency was observed in the absence of a second nick gRNA (FIG. 42).


Example 37: Delivery of DNA-Free Gene Writer Systems Through Lipid-Based Transfection of Human Cells

This example describes the application of a Gene Writer system to edit the genome in human cells via delivery of RNA components, e.g., mRNA encoding the Gene Writer polypeptide and an RNA template. Without wishing to be bound by theory, the ability to deliver only RNA components in the absence of DNA is expected to confer major advantages to this system, including a reduction in immunogenicity and cellular toxicity linked to the detection of DNA in the cytoplasm and the availability of lipid nanoparticles systems described herein, the majority of which are optimized for RNA delivery, that can circumvent issues associated with viral delivery of nucleic acid therapeutics (e.g., manufacturing challenges, pre-existing immunity, immunogenic response to viral proteins). The reduction in cellular toxicity through use of an RNA system may be especially important for the modification of more sensitive cell types, such as primary cells. Lipid transfection reagents may be utilized directly for ex vivo cell engineering and lipid-based nanoparticles are suitable for in vivo RNA delivery to a patient's cells. Thus, it is of significant value to demonstrate the capacity of a Gene Writing system to function appropriately when delivered as all RNA and in the absence of DNA. Specifically, this example demonstrates delivery of an all RNA Gene Writing system to modify the genome of HEK293T cells using the lipid-based transfection reagents Lipofectamine 3000 and MessengerMAX (Invitrogen). To demonstrate RNA-based Gene Writing is not limited to a single composition, two versions of a Cas-RT fusion polypeptide are employed that comprise an RT domain derived from either Moloney murine leukemia virus (Cas9-RT(MMLV)) or porcine endogenous retrovirus (Cas9-RT(PERV)) (Table 46).


Gene Writer polypeptide-encoding mRNAs (1) were generated using T7 polymerase-driven in vitro transcription. In general, plasmids encoding the mRNA constructs comprised a transcriptional cassette comprising the following components: T7 promoter, 5′UTR, Gene Writer coding sequence (Cas9 nickase fused with a reverse transcriptase by a peptide linker and further comprising a nuclear localization signal), 3′UTR, and an 80 nt polyA tail (SEQ ID NO: 3666). In this example, RNA molecules were prepared using unmodified nucleotides from either linearized plasmid template or using a PCR amplicon of the transcriptional cassette described above. The mRNA encoding Cas9-RT(MMLV) was capped either co-transcriptionally with CleanCap AG (TriLink BioTechnologies) or post-transcriptionally via enzymatic capping (2′O methylated, Cap1) (Table 49). The mRNA encoding Cas9-RT(PERV) was generated from plasmid template and co-transcriptionally capped with CleanCap AG (TriLink BioTechnologies) (Table 47).


Gene Writer Template RNAs (2) encoding genomic edits were generated by chemical synthesis and purified by standard desalting. The first and last three bases of each Template RNA comprised 2′-O-methyl phosphorothioate modifications. Here, Template 1 was used to introduce a CTT insertion into the human HEK3 locus (Table 48).


Where indicated, second nick gRNAs (3) were generated by chemical synthesis and comprised the following sequence modifications:











(SEQ ID NO: 3588)



mG*mC*mA*rGrArArArUrArGrArCrUrArArUrUrGr






CrArGrUrUrUrUrArGrArGrCrUrArGrArArArUrAr






GrCrArArGrUrUrArArArArUrArArGrGrCrUrArGr






UrCrCrGrUrUrArUrCrArArCrUrUrGrArArArArAr






GrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrCmU*mU






*mU*rU.






To assay the RNA Gene Writing systems described herein, approximately 50,000 HEK293T cells were plated in 24-well plates 1 day before lipofection. RNAs were mixed according to the following combinations: i) Cas9-RT mRNA (1) only; ii) Cas9-RT mRNA (1), template RNA (2), and second nick gRNA (3); or iii) Cas9-RT mRNA (1) and template RNA (2). RNA mixes comprised 0.45 μg of the Cas9-RT mRNA (1), 2.5 μM final concentration of template RNA (2), and 1.0 μM final concentration of second nick gRNA (3). RNAs were mixed with Opti-MEM media (Gibco) and Lipofectamine 3000 or MessengerMAX reagent (Invitrogen) and added to cells. Cells were then lysed and genomic DNA was collected 72 hours after nucleofection. Amplicon sequencing libraries were prepared using primers to amplify across the target site and Illumina sequencing was performed. Precise correction and indel rates were analyzed using the CRISPResso2 pipeline (Clement et al Nat Biotechnol 37(3):224-226 (2019)).


