Patent Application
20230051661
This application claims the benefit of priority of Singapore provisional application No. 10201913340Q, filed on 26 Dec. 2019, the contents of it being hereby incorporated by reference in its entirety for all purposes.
The present invention relates to the field of molecular biology biotechnology, specifically the field of gene editing and more specifically nucleobase editing.
Many human genetic diseases are caused by single-nucleotide polymorphisms (SNPs), in which the disease and healthy alleles differ by a single DNA base. CRISPR-Cas9 nuclease is commonly used to edit genomic DNA in a targetable fashion. Following DNA cleavage, three major repair mechanisms can be involved in fixing that break—homology directed repair (HDR), micro-homology mediated end joining (MMEJ), and non-homologous end joining (NHEJ). In the presence of a donor DNA/RNA, HDR may occur. However, previous studies were only able to achieve low levels of precise gene editing (0.1 to 5%). MMEJ requires that the double stranded break be formed at a region with micro-homology and hence restricts the targeting range of CRISPR-Cas9. NHEJ is the predominant pathway in repairing Cas9-induced double stranded breaks. Unfortunately, it introduces a variety of random indels. For therapeutic applications where precise point mutations are necessary, NHEJ is unable to restore a defective gene and is hence inadequate.
Base editors can correct these SNPs by converting the targeted DNA bases into another base in a controllable and efficient fashion. Current technology enables the conversion of CG base pairs to T·A base pairs using cytosine base editors (CBEs) (A. C. Komor, et al., Nature 533, 420-424 (2016); K. Nishida et al., Science 353, aaf8729 (2016); A. C. Komor, et al., Sci. Adv. 3, eaao4774 (2017)) and AT base pairs to GC base pairs using adenine base editors (ABEs) (N. M. Gaudelli, et al., Nature 551, 464-471 (2017)), which together represent half of all known disease-associated SNPs. CBEs and ABEs are also known to effect some CG to GC edits as byproducts, but they cannot effect such transversions at efficiencies or purities necessary for therapeutic use, and hence the remaining half of these SNPs are not addressable by current base-editors.
Therefore, there is a need to provide novel nucleobase editors which facilitate C:G to G:C editing (CGBEs) with high efficiency, specificity, and purity.
In one aspect, the present disclosure refers to a fusion protein or a protein complex comprising a DNA binding protein (DnaBP), a nucleobase modifying protein (NMP), and a Base Excision Repair associated protein (BERAP); wherein the fusion protein or protein complex does not comprise a Uracil binding protein or a catalytically active DNA polymerase.
In another aspect, the present disclosure refers to a fusion protein comprising:
In another aspect, the present disclosure refers to a fusion protein comprising a sequence of any one of SEQ ID NOs: 42 to 72.
In yet another aspect, the present disclosure refers to protein complex comprising:
In yet another aspect, the present disclosure refers to a protein-nucleic acid complex comprising a nucleic acid molecule and any one of:
In yet another aspect, the present disclosure refers to a pharmaceutical composition comprising the fusion protein or protein complex as disclosed herein, the fusion protein as disclosed herein, the protein complex as disclosed herein, or the protein-nucleic acid complex as disclosed herein.
In yet another aspect, the present disclosure refers to a method of replacing a cytosine with a guanine on a DNA strand in a cell, said method comprises introducing to the cell the fusion protein or protein complex as disclosed herein, the fusion protein as disclosed herein, the protein complex as disclosed herein, or the protein-nucleic acid complex as disclosed herein.
In yet another aspect, the present disclosure refers to a polynucleotide encoding the fusion protein or protein complex as disclosed herein, the fusion protein as disclosed herein, the protein complex as disclosed herein, or the protein-nucleic acid complex as disclosed herein.
In yet another aspect, the present disclosure refers to a vector comprising the polynucleotide as disclosed herein.
In yet another aspect, the present disclosure refers to a cell comprising the fusion protein or protein complex as disclosed herein, the fusion protein as disclosed herein, the protein complex as disclosed herein, or the protein-nucleic acid complex as disclosed herein.
In yet another aspect, the present disclosure refers to a method of treating a subject having or suspected of having a disease or disorder comprising administering the fusion protein or protein complex as disclosed herein, the fusion protein as disclosed herein, the protein complex as disclosed herein, the protein-nucleic acid complex as disclosed herein, the pharmaceutical composition as disclosed herein, the polynucleotide as disclosed herein, or the vector as disclosed herein to the subject.
In yet another aspect, the present disclosure refers to a method for editing a target nucleobase pair of a double-stranded DNA sequence, the method comprising:
The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:
Several terms that are employed throughout the specification are generally defined in the following paragraphs. Other definitions may also found within the body of the specification.
As used herein, the terms “about” and “approximately,” in reference to a number, is used herein to include numbers that fall within a range of 20%, 10%, 5%, 2.5%, 2%, 1.5% or 1% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
The terms “polynucleotide”, “nucleic acid” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogues thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogues. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labelling component. The term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. As used herein, the term “polypeptide” generally has its art-recognized meaning of a polymer of amino acids. The term is also used to refer to specific functional classes of polypeptides, such as, for example, nucleases, antibodies, etc.
As used herein, the term “variant” refers to an entity that shows significant structural identity with a reference entity but differs structurally from the reference entity in the presence or level of one or more chemical moieties as compared with the reference entity. In many embodiments, a variant also differs functionally from its reference entity. In general, whether a particular entity is properly considered to be a “variant” of a reference entity is based on its degree of structural identity with the reference entity. As will be appreciated by those skilled in the art, any biological or chemical reference entity has certain characteristic structural elements. A variant, by definition, is a distinct chemical entity that shares one or more such characteristic structural elements. To give but a few examples, a polypeptide may have a characteristic sequence element comprising a plurality of amino acids having designated positions relative to one another in linear or three-dimensional space and/or contributing to a particular biological function; a nucleic acid may have a characteristic sequence element comprised of a plurality of nucleotide residues having designated positions relative to on another in linear or three-dimensional space. For example, a variant polypeptide may differ from a reference polypeptide as a result of one or more differences in amino acid sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, etc.) covalently attached to the polypeptide backbone. In some embodiments, a variant polypeptide shows an overall sequence identity with a reference polypeptide (e.g., a nucleic acid modifying enzyme described herein) that is at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. Alternatively or additionally, in some embodiments, a variant polypeptide does not share at least one characteristic sequence element with a reference polypeptide. In some embodiments, the reference polypeptide has one or more biological activities. In some embodiments, a variant polypeptide shares one or more of the biological activities of the reference polypeptide, e.g., enzymatic activity. In some embodiments, a variant polypeptide lacks one or more of the biological activities of the reference polypeptide. In some embodiments, a variant polypeptide shows a reduced level of one or more biological activities (e.g., enzymatic activity) as compared with the reference polypeptide. In some embodiments, a polypeptide of interest is considered to be a “variant” of a parent or reference polypeptide if the polypeptide of interest has an amino acid sequence that is identical to that of the parent but for a small number of sequence alterations at particular positions. Typically, fewer than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% of the residues in the variant are substituted as compared with the parent. In some embodiments, a variant has 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substituted residue as compared with a parent. Often, a variant has a very small number (e.g., fewer than 5, 4, 3, 2, or 1) number of substituted functional residues (i.e., residues that participate in a particular biological activity). Furthermore, a variant typically has not more than 5, 4, 3, 2, or 1 additions or deletions, and often has no additions or deletions, as compared with the parent. Moreover, any additions or deletions are typically fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly are fewer than about 5, about 4, about 3, or about 2 residues. In some embodiments, the parent or reference polypeptide is one found in nature.
As used herein, the term “expression” of a nucleic acid sequence refers to the generation of any gene product from the nucleic acid sequence. In some examples, a gene product can be an RNA transcript. In some embodiments, a gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or 3′ end formation); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.
Unless specified otherwise, all amino acid sequences are shown with the NH2 end on the left and the COOH end on the right, and all DNA/RNA acid sequences are shown with the 5′ end on the left and the 3′ end on the right.
Disclosed herein are compositions and methods of editing a nucleobase, for example, generating a cytosine to guanine mutation (or conversion) in a polynucleotide. The inventors have developed a new class of C:G to G:C Base Editors (CGBEs) which utilize or manipulate the Base Excision Repair (BER) pathway downstream of abasic site creation. When a cytosine to guanine conversion is effected on one strand of a double stranded DNA polynucleotide, the opposing guanine on the opposing strand may also be converted into a cytosine by the intrinsic DNA repair mechanisms. Therefore, this new class of CGBEs edits C:G to G:C (
In one aspect, the present disclosure refers to a fusion protein or a protein complex comprising a DNA binding protein (DnaBP), a nucleobase modifying protein (NMP), and a Base Excision Repair associated protein (BERAP); wherein the fusion protein or protein complex does not comprise a Uracil binding protein or a catalytically active DNA polymerase.
In this aspect, the nucleobase editor comprises at least three components: a DNA binding protein (DnaBP), a nucleobase modifying protein (NMP), and a Base Excision Repair associated protein (BERAP). The nucleobase editor can be a fusion protein (a single polypeptide translated from a fusion gene) or a protein complex. As used herein, the term “protein complex” refers to a composite unit that is a combination of two or more proteins formed by interaction between the proteins. Typically but not necessarily, a “protein complex” is formed by the binding of two or more proteins together through specific non-covalent binding affinities. However, covalent bonds may also be present between the interacting partners. For instance, the two interacting partners can be covalently crosslinked so that the protein complex becomes more stable.