In these experiments, up to approximately 17% precise Writing activity was achieved using an all RNA Gene Writing system delivered by lipid-based transfection, approaching efficiencies similar to nucleofection (FIG. 43 B; see Example 35 for nucleofection). Lipofectamine 3000 was also used (FIG. 43 A). In contrast to nucleofection (Example 35), there was not an observable reduction when utilizing the 20 nt longer Template 4 as compared to Template 1 (Table 48; FIG. 43 C). Further, the all-RNA lipofection of the Gene Writer Cas9-RT(PERV) with Template 1 resulted in precise Writing of the desired edit with an efficiency of approximately 3.5% (FIG. 44).


Example 38: RNA Gene Writing Enables DNA-Free Precise Editing of Primary T Cells

This example describes the use of a Cas9-RT fusion polypeptide-based Gene Writer system for the genomic editing of target DNA sequences. More specifically, this example describes nucleofection of an all-RNA system into primary CD4+ T cells for Gene Rewriting in primary human cells, e.g., as a means of demonstrating the Gene Rewriter system for ex vivo application.


The all RNA system described here comprised: 1) Gene Writer polypeptide-encoding mRNA, e.g., an RNA encoding the Cas9-RT fusion polypeptide as a driver for programmed gene editing through a targeted nicking and reverse transcription process as described in this invention; 2) a template RNA molecule, e.g., an RNA comprising (i) a gRNA spacer sequence for guiding the driver to the targeted region, e.g., a sequence complementary to a 20-nucleotide sequence in the HEK3 locus; (ii) a primer-binding sequence capable of complementary base pairing with a single strand of the nicked DNA for target-primed reverse transcription; (iii) a heterologous object sequence providing a template for reverse transcription that further comprises the intended final target sequence; and (iv) a gRNA scaffold sequence to associate with the Cas9 domain of the Cas9-RT polypeptide fusion; and 3) an optional additional gRNA to promote second-strand nicking near the target site, e.g., an RNA comprising (i) a spacer sequence for targeting the driver to induce a second nick, on the opposite strand of the first nick guided by the template RNA, at a site proximal to the target site (e.g., within 50-150 nt from the first nick); and (ii) a gRNA scaffold sequence mediating an association with the Cas9 domain of the driver. In this example, the Cas-RT fusion polypeptide (1) (Table 46) comprises a Cas9(N863A) nickase fused to an MMLV reverse transcriptase domain. The template RNAs (2) employed here specifically follow the 5′ to 3′ orientation (i), (iv), (iii), (ii), as listed in the description thereof and are detailed in Table 48 and Example 35.


To deliver the RNA Gene Writer system into primary human CD4+ T cells and validate protein expression, 1,000,000 cells (Human Peripheral Blood CD4+ T Cells, Lonza catalog #2W-200) were stimulated by CD3/CD28 for two days and then nucleofected with 0, 2.5, 5, or 10 μg of mRNA encoding the Cas-RT polypeptide using a Nucleofector 96-well Shuttle System (Lonza) with the EO-115 nucleofection program, as according to manufacturer's protocols. One day post-nucleofection, the efficiency of delivery was assessed by immunoblotting with a Cas9 antibody (Cell Signaling) to measure protein expression of the Gene Writer polypeptide from the nucleofected mRNA (FIG. 45 A).


Subsequently, primary human CD4+ T cells were nucleofected with either: (1) 5 μg Gene Writer polypeptide mRNA (Writer only control); (2) 5 μg Gene Writer polypeptide mRNA and 5 μM template RNA, e.g., one of six template RNAs from Table 48 that target the same site of the HEK3 locus, but differ in editing result or design; or (3) 5 μg Gene Writer polypeptide mRNA, 5 μM template RNA, e.g., one of six template RNAs from Table 48, and 2.075 μM of an additional gRNA for generating a second-strand nick, e.g., the second-nick gRNA targeting a sequence 108 nt upstream of the HEK3 target site described in Example 35. Three days post-nucleofection, cells were harvested to examine 1) cell viability after RNA delivery of the Gene Writer system, and 2) editing efficiency on the target site of the genome. To assess the cell viability, the percentage of live cells was measured by flow cytometry after staining cells with a fluorescent live/dead dye (BioLegend). Cell viability was comparable in experimental conditions and in the absence of nucleofection (Untreated control) (FIG. 45 B). To evaluate efficiency of editing by the Gene Writing systems, genomic DNA was analyzed by PCR-based amplicon sequencing assay, as described in Example 35. The efficiency of the desired editing (Perfect Write) reached approximately 6.3% using Template 1 (Table 48) with the Gene Writer polypeptide mRNA (FIGS. 46 A and B). Here, the addition of a second-nick gRNA (FIG. 46 B) resulted in similar levels of editing. Thus, this example demonstrates the use of Gene Writing systems for highly specific editing in primary T cells and further shows the successful application of DNA-free, all-RNA Gene Writing in these cells.


Example 39: Detection of Retrotransposase-Mediated Integration in Human Cells

This example describes the identification of retrotransposons demonstrating functionality in human cells. By assaying native or modified retrotransposons for integration activity, this example demonstrates a method for the selection of retrotransposases comprising protein domains that can be used to recreate retrotransposases in their native domain composition or as components of chimeric or synthetic Gene Writers for engineering the genome of human cells. For example, a retrotransposon successfully producing an integration signal is expected to comprise functional DNA binding, endonuclease, reverse transcriptase, and, optionally, second-strand synthesis activities. In some embodiments, a reverse transcriptase domain from a retrotransposon that has been shown to demonstrate activity as described in this example is used to provide the reverse transcriptase activity in a Gene Writer polypeptide, e.g., as the RT of a Cas-RT fusion polypeptide. The screen described here employs the nucleofection of a two-plasmid system comprising a retrotransposon polypeptide and an inactivated reporter template into human cells to characterize the RT-dependent retrotransposition efficiency of computationally selected retrotransposons.


In this example, a two-plasmid system was employed comprising: 1) a retrotransposon-encoded protein expression driver plasmid, e.g., a plasmid encoding a retrotransposase polypeptide from Table 50, comprising a human codon-optimized retrotransposase coding sequence fused with a HiBit tag for detection of protein expression and driven by the mammalian CMV promoter, and 2) a template plasmid, e.g., a plasmid comprising (i) a promoter for expression in mammalian cells to drive transcription of the RNA template molecule, e.g., a CMV promoter, with the template molecule further comprising (ii) a reporter cassette that is inactive in the context of plasmid-derived expression, e.g., an EGFP expression cassette with coding sequence disrupted by an intron encoded in the opposite orientation (GFPai) flanked by (iii) the untranslated regions (UTRs) of the native retrotransposon that naturally comprises the retrotransposase of (1) (see FIG. 48). Here, the GFP reporter is encoded in the absence of a promoter to drive its expression to avoid any loss of signal due to GFP toxicity (see FIG. 48).


To deliver the two-plasmid system into U2OS cells, ˜400,000 cells were nucleofected with 88.3 ng driver plasmid (1) and 161.7 ng template plasmid (2) using the Lonza SE Cell Line 96-well Nucleofector™ Kit as per manufacturer's instructions. Three days post-nucleofection, integration efficiency was measured using ddPCR to determine the copy number of integrations per genome. Reverse transcription-dependent retrotransposition activity was measured by using a ddPCR approach that utilized the antisense intron as described below. Expression of the driver protein was measured by HiBit-based bioluminescence assay.


When employing an antisense intron reporter containing intronic sequence within the reporter cassette of the template plasmid, e.g., the GFPai system described here, the intron is present in the plasmid but is spliced out during transcription, thus only reporter DNA derived from the transcript by reverse transcription would lack the intron sequence (FIG. 48). To limit detection to only events derived from reverse transcription, a ddPCR Taqman probe was designed to span the splicing junction to hybridize to DNA lacking the intron but not to plasmid DNA still containing the intact intron. The forward and reverse primers were designed upstream and downstream of the probe and within the GFP sequence. This design avoids the possible background from template plasmid directly recombined into the genome without a first transcription step, or from intact template plasmid contaminating the gDNA extraction samples.


Gene Writing systems derived from retrotransposases in Table 50 were assayed as following this example to determine activity in human cells. Analysis of the integration efficiency of 163 candidate retrotransposon systems by ddPCR is shown in FIG. 49. From the assay described in this example, 25 retrotransposase candidates demonstrated successful trans-integration of the retrotransposon UTR-flanked Template sequence at greater than 0.01 copies/genome on average.


Example 40: Selection of Lipid Reagents with Reduced Aldehyde Content

In this example, lipids are selected for downstream use in lipid nanoparticle formulations containing Gene Writing component nucleic acid(s), and lipids are selected based at least in part on having an absence or low level of contaminating aldehydes. Reactive aldehyde groups in lipid reagents may cause chemical modifications to component nucleic acid(s), e.g., RNA, e.g., template RNA, during LNP formulation. Thus, in some embodiments, the aldehyde content of lipid reagents is minimized.


Liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) can be used to separate, characterize, and quantify the aldehyde content of reagents, e.g., as described in Zurek et al. The Analyst 124(9):1291-1295 (1999), incorporated herein by reference. Here, each lipid reagent is subjected to LC-MS/MS analysis. The LC/MS-MS method first separates the lipid and one or more impurities with a C8 HPLC column and follows with the detection and structural determination of these molecules with the mass spectrometer. If an aldehyde is present in a lipid reagent, it is quantified using a staple-isotope labeled (SIL) standard that is structurally identical to the aldehyde, but is heavier due to C13 and N15 labeling. An appropriate amount of the SIL standard is spiked into the lipid reagent. The mixture is then subjected to LC-MS/MS analysis. The amount of contaminating aldehyde is determined by multiplying the amount of SIL standard and the peak ratio (unknown/SIL). Any identified aldehyde(s) in the lipid reagents is quantified as described. In some embodiments, lipid raw materials selected for LNP formulation are not found to contain any contaminating aldehyde content above a chosen level. In some embodiments, one or more, and optionally all, lipid reagents used for formulation comprise less than 3% total aldehyde content. In some embodiments, one or more, and optionally all, lipid reagents used for formulation comprise less than 0.3% of any single aldehyde species. In some embodiments, one or more, and optionally all, lipid reagents used in formulation comprise less than 0.3% of any single aldehyde species and less than 3% total aldehyde content.


Example 41: Quantification of RNA Modification Caused by Aldehydes During Formulation

In this example, the RNA molecules are analyzed post-formulation to determine the extent of any modifications that may have happened during the formulation process, e.g., to detect chemical modifications caused by aldehyde contamination of the lipid reagents (see, e.g., Example 40).


RNA modifications can be detected by analysis of ribonucleosides, e.g., as according to the methods of Su et al. Nature Protocols 9:828-841 (2014), incorporated herein by reference in its entirety. In this process, RNA is digested to a mix of nucleosides, and then subjected to LC-MS/MS analysis. RNA post-formulation is contained in LNPs and must first be separated from lipids by coprecipitating with GlycoBlue in 80% isopropanol. After centrifugation, the pellets containing RNA are carefully transferred to a new Eppendorf tube, to which a cocktail of enzymes (benzonase, Phosphodiesterase type 1, phosphatase) is added to digest the RNA into nucleosides. The Eppendorf tube is placed on a preheated Thermomixer at 37° C. for 1 hour. The resulting nucleosides mix is directly analyzed by a LC-MS/MS method that first separates nucleosides and modified nucleosides with a C18 column and then detects them with mass spectrometry.


If aldehyde(s) in lipid reagents have caused chemical modification, data analysis will associate the modified nucleoside(s) with the aldehyde(s). A modified nucleoside can be quantified using a SIL standard which is structurally identical to the native nucleoside except heavier due to C13 and N15 labeling. An appropriate amount of the SIL standard is spiked into the nucleoside digest, which is then subjected to LC-MS/MS analysis. The amount of the modified nucleoside is obtained by multiplying the amount of SIL standard and the peak ratio (unknown/SIL). LC-MS/MS is capable of quantifying all the targeted molecules simultaneously. In some embodiments, the use of lipid reagents with higher contaminating aldehyde content results in higher levels of RNA modification as compared to the use of higher purity lipid reagents as materials during the lipid nanoparticle formulation process. Thus, in preferred embodiments, higher purity lipid reagents are used that result in RNA modification below an acceptable level.


Example 42: Gene Writer™ Enabling Large Insertion into Genomic DNA

This example describes the use of a Gene Writer™ gene editing system to alter a genomic sequence by insertion of a large string of nucleotides.


In this example, the Gene Writer™ polypeptide, gRNA, and writing template are provided as DNA transfected into HEK293T cells. The Gene Writer™ polypeptide uses a Cas9 nickase for both DNA-binding and endonuclease functions. The reverse transcriptase function is derived from the highly processive RT domain of an R2 retrotransposase. The writing template is designed to have homology to the target sequence, while incorporating the genetic payload at the desired position, such that reverse transcription of the template RNA results in the generation of a new DNA strand containing the desired insertion.