DNA Binding Proteins (DnaBP)
The term “DNA binding protein (DnaBP)” refers to a protein which is capable of binding with a DNA. In some examples, the DNA binding protein is a programmable DNA binding protein, which can be designed or programmed to bind with a specific DNA sequence. In some examples, the programmable DNA binding protein is an RNA-guided DNA binding protein. As used herein, an RNA-guided DNA binding protein interacts or forms a complex with a guide RNA, and can specifically target or bind with a polynucleotide of a specific sequence which usually comprises a sequence complementary to the targeting domain of the gRNA. Upon binding with the target polynucleotide, the DNA binding protein may remain bound with the target polynucleotide, or it may modify the target polynucleotide. In one example, the DNA binding protein is a CRISPR-associated protein (Cas). Many Cas proteins possess endonuclease activity and are also termed Cas nucleases. In a specific example, the DNA binding protein is a Cas protein. In some examples, the Cas protein is selected from the group including but not limited to Cas3, a Cas9, a xCas9, a SpRY Cas9, a HF-Cas9, a Cas9-NG, a circularly permutated Cas9, a codon-optimised Cas9, a domain-fused Cas9, a Cas10 and a Cas12 (also known as Cpf1), a Cas14, a CasX, a Casφ, and variants thereof. In some examples, the DnaBP is a nickase variant of any of the Cas proteins aforementioned. Therefore in some examples, the Cas domain is a Cas nickase (nCas). In some examples, the DnaBP is a Cas9 nickase (or nCas9). The term “Cas9 nickase,” as used herein, refers to a Cas9 protein that is capable of cleaving only one strand of a duplexed nucleic acid molecule (e.g., a duplexed DNA molecule). In some examples, the Cas domain is a nuclease inactive Cas (dCas).
The terms “guide RNA” and “gRNA” refer to any nucleic acid that promotes the specific association (or “targeting”) of a DNA binding protein to a target sequence either in a cell or in a cell free environment. gRNAs can be unimolecular (comprising a single RNA molecule, and referred to alternatively as chimeric), or modular (comprising more than one, and typically two, separate RNA molecules, such as a crRNA and a tracrRNA, which are usually associated with one another, for instance by duplexing).
Base Excision Repair Associated Proteins (BERAP)
As used herein, the term “Base Excision Repair associated protein (BERAP)” refers to any protein that is involved in the Base Excision Repair pathway. BERAP may also be referred to as “BER protein”. In some examples, the BERAP is an enzyme functioning in one or more steps of the BER pathway; in other examples, the BERAP is a co-factor or a scaffold protein of enzymes functioning in the BER pathway. Scaffold proteins are understood to be proteins which regulate the function or activity of other proteins or pathways by interacting or binding with one or more members of the pathways. In some cases, a scaffold protein may tether multiple members of a pathway into complexes. BERAPs can be identified by referring to proteins listed in the KEGG (Kyoto Encyclopedia of Genes and Genomes) BER pathway (https://www.genome.jp/kegg⋅bin/show_pathway?map=ko03410), or by searching the term ‘base excision repair’ in protein databases such as Uniprot (www.uniprot.org).
In some examples of the fusion protein or protein complex as disclosed herein, the BERAP is selected from the group including but not limited to: an AP endonuclease, an end processing enzyme, a catalytically inactive DNA polymerase, a lyase domain, a Flap endonuclease, a DNA ligase, and a scaffold protein involved in the BER pathway. In some examples, the BERAP is selected from the group including but not limited to: a DNA ligase III (LIG3), an XRCC1, a DNA binding or lyase domain of DNA Polymerase beta (PB), a DNA binding or lyase domain of DNA Polymerase delta, a DNA binding or lyase domain of DNA Polymerase epsilon, an AP endonuclease (APE1), Proliferating cell nuclear antigen (PCNA), DNA-(apurinic or apyrimidinic site) lyase (APEX), Poly (ADP-ribose) polymerase (PARP), Flap endonuclease 1 (FEN1), and DNA ligase I (LIG1). In one example, the BERAP is an XRCC1. In some examples, the BERAP is a rat XRCC1 (rXRCC1) or a variant thereof. In some examples, the BERAP is a human XRCC1 (hXRCC1) or a variant thereof. In one example, the BERAP is a rXRCC1 with the amino acid sequence of SEQ ID NO: 4. In one example, the BERAP is a hXRCC1 with the amino acid sequence of SEQ ID NO: 5.
In some examples of the fusion protein or protein complex as disclosed herein, the BERAP is a DNA binding or lyase domain of DNA Polymerase beta (POLB, or PB). In some specific examples, the DNA binding or lyase domain of DNA Polymerase beta corresponds to a region contained within amino acids 1-140, 1-120, 1-100, or 1-87 of the full DNA Polymerase beta sequence. In one example, the DNA binding or lyase domain of DNA Polymerase beta corresponds to a region contained within amino acids 1-140, 1-120, 1-100, or 1-87 of the full human DNA Polymerase beta sequence (SEQ ID NO: 12). In another example, the DNA binding or lyase domain of DNA Polymerase beta corresponds to a region contained within amino acids 1-140, 1-120, 1-100, or 1-87 of the full rat DNA Polymerase beta sequence (SEQ ID NO: 13). In some examples, the BERAP is a human DNA binding or lyase domain of DNA Polymerase beta (PB) or a variant thereof. In some examples, the BERAP is a rat DNA binding or lyase domain of DNA Polymerase beta (PB) or a variant thereof. In one example, the BERAP is a DNA binding or lyase domain of rat Polymerase beta (rPB), with an amino acid sequence of SEQ ID NO: 6. In one example, the BERAP is a DNA binding or lyase domain of rat Polymerase beta (rPB), with an amino acid sequence of SEQ ID NO: 7. In another example, the BERAP is a DNA binding or lyase domain of human Polymerase beta (hPB), with an amino acid sequence of SEQ ID NO: 8. In another example, the BERAP is a DNA binding or lyase domain of human Polymerase beta (hPB), with an amino acid sequence of SEQ ID NO: 9.
In some examples of the fusion protein or protein complex as disclosed herein, the BERAP is a DNA Ligase III (LIG3). In one example, the BERAP is a rat DNA Ligase III (LIG3), with an amino acid sequence of SEQ ID NO: 10. In another example, the BERAP is a human DNA Ligase III (LIG3), with an amino acid sequence of SEQ ID NO: 11.
Nucleobase Modifying Proteins (NMP)
The term “nucleobase modifying protein (NMP)” refers to any protein domain that is capable of modifying a nucleobase (such as adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U)). The modification can be any chemical or physical changes to the nucleobase, and the NMP includes but not limited to a deaminase, a nuclease, a nickase, a recombinase, a methyltransferase, a methylase, an acetylase, and an acetyltransferase.
In some examples of the fusion protein or protein complex as disclosed herein, the nucleobase modifying protein (NMP) is a cytosine deaminase domain. A cytosine deaminase domain is the functional domain of a cytosine deaminase that has deaminase activity. As the deamination occurs on the cytosine nucleobase that is comprised in a cytidine or deoxycytidine, a cytidine deaminase may also be referred to as a cytosine deaminase. In the present disclosure, the terms “cytidine deaminase” is used interchangeably with “cytosine deaminase”. For example, the apolipoprotein B mRNA-editing complex (APOBEC) family of deaminases are conventionally referred to as cytidine deaminases, but they are capable of deaminating the cytosine in cytidine or deoxycytidine. Accordingly, the deaminase domain from an APOBEC cytidine deaminase protein can also be referred to as a cytosine deaminase domain. Therefore in some examples, the cytidine deaminase domain is a deaminase domain from an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some examples, the APOBEC family deaminase is selected from the group consisting of APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, APOBEC3H deaminase, and any derivatives thereof. In some examples, the cytidine deaminase domain is an activation-induced deaminase (AID). In some examples, the cytidine deaminase domain is a cytidine deaminase 1 from Petromyzon marinus (pmCDA1). In some examples, the cytidine deaminase domain has a higher activity on methylated Cs. In some examples, the cytidine deaminase domain has a narrower targeting window.
The Exclusion of Uracil Binding Protein
The fusion protein or a protein complex as disclosed herein does not comprise a Uracil binding protein. As used herein, the term “uracil binding protein” or “UBP” refers to a protein that is capable of binding to uracil. In some examples, the UBP is a uracil modifying enzyme, a uracil base excision enzyme or a uracil DNA glycosylase (UDG or UNG). Therefore, while uracil DNA glycosylase is considered to be involved in the BER pathway and is responsible for removing the creating the abasic site, it is not comprised in the fusion protein or a protein complex (the CGBE) as defined in claim 1. Without being bound by theory, the presence of a uracil binding protein (such as UDG) as a component of the CGBE may maintain the abasic site (created by the removal of the uracil base) and hinder the downstream BER pathway which repairs the abasic site. The CGBE of the present disclosure is designed to promote the Base Excision Repair (BER) pathway, it does not comprise a uracil binding protein.
Architecture of the CGBEs
In some examples of the fusion protein or a protein complex as disclosed herein, the DNA binding protein (DnaBP) is a nickase Cas protein such as nCas9; the Base Excision Repair associated protein (BERAP) is selected from the group consisting of an XRCC1, a DNA Ligase III (LIG3), and a DNA binding or lyase domain of DNA Polymerase beta; and the nucleobase modifying protein (NMP) is a deaminase domain from an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some examples, the DNA binding protein (DnaBP) is a nickase Cas9 (nCas9); the Base Excision Repair associated protein (BERAP) an XRCC1; and the nucleobase modifying protein (NMP) is a deaminase domain from an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some examples, the DNA binding protein (DnaBP) is a nickase Cas9 (nCas9); the Base Excision Repair associated protein (BERAP) is a DNA binding or lyase domain of DNA Polymerase beta; wherein the nucleobase modifying protein (NMP) is a deaminase domain from an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some examples, the DNA binding protein (DnaBP) is a nickase Cas9 (nCas9); the Base Excision Repair associated protein (BERAP) is a DNA Ligase III (LIG3); and the nucleobase modifying protein (NMP) is a deaminase domain from an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase.
Different architectures or orientations of the fusion protein as disclosed herein are possible. In some examples of the fusion protein as disclosed herein, the orientation of the DnaBP, NMP and BERAP within the fusion protein is selected from the group consisting of: [NMP]-[DnaBP]-[BERAP], [NMP]-[BERAP]-[DnaBP] and [BERAP]-[NMP]-[DnaBP]; wherein each instance of “]-[” comprises an optional linker. In one example, the orientation of the DnaBP, NMP and BERAP within the fusion protein is [NMP]-[DnaBP]-[BERAP]. In one example, the orientation of the DnaBP, NMP and BERAP within the fusion protein is [NMP]-[DnaBP]-[BE RAP].