To create a large insertion in the human HEK293T cell DNA, the Gene Writer™ polypeptide is used in conjunction with a specific gRNA, which targets the Cas9-containing Gene Writer™ to the target locus, and a template RNA for reverse transcription, which contains an RT-binding motif (3′ UTR from an R2 element) for associating with the reverse transcriptase, a region of homology to the target site for priming reverse transcription, and a genetic payload (GFP expression unit). This complex nicks the target site and then performs TPRT on the template, initiating the reaction by using priming regions on the template that are complementary to the sequence immediately adjacent to the site of the nick and copying the GFP payload into the genomic DNA.


After transfection, cells are incubated for three days to allow for expression of the Gene Writing™ system and conversion of the genomic DNA target. After the incubation period, genomic DNA is extracted from cells. Genomic DNA is then subjected to PCR-based amplification using site-specific primers and amplicons are sequenced on an Illumina MiSeq according to manufacturer's protocols. Sequence analysis is then performed to determine the frequency of reads containing the desired edit.


Example 43: Gene Writers can Integrate Genetic Cargo Independently of the Single-Stranded Template Repair Pathway

This example describes the use of a Gene Writer system in a human cell wherein the single-stranded template repair (SSTR) pathway is inhibited.


In this example, the SSTR pathway will be inhibited using siRNAs against the core components of the pathway: FANCA, FANCD2, FANCE, USP1. Control siRNAs of a non-target control will also be included. 200k U2OS cells will be nucleofected with 30 pmols (1.5 μM) siRNAs, as well as R2Tg driver and transgene plasmids (trans configuration). Specifically, 250 ng of Plasmids expressing R2Tg, control R2Tg with a mutation in the RT domain, or control R2Tg with an endonuclease inactivating mutation) are used in conjunction with transgene at a 1:4 molar ratio (driver to transgene). Transfections of U2OS cells is performed in SE buffer using program DN100. After nucleofection, cells are grown in complete medium for 3 days. gDNA is harvested on day 3 and ddPCR is performed to assess integration at the rDNA site. Transgene integration at rDNA is detected in the absence of core SSTR pathway components.


Example 44: Formulation of Lipid Nanoparticles Encapsulating Firefly Luciferase mRNA

In this example, a reporter mRNA encoding firefly luciferase was formulated into lipid nanoparticles comprising different ionizable lipids. Lipid nanoparticle (LNP) components (ionizable lipid, helper lipid, sterol, PEG) were dissolved in 100% ethanol with the lipid component. These were then prepared at molar ratios of 50:10:38.5:1.5 using ionizable lipid LIPIDV004 or LIPIDV005 (Table A1), DSPC, cholesterol, and DMG-PEG 2000, respectively. Firefly Luciferase mRNA-LNPs containing the ionizable lipid LIPIDV003 (Table A1) were prepared at a molar ratio of 45:9:44:2 using LIPIDV003, DSPC, cholesterol, and DMG-PEG 2000, respectively. Firefly luciferase mRNA used in these formulations was produced by in vitro transcription and encoded the Firefly Luciferase protein, further comprising a 5′ cap, 5′ and 3′ UTRs, and a polyA tail. The mRNA was synthesized under standard conditions for T7 RNA polymerase in vitro transcription with co-transcriptional capping, but with the nucleotide triphosphate UTP 100% substituted with N1-methyl-pseudouridine triphosphate in the reaction. Purified mRNA was dissolved in 25 mM sodium citrate, pH 4 to a concentration of 0.1 mg/mL.


Firefly Luciferase mRNA was formulated into LNPs with a lipid amine to RNA phosphate (N:P) molar ratio of 6. The LNPs were formed by microfluidic mixing of the lipid and RNA solutions using a Precision Nanosystems NanoAssemblr™ Benchtop Instrument, using the manufacturer's recommended settings. A 3:1 ratio of aqueous to organic solvent was maintained during mixing using differential flow rates. After mixing, the LNPs were collected and dialyzed in 15 mM Tris, 5% sucrose buffer at 4° C. overnight. The Firefly Luciferase mRNA-LNP formulation was concentrated by centrifugation with Amicon 10 kDa centrifugal filters (Millipore). The resulting mixture was then filtered using a 0.2 m sterile filter. The final LNP was stored at −80° C. until further use.