The term “linker,” as used herein, refers to a bond (e.g., covalent bond), chemical group, or a molecule linking two molecules or moieties, e.g., two domains of a fusion protein, such as, for example, a nuclease-inactive Cas9 domain and a nucleic acid-editing domain (e.g., an adenosine deaminase). In some embodiments, a linker joins a gRNA binding domain of an RNA-programmable nuclease, including a Cas9 nuclease domain, and the catalytic domain of a nucleic-acid editing protein. In some embodiments, a linker joins a dCas9 and a nucleic-acid editing protein. Typically, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond, thus connecting the two. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g. a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5-100 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated. In some embodiments, a linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 101), which may also be referred to as the XTEN linker. In some embodiments, a linker comprises the amino acid sequence SGGS (SEQ ID NO: 102). In some embodiments, a linker comprises SGGSGGGS (SEQ ID NO: 103), GGGGS (SEQ ID NO: 104), G, EAAAK (SEQ ID NO: 105), GGS, SGSETPGTSESATPES (SEQ ID NO: 101) or XP motif, or a combination of any of these. In some embodiments, a linker comprises repeats of SGGSGGGS (SEQ ID NO: 103), GGGGS (SEQ ID NO: 104), G, EAAAK (SEQ ID NO: 105), GGS, or XP, wherein the repeats is denoted by n, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.
In another aspect, the present disclosure provides a fusion protein comprising:
In one example of the fusion protein as disclosed herein, the fusion protein comprises one of the below structures:
In one example of the fusion protein as disclosed herein, any linker is independently 1-50 amino acids in length. In one example of the fusion protein as disclosed herein, any linker is independently 1-25 amino acids in length. In one example of the fusion protein as disclosed herein, any linker is independently 5-20 amino acids in length. In one example of the fusion protein as disclosed herein, any linker is independently 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.
In one example of the fusion protein as disclosed herein, any linker independently comprises one or more amino acid sequences selected from the group consisting of: SGSETPGTSESATPES (XTEN linker) (SEQ ID NO:101, SGGS (SEQ ID NO:102) and GGGGS (SEQ ID NO:104).
In another aspect, the present disclosure provides a protein complex comprising:
In some examples, the fusion protein as disclosed herein comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, and SEQ ID NO:72. In one example, the fusion protein as disclosed herein has an amino acid sequence of SEQ ID NO: 44. In another example, the fusion protein as disclosed herein has an amino acid sequence of SEQ ID NO: 48. In another example, the fusion protein as disclosed herein has an amino acid sequence of SEQ ID NO: 69. In another example, the fusion protein as disclosed herein has an amino acid sequence of SEQ ID NO: 70.
In another aspect, the present disclosure provides a protein-nucleic acid complex comprising a nucleic acid molecule and any one of:
In one example of the protein-nucleic acid complex as disclosed herein, the nucleic acid molecule is an RNA. In some examples, the RNA is a guide RNA (gRNA), or more specifically a single guide RNA (sgRNA). In some examples, the single guide RNA (sgRNA) as disclosed herein comprises a sequence selected from the group consisting of: SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41.
As used herein, the term “protein-nucleic acid complex” means a complex unit that is a combination of at least one protein and at least one nucleic acid formed by an interaction, including an interaction between the protein and the nucleic acid. Typically, “protein-nucleic acid complexes” are formed by, but not necessarily, the binding of proteins and nucleic acids through non-covalent affinity. In some examples, the gene editing complex is a protein-nucleic acid complex such as a ribonucleoprotein (RNP). A non-limiting example of an RNP is CRISPR-Cas RNP that includes a Cas protein and gRNA.
In one example of the protein-nucleic acid complex as disclosed herein, the nucleic acid molecule comprises a sequence which is about 80%, 90%, or 95% identical or reverse complementary to any of the target sequences listed in Table 2. In some examples, the nucleic acid molecule comprises a sequence which is identical or reverse complementary to any of the target sequences listed in Table 2.
CACACACACTTAGAATCTGTGGG
In another aspect, the present disclosure provides a method of replacing a cytosine with a guanine on a DNA strand in a cell, said method comprises introducing to the cell the fusion protein or protein complex as disclosed herein, the fusion as disclosed herein, the protein complex as disclosed herein, or the protein-nucleic acid complex as disclosed herein.
In some examples, the cell is a eukaryotic cell. In some examples, the cell is an animal cell. In some specific examples, the cell is a human cell. In some examples, the method is performed in vivo or in vitro.
In some examples, the BERAP and the NMP interact with the same strand of a target DNA molecule.
In another aspect, the present disclosure provides a vector comprising the polynucleotide as disclosed herein.
In another aspect, the present disclosure provides a kit comprising the fusion protein or protein complex as disclosed herein, the fusion protein as disclosed herein, the protein complex as disclosed herein, or the protein-nucleic acid complex as disclosed herein.
In another aspect, the present disclosure provides a cell comprising the fusion protein or protein complex as disclosed herein, the fusion protein as disclosed herein, the protein complex as disclosed herein, or the protein-nucleic acid complex as disclosed herein.
In another aspect, the present disclosure provides a cell comprising one or more nucleic acid molecules that encode the fusion protein or protein complex as disclosed herein, the fusion protein as disclosed herein, the protein complex as disclosed herein, or the protein-nucleic acid complex as disclosed herein.
In another aspect, the present disclosure provides a method of treating a subject having or suspected of having a disease or disorder comprising administering the fusion protein or protein complex as disclosed herein, the fusion protein as disclosed herein, the protein complex as disclosed herein, or the protein-nucleic acid complex as disclosed herein, the pharmaceutical composition as disclosed herein, the polynucleotide as disclosed herein, or the vector as disclosed herein to the subject. In one example, the disease or disorder comprises one or more C to G (C>G) mutations. In another example, the disease or disorder comprises one or more G to C (G>C) mutations. In another example, the disease or disorder comprises C>G and G>C mutations. In one example, the disease or disorder is inclusive of, but not limited to any of the conditions listed in https://www.ncbi.nlm.nih.gov/clinvar/?term=C%3EG or https://www.ncbi.nlm.nih.gov/clinvar/?term=G%3EC, and filtering based on the selection of the following clinical significance: pathogenic, risk factor or likely pathogenic. In one example, the disease or disorder is selected from a group consisting of skin fibrosis, bladder cancer, liver cancer, Myasthenic syndrome, Spondyloepimetaphyseal dysplasia, Parkinson's disease, Deafness, blood disorders, and Schnyder crystalline corneal dystrophy.
In another aspect, the present disclosure provides a fusion protein or protein complex as disclosed herein, the fusion protein as disclosed herein, the protein complex as disclosed herein, or the protein-nucleic acid complex as disclosed herein, the pharmaceutical composition as disclosed herein, the polynucleotide as disclosed herein, or the vector as disclosed herein for use in treating a subject having or suspected of having a disease or disorder. In one example, the disease or disorder comprises one or more C to G (C>G) mutations. In another example, the disease or disorder comprises one or more G to C (G>C) mutations. In another example, the disease or disorder comprises C>G and G>C mutations. In one example, the disease or disorder is inclusive of, but not limited to any of the conditions listed in https://www.ncbi.nlm.nih.gov/clinvar/?term=C%3EG or https://www.ncbi.nlm.nih.gov/clinvar/?term=G%3EC, and filtering based on the selection of the following clinical significance: pathogenic, risk factor or likely pathogenic. In one example, the disease or disorder is selected from a group consisting of skin fibrosis, bladder cancer, liver cancer, Myasthenic syndrome, Spondyloepimetaphyseal dysplasia, Parkinson's disease, Deafness, blood disorders, and Schnyder crystalline corneal dystrophy.
In another aspect, the present disclosure provides a fusion protein or protein complex as disclosed herein, the fusion protein as disclosed herein, the protein complex as disclosed herein, or the protein-nucleic acid complex as disclosed herein, the pharmaceutical composition as disclosed herein, the polynucleotide as disclosed herein, or the vector as disclosed herein in the manufacture of a medicament for treating a subject having or suspected of having a disease or disorder. In one example, the disease or disorder comprises one or more C to G (C>G) mutations. In another example, the disease or disorder comprises one or more G to C (G>C) mutations. In another example, the disease or disorder comprises C>G and G>C mutations. In one example, the disease or disorder is inclusive of, but not limited to any of the conditions listed in https://www.ncbi.nlm.nih.gov/clinvar/?term=C%3EG or https://www.ncbi.nlm.nih.gov/clinvar/?term=G%3EC, and filtering based on the selection of the following clinical significance: pathogenic, risk factor or likely pathogenic. In one example, the disease or disorder is selected from a group consisting of skin fibrosis, bladder cancer, liver cancer, Myasthenic syndrome, Spondyloepimetaphyseal dysplasia, Parkinson's disease, Deafness, blood disorders, and Schnyder crystalline corneal dystrophy.
In another aspect, the present disclosure provides a method for editing a target nucleobase pair of a double-stranded DNA sequence, the method comprising:
In one example, the method as disclosed herein further comprises converting a fourth nucleobase that is complementary to the third nucleobase, thereby generating an intended edited base pair.
In one example of the method as disclosed herein, the efficiency of generating the intended edited base pair is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 35%, at least 40%, at least 45% or at least 50%.
In one example of the method as disclosed herein, the ratio of intended edited base pairs to unintended edited base pairs is at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1, or at least 10:1.
In one example of the method as disclosed herein, the first nucleobase is cytosine. In one example of the method as disclosed herein, the second nucleobase is uracil. In one example of the method as disclosed herein, the third nucleobase is guanine. In one example of the method as disclosed herein, the fourth nucleobase is cytosine.
In some examples of the method as disclosed herein, the nucleobase editor comprises nickase activity.
In some examples of the method as disclosed herein, the target region is 5-40, 5-30, 5-20, or 20 amino acids in length.
In some examples of the method as disclosed herein, the intended edited base pair resides within or proximal to the CGBE-binding site (protospacer). In some examples, the intended edited base pair is located 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides upstream, within, or downstream of the CBGE-binding site. In some examples, the intended edited base pair is upstream of a protospacer adjacent motif (PAM) site. In some examples, the intended edited base pair is located 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides upstream of the PAM site. As used herein, the protospacer adjacent motif (or PAM for short) is a short DNA sequence (usually 2-6 base pairs in length) that follows the DNA region targeted for cleavage by the CRISPR system, such as CRISPR-Cas9 (which has the PAM site sequence of NGG). In some examples, the intended edited base pair is located 14, 15, or 16 or 17 nucleotides upstream of the PAM site. Unless explicitly stated otherwise, the term “upsteam of the PAM site” describes nucleotides/base pairs to the 5′ direction of the PAM site, on the non-complementary strand (the strand not bound by the guide RNA).
In some examples of the method as disclosed herein, the intended edited base pair is downstream of a protospacer adjacent motif (PAM) site. In some examples, the intended edited base pair is located 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides downstream of the PAM site. As used herein, the protospacer adjacent motif (or PAM for short) is a short DNA sequence (usually 2-6 base pairs in length) that follows the DNA region targeted for cleavage by the CRISPR system, such as CRISPR-Cas9 (which has the PAM site sequence of NGG). In some examples, the intended edited base pair is located 14, 15, 16, 17, 18, 19 or 20 nucleotides downstream of the PAM site. Unless explicitly stated otherwise, the term “downstream of the PAM site” describes nucleotides/base pairs to the 3′ direction of the PAM site, on the non-complementary strand (the strand not bound by the guide RNA).
In some examples of the method as disclosed herein, the nucleobase editor comprises a linker. In some examples, the linker is 1-25, 5-20, 10-15 amino acids in length.
In some examples of the method as disclosed herein, the target region comprises a target window, wherein the target window comprises the target nucleobase pair. As used herein, the target region is a region on double-stranded DNA sequence which the fusion protein or protein complex (the CGBE) is designed to recognize or bind with. In examples wherein the DNA binding protein is guided by a guide RNA (such is the case for the Cas family proteins), the target region may be the region bound by the guide RNA. As used herein, the target window refers to a sequence window within the target region that is subject to efficient C to G editing of the CBGE. For optimal C to G editing, the target “C” is ideally located within the targeting window. In some examples, the target window is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some examples, the target window is 3, 4, 5, 6, or 7 nucleotides in length. In some examples, the target window comprises the 14th, 15th, 16th and 17th nucleotides upstream of the PAM site. In one example, the target window is the 15th nucleotides upstream of the PAM site.
In some examples of the method as disclosed herein, the nucleobase editor comprises the fusion protein or protein complex as disclosed herein, the fusion protein as disclosed herein, or the protein complex as disclosed herein.
In some examples of the method as disclosed herein, the first nucleobase of the target nucleobase pair is a cytosine, and wherein said cytosine is in a DNA motif characterized by any one of the group consisting of WCW, ACC and GCT; wherein “C” is said Cytosine, W indicates an Adenine(A) or a Thymine (T).
In some examples, the method as disclosed herein is performed in vivo or in vitro.
GAGTCCGAGCAGAAGAAGAAGTTTTAGAGCTAGAAATAGCAAG
GGAATCCCTTCTGCAGCACCGTTTTAGAGCTAGAAATAGCAAG
GAACACAAAGCATAGACTGCGTTTTAGAGCTAGAAATAGCAAGT
GGAAACGGATAGTTCTGAAAGTTTTAGAGCTAGAAATAGCAAGT
CTTAACTATTTGTATTCCACGTTTTAGAGCTAGAAATAGCAAGTT
CTTCCCAAGTGAGAAGCCAGGTTTTAGAGCTAGAAATAGCAAG
CCAGCCCGCTGGCCCTGTAAGTTTTAGAGCTAGAAATAGCAAG
CATTCCGTTATTTTACATATGTTTTAGAGCTAGAAATAGCAAGTT
GTTTCCTTTACAGGGCCAGCGTTTTAGAGCTAGAAATAGCAAGT
ATACGCACAGTTTGACAGATGTTTTAGAGCTAGAAATAGCAAGT
GCTGGCCCTGTAAAGGAAACGTTTTAGAGCTAGAAATAGCAAG
GCATGCGTGTGTGTTTAAGCGTTTTAGAGCTAGAAATAGCAAGT
TTGGGCTGCAGTAACTTGAAGTTTTAGAGCTAGAAATAGCAAGT
TCTTTCAAGCAGGTGATTACGTTTTAGAGCTAGAAATAGCAAGT
AGTTTCCTTTACAGGGCCAGGTTTTAGAGCTAGAAATAGCAAGT
GAGGTCGTGGCTGAGCACAAGTTTTAGAGCTAGAAATAGCAAG
GGCCTCTATTGTTGGTAGAAGTTTTAGAGCTAGAAATAGCAAGT
GGCCCAGACTGAGCACGTGAGTTTTAGAGCTAGAAATAGCAAG
GGCACTGCGGCTGGAGGTGGGTTTTAGAGCTAGAAATAGCAA
GTCATCTTAGTCATTACCTGGTTTTAGAGCTAGAAATAGCAAGT
CACACACACTTAGAATCTGTGTTTTAGAGCTAGAAATAGCAAGT
ACACACACACTTAGAATCTGGTTTTAGAGCTAGAAATAGCAAGT
GGACACGAAGATCAGCTGCAGTTTTAGAGCTAGAAATAGCAAG
CCCTTTCCTGCGTGACGTCGGTTTTAGAGCTAGAAATAGCAAG
CCCTTTCCTGCGTGACGTCGGTTTTAGAGCTAGAAATAGCAAG
GAAGTCGTTGTCAAACAGGAGTTTTAGAGCTAGAAATAGCAAGT
GATGTCTGCAGGCCAGATGAGTTTTAGAGCTAGAAATAGCAAG
In addition to the table above, full amino acid sequences of specific fusion proteins (CGBEs) as disclosed herein are provided in Table 4 below:
MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSW
SPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLIS
SGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVL
ELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATG
LKSGSETPGTSESATPES[DKKYSIGLAIGTNSVGWAVITDEYKVP
AKYPHKIKSGAEAKKLPGVGTKIAEKIDEFLATGKLRKLEKSGGSP
MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSW
SPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLIS
SGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVL
ELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATG
LKSGSETPGTSESATPES[DKKYSIGLAIGTNSVGWAVITDEYKVP
AKYPHKIKSGAEAKKLPGVGTKIAEKIDEFLATGKLRKLEKIRQDDT
SSSINFLTRVSGIGPSAARKFVDEGIKTLEDLRKNEDKLNHHQRIGL
KSGGSPKKKRKV
MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSW
SPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLIS
SGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVL
ELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATG
LKSGSETPGTSESATPES[DKKYSIGLAIGTNSVGWAVITDEYKVP
AKYPHKIKSGAEAKKLPGVGTKIAEKIDEFLATGKLRKLEKSGGSP
MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSW
SPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLIS
SGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVL
ELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATG
LKSGSETPGTSESATPES[DKKYSIGLAIGTNSVGWAVITDEYKVP
AKYPHKIKSGAEAKKLPGVGTKIAEKIDEFLATGKLRKLEKIRQDDT
SSSINFLTRVTGIGPSAARKLVDEGIKTLEDLRKNEDKLNHHQRIGL
KSGGSPKKKRKV
MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSW
SPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLIS
SGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVL
ELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATG
LKSGSETPGTSESATPES[DKKYSIGLDIGTNSVGWAVITDEYKVP
AKYPHKIKSGAEAKKLPGVGTKIAEKIDEFLATGKLRKLEKSGGSP
MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSW
SPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLIS
SGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVL
ELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATG
LKSGSETPGTSESATPES[DKKYSIGLDIGTNSVGWAVITDEYKVP
AKYPHKIKSGAEAKKLPGVGTKIAEKIDEFLATGKLRKLEKIRQDDT
SSSINFLTRVSGIGPSAARKFVDEGIKTLEDLRKNEDKLNHHQRIGL
KSGGSPKKKRKV
MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSW
SPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLIS
SGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVL
ELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATG
LKSGSETPGTSESATPES[DKKYSIGLDIGTNSVGWAVITDEYKVP
AKYPHKIKSGAEAKKLPGVGTKIAEKIDEFLATGKLRKLEKSGGSP
MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSW
SPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLIS
SGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVL
ELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATG
LKSGSETPGTSESATPES[DKKYSIGLDIGTNSVGWAVITDEYKVP
AKYPHKIKSGAEAKKLPGVGTKIAEKIDEFLATGKLRKLEKIRQDDT
SSSINFLTRVTGIGPSAARKLVDEGIKTLEDLRKNEDKLNHHQRIGL
KSGGSPKKKRKV
MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSW
SPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLIS
SGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVL
ELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATG
LKSGGSSKRKAPQETLNGGITDMLTELANFEKNVSQAIHKYNAYR
KAASVIAKYPHKIKSGAEAKKLPGVGTKIAEKIDEFLATGKLRKLEK
MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSW
SPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLIS
SGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVL
ELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATG
LKSGGSSKRKAPQETLNGGITDMLTELANFEKNVSQAIHKYNAYR
KAASVIAKYPHKIKSGAEAKKLPGVGTKIAEKIDEFLATGKLRKLEKI
RQDDTSSSINFLTRVSGIGPSAARKFVDEGIKTLEDLRKNEDKLNH
HQRIGLKSGSETPGTSESATPESfDKKYSIGLAIGTNSVGWAVITDE
MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSW
SPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLIS
SGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVL
ELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATG
LKSGGSSKRKAPQETLNGGITDMLVELANFEKNVSQAIHKYNAYR
KAASVIAKYPHKIKSGAEAKKLPGVGTKIAEKIDEFLATGKLRKLEK
MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSW
SPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLIS
SGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVL
ELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATG
LKSGGSSKRKAPQETLNGGITDMLVELANFEKNVSQAIHKYNAYR
KAASVIAKYPHKIKSGAEAKKLPGVGTKIAEKIDEFLATGKLRKLEKI
RQDDTSSSINFLTRVTGIGPSAARKLVDEGIKTLEDLRKNEDKLNH
HQRIGLKSGSETPGTSESATPESfDKKYSIGLAIGTNSVGWAVITDE
MSKRKAPQETLNGGITDMLTELANFEKNVSQAIHKYNAYRKAASVI
AKYPHKIKSGAEAKKLPGVGTKIAEKIDEFLATGKLRKLEKSGGSS
SETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGR
HSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSP
CGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSG
VTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLEL
YCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK
MSKRKAPQETLNGGITDMLTELANFEKNVSQAIHKYNAYRKAASVI
AKYPHKIKSGAEAKKLPGVGTKIAEKIDEFLATGKLRKLEKIRQDDT
SSSINFLTRVSGIGPSAARKFVDEGIKTLEDLRKNEDKLNHHQRIGL
KSGGSSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEI
NWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWF
LSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLR
DLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVR
LYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHIL
WATGLKSGSETPGTSESATPES[DKKYSIGLAIGTNSVGWAVITDE
MSKRKAPQETLNGGITDMLVELANFEKNVSQAIHKYNAYRKAASVI
AKYPHKIKSGAEAKKLPGVGTKIAEKIDEFLATGKLRKLEKSGGSS
SETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGR
HSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSP
CGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSG
VTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLEL
YCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK
MSKRKAPQETLNGGITDMLVELANFEKNVSQAIHKYNAYRKAASVI
AKYPHKIKSGAEAKKLPGVGTKIAEKIDEFLATGKLRKLEKIRQDDT
SSSINFLTRVTGIGPSAARKLVDEGIKTLEDLRKNEDKLNHHQRIGL
KSGGSSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEI
NWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWF
LSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLR
DLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVR
LYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHIL
WATGLKSGSETPGTSESATPES[DKKYSIGLAIGTNSVGWAVITDE
MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSW
SPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLIS
SGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVL
ELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATG
LKSGSETPGTSESATPES[DKKYSIGLAIGTNSVGWAVITDEYKVP
VVLQLEKEEQIHSVDIGNDGSAFVEVLVGSSAGGAGEQDYEVLLV
TSSFMSPSESRSGSNPNRVRMFGPDKLVRAAAEKRWDRVKIVCS
QPYSKDSPFGLSFVRFHSPPDKDEAEAPSQKVTVTKLGQFRVKEE
DESANSLRPGALFFSRINKTSPVTASDPAGPSYAAATLQASSAASS
ASPVSRAIGSTSKPQESPKGKRKLDLNQEEKKTPSKPPAQLSPSV
PKRPKLPAPTRTPATAPVPARAQGAVTGKPRGEGTEPRRPRAGP
EELGKILQGVVVVLSGFQNPFRSELRDKALELGAKYRPDWTRDST
HLICAFANTPKYSQVLGLGGRIVRKEWVLDCHRMRRRLPSQRYLM
AGPGSSSEEDEASHSGGSGDEAPKLPQKQPQTKTKPTQAAGPSS
PQKPPTPEETKAASPVLQEDIDIEGVQSEGQDNGAEDSGDTEDEL
RRVAEQKEHRLPPGQEENGEDPYAGSTDENTDSEEHQEPPDLPV
PELPDFFQGKHFFLYGEFPGDERRKLIRYVTAFNGELEDYMSDRV
QFVITAQEWDPSFEEALMDNPSLAFVRPRWIYSCNEKQKLLPHQL
YGVVPQASGGSPKKKRKV
MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSW
SPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLIS
SGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVL
ELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATG
LKSGSETPGTSESATPES[DKKYSIGLDIGTNSVGWAVITDEYKVP
VVLQLEKEEQIHSVDIGNDGSAFVEVLVGSSAGGAGEQDYEVLLV
TSSFMSPSESRSGSNPNRVRMFGPDKLVRAAAEKRWDRVKIVCS
QPYSKDSPFGLSFVRFHSPPDKDEAEAPSQKVTVTKLGQFRVKEE
DESANSLRPGALFFSRINKTSPVTASDPAGPSYAAATLQASSAASS
ASPVSRAIGSTSKPQESPKGKRKLDLNQEEKKTPSKPPAQLSPSV
PKRPKLPAPTRTPATAPVPARAQGAVTGKPRGEGTEPRRPRAGP
EELGKILQGVVVVLSGFQNPFRSELRDKALELGAKYRPDWTRDST
HLICAFANTPKYSQVLGLGGRIVRKEWVLDCHRMRRRLPSQRYLM
AGPGSSSEEDEASHSGGSGDEAPKLPQKQPQTKTKPTQAAGPSS
PQKPPTPEETKAASPVLQEDIDIEGVQSEGQDNGAEDSGDTEDEL
RRVAEQKEHRLPPGQEENGEDPYAGSTDENTDSEEHQEPPDLPV
PELPDFFQGKHFFLYGEFPGDERRKLIRYVTAFNGELEDYMSDRV
QFVITAQEWDPSFEEALMDNPSLAFVRPRWIYSCNEKQKLLPHQL
YGVVPQASGGSPKKKRKV
MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSW
SPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLIS
SGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVL
ELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATG
LKSGGSGPEIRLRHVVSCSSQDSTHCAENLLKADTYRKWRAAKA
GEKTISVVLQLEKEEQIHSVDIGNDGSAFVEVLVGSSAGGAGEQD