TABLE A1







Ionizable Lipids used in Example 44 (Formula (ix), (vii), and (iii))












Molecular



LIPID ID
Chemical Name
Weight
Structure





LIPIDV003
(9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl) oxy)-2-((((3-(diethylamino) propoxy)carbonyl)oxy) methyl)propyl octadeca-9,12-dienoate
852.29


embedded image







LIPIDV004
Heptadecan-9-yl 8-((2- hydroxyethyl)(8-(nonyloxy)-8- oxooctyl)amino)octanoate
710.18


embedded image







LIPIDV005

919.56


embedded image









text missing or illegible when filed








Prepared LNPs were analyzed for size, uniformity, and % RNA encapsulation. The size and uniformity measurements were performed by dynamic light scattering using a Malvern Zetasizer DLS instrument (Malvern Panalytical). LNPs were diluted in PBS prior to being measured by DLS to determine the average particle size (nanometers, nm) and polydispersity index (pdi). The particle sizes of the Firefly Luciferase mRNA-LNPs are shown in Table A2.









TABLE A2







LNP particle size and uniformity












LNP ID
Ionizable Lipid
Particle Size (nm)
pdi
















LNPV019-002
LIPIDV005
77
0.04



LNPV006-006
LIPIDV004
71
0.08



LNPV011-003
LIPIDV003
87
0.08










The percent encapsulation of luciferase mRNA was measured by the fluorescence-based RNA quantification assay Ribogreen (ThermoFisher Scientific). LNP samples were diluted in 1× TE buffer and mixed with the Ribogreen reagent per manufacturer's recommendations and measured on a i3 SpectraMax spectrophotomer (Molecular Devices) using 644 nm excitation and 673 nm emission wavelengths. To determine the percent encapsulation, LNPs were measured using the Ribogreen assay with intact LNPs and disrupted LNPs, where the particles were incubated with 1× TE buffer containing 0.2% (w/w) Triton-X100 to disrupt particles to allow encapsulated RNA to interact with the Ribogreen reagent. The samples were again measured on the i3 SpectraMax spectrophotometer to determine the total amount of RNA present. Total RNA was subtracted from the amount of RNA detected when the LNPs were intact to determine the fraction encapsulated. Values were multiplied by 100 to determine the percent encapsulation. The Firefly Luciferase mRNA-LNPs that were measured by Ribogreen and the percent RNA encapsulation is reported in Table A3.









TABLE A3







RNA encapsulation after LNP formulation









LNP ID
Ionizable Lipid
% mRNA encapsulation





LNPV019-002
LIPIDV005
98


LNPV006-006
LIPIDV004
92


LNPV011-003
LIPIDV003
97









Example 45: In Vitro Activity Testing of mRNA-LNPs in Primary Hepatocytes

In this example, LNPs comprising the luciferase reporter mRNA were used to deliver the RNA cargo into cells in culture. Primary mouse or primary human hepatocytes were thawed and plated in collagen-coated 96-well tissue culture plates at a density of 30,000 or 50,000 cells per well, respectively. The cells were plated in 1× William's Media E with no phenol red and incubated at 37° C. with 5% CO2. After 4 hours, the medium was replaced with maintenance medium (1× William's Media E with no phenol containing Hepatocyte Maintenance Supplement Pack (ThermoFisher Scientific)) and cells were grown overnight at 37° C. with 5% CO2. Firefly Luciferase mRNA-LNPs were thawed at 4° C. and gently mixed. The LNPs were diluted to the appropriate concentration in maintenance media containing 7.5% fetal bovine serum. The LNPs were incubated at 37° C. for 5 minutes prior to being added to the plated primary hepatocytes. To assess delivery of RNA cargo to cells, LNPs were incubated with primary hepatocytes for 24 hours and cells were then harvested and lysed for a Luciferase activity assay. Briefly, medium was aspirated from each well followed by a wash with 1×PBS. The PBS was aspirated from each well and 200 μL passive lysis buffer (PLB) (Promega) was added back to each well and then placed on a plate shaker for 10 minutes. The lysed cells in PLB were frozen and stored at −80° C. until luciferase activity assay was performed.


To perform the luciferase activity assay, cellular lysates in passive lysis buffer were thawed, transferred to a round bottom 96-well microtiter plate and spun down at 15,000 g at 4° C. for 3 min to remove cellular debris. The concentration of protein was measured for each sample using the Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific) according to the manufacturer's instructions. Protein concentrations were used to normalize for cell numbers and determine appropriate dilutions of lysates for the luciferase assay. The luciferase activity assay was performed in white-walled 96-well microtiter plates using the luciferase assay reagent (Promega) according to manufacturer's instructions and luminescence was measured using an i3X SpectraMax plate reader (Molecular Devices). The results of the dose-response of Firefly luciferase activity mediated by the Firefly mRNA-LNPs are shown in FIGS. 50A and B and indicate successful LNP-mediated delivery of RNA into primary cells in culture. As shown in FIG. 50A, LNPs formulated as according to Example 44 were analyzed for delivery of cargo to primary human (A) and mouse (B) hepatocytes, as according to Example 45. The luciferase assay revealed dose-responsive luciferase activity from cell lysates, indicating successful delivery of RNA to the cells and expression of Firefly luciferase from the mRNA cargo.