YEVLLVTSSFMSPSESRSGSNPNRVRMFGPDKLVRAAAEKRWDR
VKIVCSQPYSKDSPFGLSFVRFHSPPDKDEAEAPSQKVTVTKLGQ
FRVKEEDESANSLRPGALFFSRINKTSPVTASDPAGPSYAAATLQA
SSAASSASPVSRAIGSTSKPQESPKGKRKLDLNQEEKKTPSKPPA
QLSPSVPKRPKLPAPTRTPATAPVPARAQGAVTGKPRGEGTEPR
RPRAGPEELGKILQGVVVVLSGFQNPFRSELRDKALELGAKYRPD
WTRDSTHLICAFANTPKYSQVLGLGGRIVRKEWVLDCHRMRRRL
PSQRYLMAGPGSSSEEDEASHSGGSGDEAPKLPQKQPQTKTKPT
QAAGPSSPQKPPTPEETKAASPVLQEDIDIEGVQSEGQDNGAEDS
GDTEDELRRVAEQKEHRLPPGQEENGEDPYAGSTDENTDSEEH
QEPPDLPVPELPDFFQGKHFFLYGEFPGDERRKLIRYVTAFNGEL
EDYMSDRVQFVITAQEWDPSFEEALMDNPSLAFVRPRWIYSCNE
KQKLLPHQLYGVVPQASGSETPGTSESATPESfDKKYSIGLAIGTN
MGPEIRLRHVVSCSSQDSTHCAENLLKADTYRKWRAAKAGEKTIS
VVLQLEKEEQIHSVDIGNDGSAFVEVLVGSSAGGAGEQDYEVLLV
TSSFMSPSESRSGSNPNRVRMFGPDKLVRAAAEKRWDRVKIVCS
QPYSKDSPFGLSFVRFHSPPDKDEAEAPSQKVTVTKLGQFRVKEE
DESANSLRPGALFFSRINKTSPVTASDPAGPSYAAATLQASSAASS
ASPVSRAIGSTSKPQESPKGKRKLDLNQEEKKTPSKPPAQLSPSV
PKRPKLPAPTRTPATAPVPARAQGAVTGKPRGEGTEPRRPRAGP
EELGKILQGVVVVLSGFQNPFRSELRDKALELGAKYRPDWTRDST
HLICAFANTPKYSQVLGLGGRIVRKEWVLDCHRMRRRLPSQRYLM
AGPGSSSEEDEASHSGGSGDEAPKLPQKQPQTKTKPTQAAGPSS
PQKPPTPEETKAASPVLQEDIDIEGVQSEGQDNGAEDSGDTEDEL
RRVAEQKEHRLPPGQEENGEDPYAGSTDENTDSEEHQEPPDLPV
PELPDFFQGKHFFLYGEFPGDERRKLIRYVTAFNGELEDYMSDRV
QFVITAQEWDPSFEEALMDNPSLAFVRPRWIYSCNEKQKLLPHQL
YGVVPQASGGSSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKE
TCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNT
RCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPR
NRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRY
PHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQ
RLPPHILWATGLKSGSETPGTSESATPES[DKKYSIGLAIGTNSVG
MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSW
SPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLIS
SGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVL
ELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATG
LKSGSETPGTSESATPES[DKKYSIGLAIGTNSVGWAVITDEYKVP
SAKPNNSGEAPSSPTPKRSLSSSKCDPRHKDCLLREFRKLCAMV
ADNPSYNTKTQIIQDFLRKGSAGDGFHGDVYLTVKLLLPGVIKTVY
NLNDKQIVKLFSRIFNCNPDDMARDLEQGDVSETIRVFFEQSKSFP
PAAKSLLTIQEVDEFLLRLSKLTKEDEQQQALQDIASRCTANDLKCI
IRLIKHDLKMNSGAKHVLDALDPNAYEAFKASRNLQDVVERVLHNA
QEVEKEPGQRRALSVQASLMTPVQPMLAEACKSVEYAMKKCPN
GMFSEIKYDGERVQVHKNGDHFSYFSRSLKPVLPHKVAHFKDYIP
QAFPGGHSMILDSEVLLIDNKTGKPLPFGTLGVHKKAAFQDANVCL
FVFDCIYFNDVSLMDRPLCERRKFLHDNMVEIPNRIMFSEMKRVTK
ALDLADMITRVIQEGLEGLVLKDVKGTYEPGKRHWLKVKKDYLNE
GAMADTADLVVLGAFYGQGSKGGMMSIFLMGCYDPGSQKWCTV
TKCAGGHDDATLARLQNELDMVKISKDPSKIPSWLKVNKIYYPDFI
VPDPKKAAVWEITGAEFSKSEAHTADGISIRFPRCTRIRDDKDWKS
ATNLPQLKELYQLSKEKADFTVVAGDEGSSTTGGSSEENKGPSG
SAVSRKAPSKPSASTKKAEGKLSNSNSKDGNMQTAKPSAMKVGE
KLATKSSPVKVGEKRKAADETLCQTKVLLDIFTGVRLYLPPSTPDF
SRLRRYFVAFDGDLVQEFDMTSATHVLGSRDKNPAAQQVSPEWI
WACIRKRRLVAPCSGGSPKKKRKV
MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSW
SPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLIS
SGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVL
ELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATG
LKSGSETPGTSESATPES[DKKYSIGLDIGTNSVGWAVITDEYKVP
SAKPNNSGEAPSSPTPKRSLSSSKCDPRHKDCLLREFRKLCAMV
ADNPSYNTKTQIIQDFLRKGSAGDGFHGDVYLTVKLLLPGVIKTVY
NLNDKQIVKLFSRIFNCNPDDMARDLEQGDVSETIRVFFEQSKSFP
PAAKSLLTIQEVDEFLLRLSKLTKEDEQQQALQDIASRCTANDLKCI
IRLIKHDLKMNSGAKHVLDALDPNAYEAFKASRNLQDVVERVLHNA
QEVEKEPGQRRALSVQASLMTPVQPMLAEACKSVEYAMKKCPN
GMFSEIKYDGERVQVHKNGDHFSYFSRSLKPVLPHKVAHFKDYIP
QAFPGGHSMILDSEVLLIDNKTGKPLPFGTLGVHKKAAFQDANVCL
FVFDCIYFNDVSLMDRPLCERRKFLHDNMVEIPNRIMFSEMKRVTK
ALDLADMITRVIQEGLEGLVLKDVKGTYEPGKRHWLKVKKDYLNE
GAMADTADLVVLGAFYGQGSKGGMMSIFLMGCYDPGSQKWCTV
TKCAGGHDDATLARLQNELDMVKISKDPSKIPSWLKVNKIYYPDFI
VPDPKKAAVWEITGAEFSKSEAHTADGISIRFPRCTRIRDDKDWKS
ATNLPQLKELYQLSKEKADFTVVAGDEGSSTTGGSSEENKGPSG
SAVSRKAPSKPSASTKKAEGKLSNSNSKDGNMQTAKPSAMKVGE
KLATKSSPVKVGEKRKAADETLCQTKVLLDIFTGVRLYLPPSTPDF
SRLRRYFVAFDGDLVQEFDMTSATHVLGSRDKNPAAQQVSPEWI
WACIRKRRLVAPCSGGSPKKKRKV
MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSW
SPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLIS
SGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVL
ELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATG
LKSGGSSKAAGTPKKKAVVQAKLTTTGQVTSPVKGASFVTSTNPR
KFSGFSAKPNNSGEAPSSPTPKRSLSSSKCDPRHKDCLLREFRKL
CAMVADNPSYNTKTQIIQDFLRKGSAGDGFHGDVYLTVKLLLPGVI
KTVYNLNDKQIVKLFSRIFNCNPDDMARDLEQGDVSETIRVFFEQS
KSFPPAAKSLLTIQEVDEFLLRLSKLTKEDEQQQALQDIASRCTAN
DLKCHRLIKHDLKMNSGAKHVLDALDPNAYEAFKASRNLQDVVER
VLHNAQEVEKEPGQRRALSVQASLMTPVQPMLAEACKSVEYAMK
KCPNGMFSEIKYDGERVQVHKNGDHFSYFSRSLKPVLPHKVAHF
KDYIPQAFPGGHSMILDSEVLLIDNKTGKPLPFGTLGVHKKAAFQD
ANVCLFVFDCIYFNDVSLMDRPLCERRKFLHDNMVEIPNRIMFSEM
KRVTKALDLADMITRVIQEGLEGLVLKDVKGTYEPGKRHWLKVKK
DYLNEGAMADTADLVVLGAFYGQGSKGGMMSIFLMGCYDPGSQ
KWCTVTKCAGGHDDATLARLQNELDMVKISKDPSKIPSWLKVNKI
YYPDFIVPDPKKAAVWEITGAEFSKSEAHTADGISIRFPRCTRIRDD
KDWKSATNLPQLKELYQLSKEKADFTVVAGDEGSSTTGGSSEEN
KGPSGSAVSRKAPSKPSASTKKAEGKLSNSNSKDGNMQTAKPSA
MKVGEKLATKSSPVKVGEKRKAADETLCQTKVLLDIFTGVRLYLPP
STPDFSRLRRYFVAFDGDLVQEFDMTSATHVLGSRDKNPAAQQV
SPEWIWACIRKRRLVAPCSGSETPGTSESATPESfDKKYSIGLAIGT
MSKAAGTPKKKAVVQAKLTTTGQVTSPVKGASFVTSTNPRKFSGF
SAKPNNSGEAPSSPTPKRSLSSSKCDPRHKDCLLREFRKLCAMV
ADNPSYNTKTQIIQDFLRKGSAGDGFHGDVYLTVKLLLPGVIKTVY
NLNDKQIVKLFSRIFNCNPDDMARDLEQGDVSETIRVFFEQSKSFP
PAAKSLLTIQEVDEFLLRLSKLTKEDEQQQALQDIASRCTANDLKCI
IRLIKHDLKMNSGAKHVLDALDPNAYEAFKASRNLQDVVERVLHNA
QEVEKEPGQRRALSVQASLMTPVQPMLAEACKSVEYAMKKCPN
GMFSEIKYDGERVQVHKNGDHFSYFSRSLKPVLPHKVAHFKDYIP
QAFPGGHSMILDSEVLLIDNKTGKPLPFGTLGVHKKAAFQDANVCL
FVFDCIYFNDVSLMDRPLCERRKFLHDNMVEIPNRIMFSEMKRVTK
ALDLADMITRVIQEGLEGLVLKDVKGTYEPGKRHWLKVKKDYLNE
GAMADTADLVVLGAFYGQGSKGGMMSIFLMGCYDPGSQKWCTV
TKCAGGHDDATLARLQNELDMVKISKDPSKIPSWLKVNKIYYPDFI
VPDPKKAAVWEITGAEFSKSEAHTADGISIRFPRCTRIRDDKDWKS
ATNLPQLKELYQLSKEKADFTVVAGDEGSSTTGGSSEENKGPSG
SAVSRKAPSKPSASTKKAEGKLSNSNSKDGNMQTAKPSAMKVGE
KLATKSSPVKVGEKRKAADETLCQTKVLLDIFTGVRLYLPPSTPDF
SRLRRYFVAFDGDLVQEFDMTSATHVLGSRDKNPAAQQVSPEWI
RELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTER
YFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLY
HHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNE
AHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIAL
QSCHYQRLPPHILWATGLKSGSETPGTSESATPES[DKKYSIGLAI
MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSW
SPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLIS
SGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVL
ELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATG
LKSGSETPGTSESATPES[DKKYSIGLAIGTNSVGWAVITDEYKVP
CGCCLLQRRKPVLSFQRGHLRPRATHLISWSGSHVGLCTGPCEM
AEQRFCVDYAKRGTAGCKKCKEKIVKGVCRIGKVVPNPFSESGG
DMKEWYHIKCMFEKLERARATTKKIEDLTELEGWEELEDNEKEQI
SQHIADLSSKTAATPKKKATVQAKLTTTGQVTSPVKGASFITSTNP
RKFSGFSAAKPNNSEQDPSSPAPKTSLSASKCDPKHKDCLLREFR
KLCAMVAENPSYNTKTQIIHDFLQKGSTGDGFRGDVYLTVKLLLPG
VIKSVYNLNDKQIVKLFSRIFNCNPDDMARDLEQGDVSETIRVFFE
QSKSFPPAAKSLLTIQEVDAFLLHLSKLTKEDEQQQALQDIASRCT
ANDLKCIIRLIKHDLKMNSGAKHVLDALDPNAYEAFKASRNLQDVV
ERVLHNEQEVEKDPGRRRALSVQASLMTPVQPMLAEACKSIEYA
MKKCPNGMFSEIKYDGERVQVHKKGDHFSYFSRSLKPVLPHKVA