Example 46: LNP-Mediated Delivery of RNA to the Mouse Liver

To measure the effectiveness of LNP-mediated delivery of firefly luciferase containing particles to the liver, LNPs were formulated and characterized as described in Example 44 and tested in vitro prior (Example 45) to administration to mice. C57BL/6 male mice (Charles River Labs) at approximately 8 weeks of age were dosed with LNPs via intravenous (i.v.) route at 1 mg/kg. Vehicle control animals were dosed i.v. with 300 μL phosphate buffered saline. Mice were injected via intraperitoneal route with dexamethasone at 5 mg/kg 30 minutes prior to injection of LNPs. Tissues were collected at necropsy at or 6, 24, 48 hours after LNP administration with a group size of 5 mice per time point. Liver and other tissue samples were collected, snap-frozen in liquid nitrogen, and stored at −80° C. until analysis. Frozen liver samples were pulverized on dry ice and transferred to homogenization tubes containing lysing matrix D beads (MP Biomedical). Ice-cold 1× luciferase cell culture lysis reagent (CCLR) (Promega) was added to each tube and the samples were homogenized in a Fast Prep-24 5G Homogenizer (MP Biomedical) at 6 m/s for 40 seconds. The samples were transferred to a clean microcentrifuge tube and clarified by centrifugation. Prior to luciferase activity assay, the protein concentration of liver homogenates was determined for each sample using the Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific) according to the manufacturer's instructions. Luciferase activity was measured with 200 μg (total protein) of liver homogenate using the luciferase assay reagent (Promega) according to manufacturer's instructions using an i3X SpectraMax plate reader (Molecular Devices). Liver samples revealed successful delivery of mRNA by all lipid formulations, with reporter activity following the ranking LIPIDV005>LIPIDV004>LIPIDV003 (FIG. 51). As shown in FIG. 51, Firefly luciferase mRNA-containing LNPs were formulated and delivered to mice by iv, and liver samples were harvested and assayed for luciferase activity at 6, 24, and 48 hours post administration. Reporter activity by the various formulations followed the ranking LIPIDV005>LIPIDV004>LIPIDV003. RNA expression was transient and enzyme levels returned near vehicle background by 48 hours. Post-administration. This assay validated the use of these ionizable lipids and their respective formulations for RNA systems for delivery to the liver.


Without wishing to be limited by example, the lipids and formulations described in this example are support the efficacy for the in vivo delivery of other RNA molecules beyond a reporter mRNA. All-RNA Gene Writing systems can be delivered by the formulations described herein. For example, all-RNA systems employing a Gene Writer polypeptide mRNA, Template RNA, and an optional second-nick gRNA are described for editing the genome in vitro by nucleofection, by using modified nucleotides, by lipofection), and editing cells, e.g., primary T cells. As described in this application, these all-RNA systems have many unique advantages in cellular immunogenicity and toxicity, which is of importance when dealing with more sensitive primary cells, especially immune cells, e.g., T cells, as opposed to immortalized cell culture cell lines. Further, it is contemplated that these all RNA systems could be targeted to alternate tissues and cell types using novel lipid delivery systems as referenced herein, e.g., for delivery to the liver, the lungs, muscle, immune cells, and others, given the function of Gene Writing systems has been validated in multiple cell types in vitro here, and the function of other RNA systems delivered with targeted LNPs is known in the art. The in vivo delivery of Gene Writing systems has potential for great impact in many therapeutic areas, e.g., correcting pathogenic mutations), instilling protective variants, and enhancing cells endogenous to the body, e.g., T cells. Given an appropriate formulation, all-RNA Gene Writing is conceived to enable the manufacture of cell-based therapies in situ in the patient.










LENGTHY TABLES




The patent application contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).