HFKDYIPKAFPGGQSMILDSEVLLIDNNTGKPLPFGTLGVHKKAAF
QDANVCLFVFDCIYFNDVSLMDRPLCERRKFLHDNMVEIRNRIMF
SEMKQVTKASDLADMINRVIREGLEGLVLKDVKGTYEPGKRHWLK
VKKDYLNEGAMADTADLVVLGAFYGQGSKGGMMSIFLMGCYDPD
SQKWCTVTKCAGGHDDATLARLQKELDMVKISKDPSKIPSWLKIN
KIYYPDFIVPDPKKAAVWEITGAEFSRSEAHTADGISIRFPRCTRIR
DDKDWKSATNLPQLKELYQLSKDKADFAVVAGDEGSSTTGGSNG
ENEGTAGSTVPRKGPKGPPSKSSASAKKTEQKLNDPSSRGGEKL
AVKSSPVKVGMKRKAADETPGLTKRRRASRQRGRRAMRTGRRS
MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSW
SPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLIS
SGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVL
ELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATG
LKSGSETPGTSESATPES[DKKYSIGLDIGTNSVGWAVITDEYKVP
CGCCLLQRRKPVLSFQRGHLRPRATHLISWSGSHVGLCTGPCEM
AEQRFCVDYAKRGTAGCKKCKEKIVKGVCRIGKVVPNPFSESGG
DMKEWYHIKCMFEKLERARATTKKIEDLTELEGWEELEDNEKEQI
SQHIADLSSKTAATPKKKATVQAKLTTTGQVTSPVKGASFITSTNP
RKFSGFSAAKPNNSEQDPSSPAPKTSLSASKCDPKHKDCLLREFR
KLCAMVAENPSYNTKTQIIHDFLQKGSTGDGFRGDVYLTVKLLLPG
VIKSVYNLNDKQIVKLFSRIFNCNPDDMARDLEQGDVSETIRVFFE
QSKSFPPAAKSLLTIQEVDAFLLHLSKLTKEDEQQQALQDIASRCT
ANDLKCIIRLIKHDLKMNSGAKHVLDALDPNAYEAFKASRNLQDVV
ERVLHNEQEVEKDPGRRRALSVQASLMTPVQPMLAEACKSIEYA
MKKCPNGMFSEIKYDGERVQVHKKGDHFSYFSRSLKPVLPHKVA
HFKDYIPKAFPGGQSMILDSEVLLIDNNTGKPLPFGTLGVHKKAAF
QDANVCLFVFDCIYFNDVSLMDRPLCERRKFLHDNMVEIRNRIMF
SEMKQVTKASDLADMINRVIREGLEGLVLKDVKGTYEPGKRHWLK
VKKDYLNEGAMADTADLVVLGAFYGQGSKGGMMSIFLMGCYDPD
SQKWCTVTKCAGGHDDATLARLQKELDMVKISKDPSKIPSWLKIN
KIYYPDFIVPDPKKAAVWEITGAEFSRSEAHTADGISIRFPRCTRIR
DDKDWKSATNLPQLKELYQLSKDKADFAVVAGDEGSSTTGGSNG
ENEGTAGSTVPRKGPKGPPSKSSASAKKTEQKLNDPSSRGGEKL
AVKSSPVKVGMKRKAADETPGLTKRRRASRQRGRRAMRTGRRS
MTLAFKTLFPRNLCALGRKELCLFSEQHHWPAIRQFSQWSETNLL
CGCCLLQRRKPVLSFQRGHLRPRATHLISWSGSHVGLCTGPCEM
AEQRFCVDYAKRGTAGCKKCKEKIVKGVCRIGKVVPNPFSESGG
DMKEWYHIKCMFEKLERARATTKKIEDLTELEGWEELEDNEKEQI
SQHIADLSSKTAATPKKKATVQAKLTTTGQVTSPVKGASFITSTNP
RKFSGFSAAKPNNSEQDPSSPAPKTSLSASKCDPKHKDCLLREFR
KLCAMVAENPSYNTKTQIIHDFLQKGSTGDGFRGDVYLTVKLLLPG
VIKSVYNLNDKQIVKLFSRIFNCNPDDMARDLEQGDVSETIRVFFE
QSKSFPPAAKSLLTIQEVDAFLLHLSKLTKEDEQQQALQDIASRCT
ANDLKCHRLIKHDLKMNSGAKHVLDALDPNAYEAFKASRNLQDVV
ERVLHNEQEVEKDPGRRRALSVQASLMTPVQPMLAEACKSIEYA
MKKCPNGMFSEIKYDGERVQVHKKGDHFSYFSRSLKPVLPHKVA
HFKDYIPKAFPGGQSMILDSEVLLIDNNTGKPLPFGTLGVHKKAAF
QDANVCLFVFDCIYFNDVSLMDRPLCERRKFLHDNMVEIRNRIMF
SEMKQVTKASDLADMINRVIREGLEGLVLKDVKGTYEPGKRHWLK
VKKDYLNEGAMADTADLVVLGAFYGQGSKGGMMSIFLMGCYDPD
SQKWCTVTKCAGGHDDATLARLQKELDMVKISKDPSKIPSWLKIN
KIYYPDFIVPDPKKAAVWEITGAEFSRSEAHTADGISIRFPRCTRIR
DDKDWKSATNLPQLKELYQLSKDKADFAVVAGDEGSSTTGGSNG
ENEGTAGSTVPRKGPKGPPSKSSASAKKTEQKLNDPSSRGGEKL
AVKSSPVKVGMKRKAADETPGLTKRRRASRQRGRRAMRTGRRS
WGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFL
SWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRD
LISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRL
YVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILW
ATGLKSGSETPGTSESATPES[DKKYSIGLAIGTNSVGWAVITDEY
MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSW
SPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLIS
SGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVL
ELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATG
LKSGSETPGTSESATPES[DKKYSIGLAIGTNSVGWAVITDEYKVP
VLQLEKEEQIHSVDIGNDGSAFVEVLVGSSAGGATAGEQDYEVLL
VTSSFMSPSESRSGSNPNRVRIFGPDKLVRAAAEKRWDRVKIVCS
QPYSKDSPYGLSFVKFHSPPDKDEAEAPSQKVTVTKLGQFRVKEE
DDSANSLRPGALFFNRINKAASASASDPAGPSYAAATLQASSAAS
SALPVPKVGGSSSKLQEPPKGKRKLDLGLEDSKPPSKPSAGPAAL
KRPKLPVPSRTPAATPASTPAQKAVPGKPRGEGTEPRGARAGPQ
ELGKILQGVVVVLSGFQNPFRSELRDKALELGAKYRPDWTPDSTH
LICAFANTPKYSQVLGLGGRIVRKEWVLDCYRMRRRLPSRRYLMA
GLGSSSEDEGDSHSESGEDEAPKLPRKRPQPKAKTQAAGPSSPP
RPPTPEETKAPSPGPQDNSDTDGEQSEGRDNGAEDSGDTEDEL
RRVAKQREQRQPPAPEENGEDPYAGSTDENTDSEAPSEADLPIP
ELPDFFQGKHFFLYGEFPGDERRKLIRYVTAFNGELEDYMSDRVQ
FVITAQEWDPNFEEALMENPSLAFVRPRWIYSCNEKQKLLPHQLY
GVVPQASGGSPKKKRKV
MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSW
SPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLIS
SGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVL
ELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATG
LKSGSETPGTSESATPES[DKKYSIGLDIGTNSVGWAVITDEYKVP
VLQLEKEEQIHSVDIGNDGSAFVEVLVGSSAGGATAGEQDYEVLL
VTSSFMSPSESRSGSNPNRVRIFGPDKLVRAAAEKRWDRVKIVCS
QPYSKDSPYGLSFVKFHSPPDKDEAEAPSQKVTVTKLGQFRVKEE
DDSANSLRPGALFFNRINKAASASASDPAGPSYAAATLQASSAAS
SALPVPKVGGSSSKLQEPPKGKRKLDLGLEDSKPPSKPSAGPAAL
KRPKLPVPSRTPAATPASTPAQKAVPGKPRGEGTEPRGARAGPQ
ELGKILQGVVVVLSGFQNPFRSELRDKALELGAKYRPDWTPDSTH
LICAFANTPKYSQVLGLGGRIVRKEWVLDCYRMRRRLPSRRYLMA
GLGSSSEDEGDSHSESGEDEAPKLPRKRPQPKAKTQAAGPSSPP
RPPTPEETKAPSPGPQDNSDTDGEQSEGRDNGAEDSGDTEDEL
RRVAKQREQRQPPAPEENGEDPYAGSTDENTDSEAPSEADLPIP
ELPDFFQGKHFFLYGEFPGDERRKLIRYVTAFNGELEDYMSDRVQ
MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSW
SPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLIS
SGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVL
ELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATG
LKSGGSPEISLRHVVSCSSQDSTHRAENLLKADTYRKWRSAKAG
EKTISVVLQLEKEEQIHSVDIGNDGSAFVEVLVGSSAGGATAGEQD
YEVLLVTSSFMSPSESRSGSNPNRVRIFGPDKLVRAAAEKRWDRV
KIVCSQPYSKDSPYGLSFVKFHSPPDKDEAEAPSQKVTVTKLGQF
RVKEEDDSANSLRPGALFFNRINKAASASASDPAGPSYAAATLQA
SSAASSALPVPKVGGSSSKLQEPPKGKRKLDLGLEDSKPPSKPSA
GPAALKRPKLPVPSRTPAATPASTPAQKAVPGKPRGEGTEPRGA
RAGPQELGKILQGVVVVLSGFQNPFRSELRDKALELGAKYRPDWT
PDSTHLICAFANTPKYSQVLGLGGRIVRKEWVLDCYRMRRRLPSR
RYLMAGLGSSSEDEGDSHSESGEDEAPKLPRKRPQPKAKTQAAG
PSSPPRPPTPEETKAPSPGPQDNSDTDGEQSEGRDNGAEDSGD
TEDELRRVAKQREQRQPPAPEENGEDPYAGSTDENTDSEAPSEA
DLPIPELPDFFQGKHFFLYGEFPGDERRKLIRYVTAFNGELEDYMS
DRVQFVITAQEWDPNFEEALMENPSLAFVRPRWIYSCNEKQKLLP
HQLYGVVPQASGSETPGTSESATPES[DKKYSIGLAIGTNSVGWA
MPEISLRHVVSCSSQDSTHRAENLLKADTYRKWRSAKAGEKTISV
VLQLEKEEQIHSVDIGNDGSAFVEVLVGSSAGGATAGEQDYEVLL
VTSSFMSPSESRSGSNPNRVRIFGPDKLVRAAAEKRWDRVKIVCS
QPYSKDSPYGLSFVKFHSPPDKDEAEAPSQKVTVTKLGQFRVKEE
DDSANSLRPGALFFNRINKAASASASDPAGPSYAAATLQASSAAS
SALPVPKVGGSSSKLQEPPKGKRKLDLGLEDSKPPSKPSAGPAAL
KRPKLPVPSRTPAATPASTPAQKAVPGKPRGEGTEPRGARAGPQ
ELGKILQGVVVVLSGFQNPFRSELRDKALELGAKYRPDWTPDSTH
LICAFANTPKYSQVLGLGGRIVRKEWVLDCYRMRRRLPSRRYLMA
GLGSSSEDEGDSHSESGEDEAPKLPRKRPQPKAKTQAAGPSSPP
RPPTPEETKAPSPGPQDNSDTDGEQSEGRDNGAEDSGDTEDEL
RRVAKQREQRQPPAPEENGEDPYAGSTDENTDSEAPSEADLPIP
ELPDFFQGKHFFLYGEFPGDERRKLIRYVTAFNGELEDYMSDRVQ
FVITAQEWDPNFEEALMENPSLAFVRPRWIYSCNEKQKLLPHQLY
GVVPQASGGSSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKET
CLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTR
CSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRN
RQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYP
HLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQR
LPPHILWATGLKSGSETPGTSESATPES[DKKYSIGLAIGTNSVGW
The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including”, “containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred examples and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a genetic marker” includes a plurality of genetic markers, including mixtures and combinations thereof.