Claims
  • 1. A fusion protein comprising: a) a reverse transcriptase (RT) domain having the amino acid sequence of SEQ ID NO: 3225; andb) a Cas9 nickase domain, wherein the RT domain is C-terminal of the Cas9 nickase domain.
  • 2. The fusion protein of claim 1, wherein the Cas9 nickase domain is a SpyCas9 nickase domain.
  • 3. The fusion protein of claim 1, wherein the Cas9 nickase domain is a SpyCas9(N863A) nickase domain.
  • 4. The fusion protein of claim 1, wherein the Cas9 nickase domain comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 3269.
  • 5. The fusion protein of claim 1, wherein the Cas9 nickase domain is an NmeCas9 domain.
  • 6. The fusion protein of claim 1, wherein the Cas9 nickase domain is an St1Cas9 domain.
  • 7. The fusion protein of claim 1, wherein the Cas9 nickase domain is a SauCas9 domain.
  • 8. The fusion protein of claim 1, which further comprises a peptide linker disposed between the RT domain and the Cas9 nickase domain.
  • 9. The fusion protein of claim 8, wherein the peptide linker is between 2-40 amino acids in length.
  • 10. The fusion protein of claim 8, wherein the peptide linker has an amino acid sequence according to SEQ ID NO: 1589.
  • 11. The fusion protein of claim 1, which further comprises a nuclear localization sequence (NLS).
  • 12. The fusion protein of claim 11, wherein the NLS is fused to the N-terminus of the Cas9 nickase domain.
  • 13. The fusion protein of claim 11, wherein the NLS is fused to the C-terminus of the fusion protein.
  • 14. The fusion protein of claim 11, wherein the NLS is a monopartite NLS or a bipartite NLS.
  • 15. The fusion protein of claim 11, which further comprises a linker disposed between the NLS and the Cas9 nickase domain.
  • 16. The fusion protein of claim 1, which comprises an amino acid sequence according to SEQ ID NO: 3561.
  • 17. The fusion protein of claim 1, wherein the Cas9 nickase domain has an activity at least 50% of that of an otherwise similar Cas9 nickase molecule that is not fused to an RT domain.
  • 18. A nucleic acid encoding the fusion protein of claim 1.
  • 19. The nucleic acid of claim 18, which is an mRNA.
  • 20. The nucleic acid of claim 18, which comprises a nucleotide sequence according to SEQ ID NO: 3566.
  • 21. A system for modifying DNA comprising: (a) the fusion protein of claim 1 or a nucleic acid encoding the fusion protein; and(b) a template RNA comprising, from 5′ to 3′ (i) a gRNA spacer that binds a target site, (ii) a sequence that binds the fusion protein, (iii) a heterologous object sequence, and (iv) a 3′ target homology domain.
  • 22. The system of claim 21, wherein (a) comprises the nucleic acid encoding the fusion protein.
  • 23. The system of claim 22, wherein the nucleic acid encoding the fusion protein is an mRNA.
  • 24. The system of claim 21, wherein the sequence that binds the fusion protein is a gRNA scaffold.
  • 25. A lipid nanoparticle (LNP) comprising the system of claim 21.
  • 26. A method for modifying a target site in genomic DNA in a cell, the method comprising contacting the cell with the system of claim 21.
  • 27. A fusion protein comprising: a) a reverse transcriptase (RT) domain having the amino acid sequence of any of SEQ ID NOs: 1548, 3136-3224, or 3226-3261; andb) a Cas9 nickase domain, wherein the RT domain is C-terminal of the Cas9 nickase domain.
  • 28. A nucleic acid encoding the fusion protein of claim 27.
  • 29. A system for modifying DNA comprising: (a) the fusion protein of claim 27 or a nucleic acid encoding the fusion protein; and(b) a template RNA comprising, from 5′ to 3′ (i) a gRNA spacer that binds a target site, (ii) a sequence that binds the fusion protein, (iii) a heterologous object sequence, and (iv) a 3′ target homology domain.
  • 30. A method for modifying a target site in genomic DNA in a cell, the method comprising contacting the cell with the system of claim 29.
RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 17/929,116, filed Sep. 1, 2022, which is a continuation of International Application No. PCT/US2021/020948, filed Mar. 4, 2021, which claims priority to U.S. Ser. No. 62/985,285 filed Mar. 4, 2020, U.S. Ser. No. 63/035,627 filed Jun. 5, 2020, and U.S. Ser. No. 63/067,828 filed Aug. 19, 2020, the entire contents of each of which is incorporated herein by reference.

Provisional Applications (3)
Number Date Country
62985285 Mar 2020 US
63035627 Jun 2020 US
63067828 Aug 2020 US
Continuations (2)
Number Date Country
Parent 17929116 Sep 2022 US
Child 18355017 US
Parent PCT/US21/20948 Mar 2021 US
Child 17929116 US