As used herein, the term “about”, in the context of concentrations of components of the formulations, typically means +/−5% of the stated value, more typically +/−4% of the stated value, more typically +/−3% of the stated value, more typically, +/−2% of the stated value, even more typically +/−1% of the stated value, and even more typically +/−0.5% of the stated value.
Throughout this disclosure, certain examples may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Certain examples may also be described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the examples with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
Other examples are within the following claims and non-limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.
The CG to GC Base Editor (CGBE) disclosed in the present application leverage the cell's innate base excision repair (BER) pathway. Among the proteins involved in the BER pathway, the inventors of the present invention have extensively characterized three major players, which are DNA polymerase 13, DNA ligase III and XRCC1.
Because the relative orientation of Cas9 fusions may affect the activity of the fusion, several versions of the CGBE candidates with the fused rAPOBEC and BER proteins at different orientations with respect to nCas9 were tested (
High-throughput sequencing of the HEK2 site revealed that the CGBE candidates were able to edit C:G to both G:C and T:A. Out of the 31 candidates tested, 20 candidates showed an increased level of C:G to G:C editing relative to BE3 at position 6 within the protospacer (
Similarly, sequencing at the HEK3 site revealed editing of C:G to both G:C and T:A. 12 out of 31 candidates gave C:G to G:C editing levels at position 5 that were higher than that achieved by BE3, with the best performing candidates effecting up to 13% C:G to G:C editing compared with 4% for BE3 (
CGBEs were further tested for C:G to G:C editing at four genomic sites known to be amenable to BE3-mediated editing—EMX1, HEK4, RNF2, and FANCF (
It is then determined if target sequence context impacts editing efficiency. In characterizing the two CGBEs with four gRNAs targeting four disease-associated sites—including dyslipidemia-associated gene ADRB2, hearing loss-associated gene GJB2, and hypertrophic cardiomyopathy-associated gene MYBPC3—it was observed that not only did CGBEs efficiently interrogate disease-associated genes, but they also gave higher levels of C:G to G:C editing at C's immediately following an A/T (
A reduction in editing levels at position 4 relative to position 6 of HEK2 was observed (
Recognizing that simply removing UGI can potentially increase C:G to G:C editing at the expense of C:G to T:A editing, the effect of fusing rXRCC1 or rPB(8 kD) to rAPOBEC-nCas9 was hence quantified. Across 28 independent treatments using a variety of gRNAs (
As with CRISPR-Cas systems, base editors have been reported to exhibit potential DNA and RNA off-target effects. Because CGBEs share the same APOBEC-nCas9 component with BE3, CGBE and BE3 activities were assessed side-by-side on 29 off-target sites using 5 gRNAs. CGBE and BE3 induced Ai % C:G to D:H edits at the same 15 positions (D is either A, G or T; H is either A, C, or T). Only at 2 out of these 15 positions did CGBE induce greater off-target editing than BE3; at the remaining 13 sites, CGBE showed lower off-target activity. While reduction of off-target activity can be attributed to lower C:G to T:A editing at off-target sites, C:G to G:C editing at the same off-target sites increased (
One limitation of BE3 is its low efficiency in some cell types. With BE3, low C:G to T:A editing was observed in H9 stem cells at five genomic sites (with a maximum of 1.2% C:G to T:A editing at HEK4;
CGBEs that target cytosine within a specified window and convert it into guanine as a predominant editing product were then developed. In separate works, Liu and Koblan designed CGBE candidates by fusing UDG (UNG), UdgX, and polymerases with rAPOBEC-nCas9 (
Koblan and Liu's work seeks to induce base excision and perform a translesion polymerization across the targeted abasic site (
Constructs and Molecular Cloning
The BE3 (Addgene plasmid #73021), prime editor 2 (Addgene plasmid #132775), pegRNA-HEK3_CTT_ins (Addgene plasmid #132778) plasmids are used in the present disclosure. The BE3 plasmid is a mammalian expression plasmid with BE3 being driven by a CMV promoter. hXRCC1 (pTXG-hXRCC1) and hLIG3 (pGEX4T-hLIG3) (Addgene plasmid #52283 and #81055 respectively) are also used. The mutation R400Q is introduced to hXRCC1 and N628K is introduced to hLIG3 via blunt-end ligation. Briefly, plasmid containing either hXRCC1 or hLIG3 is amplified via PCR using Q5 Hot Start HiFi 2× Master Mix (NEB, M0494). PCR product is then treated with DpnI (NEB, R0176) and T4 Polynucleotide Kinase (NEB, M0201) at 37° C. for 30 minutes and inactivated at 65° C. for 20 minutes before being ligated using T4 DNA Ligase (NEB, M0202; room temperature for 2 hours). Ligated product is then transformed into chemically competent 5-alpha Escherichia Coli (NEB, C2987). rXRCC1, rLIG3, hPBs, and rPBs were obtained as human codon-optimized de novo synthesized gene fragments (Twist Biosciences). All other oligonucleotides used in the study were de novo synthesized (IDT DNA). To fuse BER proteins with rAPOBEC-nCas9, Q5 Hot Start HiFi 2× Master Mix was used to generate Gibson fragments of the BER proteins as Gibson inserts. After checking PCR products on a gel, Gibson insert and vector were incubated with NEBuilder HiFi DNA Assembly Master Mix (NEB, E2621) for 1 hour at 50° C. The Gibson reaction is then transformed into chemically competent Escherichia Coli.
All assembled plasmids were Sanger sequenced for sequence verification and were prepared using either the PureYield Plasmid Miniprep System (Promega, A1223) or Plasmid Plus Maxi Kit (Qiagen, 12965).
Cell Culture, Transfection, and Genomic DNA Harvest
HEK293AAV cells (Agilent, 240073) were maintained in DMEM with GlutaMAX and sodium pyruvate (Thermo Fisher, 10569-010) supplemented with 10% HI FBS (Thermo Fisher) at 37° C. and 5% CO2. HTB-9 cells (ATCC, 5637) were maintained in RPMI-1640 with L-glutamine and sodium bicarbonate (Sigma, R8758) supplemented with 10% HI FBS (Thermo Fisher) and 1% MEM Non-Essential Amino Acids Solution (Thermo Fisher, 11140050) at 37° C. and 5% CO2. Both HTB9 and HEK cells were transfected via lipofection. After cells reached ˜80% confluency, they were washed with PBS, pH 7.2 (Thermo Fisher, 20012-027) before being treated with TrypLE Express (Thermo Fisher, 12604). 30,000 cells were added to each well of a 48-well plate one day before transfection. For each well, 750 ng of base-editor plasmid, 250 ng of gRNA plasmid, and 20 ng of GFP plasmid were transfected into these cells using Lipofectamine 3000 (Invitrogen, L3000015) according to the manufacturer's protocol. The media were replaced with fresh media 24 hours after transfection. For prime editing, 750 ng of PE plasmid, 250 ng of pegRNA, and 83 ng of sgRNA were used for transfection. 72 hours after transfection, media were removed; cells were washed with 50 μL PBS, pH 7.2, and genomic DNA was extracted using 50 μL of Quick Extract DNA Extract Solution (Lucigen, QE09050) per well according to manufacturer's protocol. All sample sizes indicate biological replicates.
Jurkat cells (ATCC, TIB-152, Clone E6-1) were maintained in RPMI-1640 with L-glutamine and sodium bicarbonate (Sigma, R8758) supplemented with 10% HI FBS (Thermo Fisher) and 1% MEM Non-Essential Amino Acids Solution (Thermo Fisher, 11140050) at 37° C. and 5% CO2. 200,000 cells were nucleofected with 750 ng of base editor and 250 ng of gRNA expression plasmids using the SE Cell Line 4D-Nucleofector X Kit S (Lonza) and program CL-120 on the 4D X-Unit.
HepG2 cells were maintained in IMDM (Thermo Fisher, 31980-030) supplemented with 10% FBS (Thermo Fisher) and 1% NEAA (Thermo Fisher, 11140050) at 37° C. and 5% CO2. 200,000 cells were nucleofected with 750 ng of base editor and 250 ng of gRNA expression plasmids using the SF Cell Line 4D-Nucleofector X Kit S (Lonza) and program EH-100 on the 4D X-Unit.
eHAP cells (Horizon Discovery, C669) were maintained in IMDM (Thermo Fisher, 31980-030) supplemented with 10% FBS at 37° C. and 5% CO2. 200,000 cells were nucleofected with 750 ng of base editor and 250 ng of gRNA expression plasmids using the SE Cell Line 4D-Nucleofector X Kit S (Lonza) and program DS-138 on the 4D X-Unit.
H9 stem cells (WiCell, WA09) were maintained in mTeSR1 (Stemcell technology, 85850). 200,000 cells were nucleofected with 1500 ng of base editor and 500 ng of gRNA expression plasmids using the P3 Primary Cell kit (Lonza, V4XP-3024) and program hES H9 program on the 4D X-Unit.
Sequencing of Genomic DNA
Sites of interest were prepared for high-throughput sequencing via two PCR amplifications—the first PCR amplifies the region of interest while the second PCR adds appropriate sequencing barcodes. The first PCR was performed in commonly used methods. Primers for the second PCR are based off Illumina adaptors. Amplicons from the second PCR were then pooled and gel extracted (Promega, A9282) to make the final library, which was quantified via Qubit fluorometer (Thermo Fisher) and sequenced on an Illumina iSeq 100 according to the manufacturer's protocol. The resultant FASTQ files were analyzed using CRISPResso2. All sample sizes indicate biological replicates.
Statistical analyses were performed on Matlab. Weblogos were created using Weblogo 3.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10201913340Q | Dec 2019 | SG | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2020/050787 | 12/28/2020 | WO |
