CRISPR-related methods and compositions with governing gRNAS

Abstract
Disclosed herein are methods and compositions useful in targeting a payload to, or editing a target nucleic acid, where a governing gRNA molecule is used to target, optionally inactivate, a Cas9 molecule or a Cas9 molecule/gRNA complex.
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 Dec. 18, 2014, is named C2159-704110_SL.txt and is 501,350 bytes in size.


FIELD OF THE INVENTION

The invention relates to CRISPR-related methods and components for editing of, or delivery of a payload to, a target nucleic acid sequence.


BACKGROUND OF THE INVENTION

CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) evolved in bacteria as an adaptive immune system to defend against viral attack. Upon exposure to a virus, short segments of viral DNA are integrated into the CRISPR locus. RNA is transcribed from a portion of the CRISPR locus that includes the viral sequence. That RNA, which contains sequence complimentary to the viral genome, mediates targeting of a Cas9 protein to a target sequence in the viral genome. The Cas9 protein cleaves and thereby silences the viral target.


Recently, the CRISPR/Cas system has been adapted for genome editing in eukaryotic cells. The introduction of site-specific double strand breaks (DSBs) allows for target sequence alteration through one of two endogenous DNA repair mechanisms—either non-homologous end-joining (NHEJ) or homology-directed repair (HDR). The CRISPR/Cas system has also been used for gene regulation including transcription repression and activation without altering the target sequence. Targeted gene regulation based on the CRISPR/Cas system uses an enzymatically inactive Cas9 (also known as a catalytically dead Cas9).


Despite the recent advances adapting the CRISPR/Cas system for genome editing in eukaryotic cells, there remains a need for improved regulation and control of these systems for use in eukaryotic cells.


SUMMARY OF THE INVENTION

Disclosed herein are methods and compositions, e.g., a Cas9 molecule complexed with a gRNA molecule, that can be used to target a specific location in a target DNA. Depending on the Cas9 molecule/gRNA molecule complex used in the disclosed methods and compositions, specific editing of a target nucleic acid, or the delivery of a payload, can be effected.


Methods and compositions that use, or include, a nucleic acid, e.g., a DNA, that encodes a Cas9 molecule or a gRNA molecule, can, in addition, use or include a “governing gRNA molecule.” The governing gRNA molecule can complex with the Cas9 molecule to inactivate or silence a component of a Cas9 system. In one aspect, the disclosure features a gRNA molecule, referred to herein as a governing gRNA molecule, comprises a targeting domain which targets a component of the Cas9 system. In an embodiment, the governing gRNA molecule targets and silences (1) a nucleic acid that encodes a Cas9 molecule (i.e., a Cas9-targeting gRNA molecule), (2) a nucleic acid that encodes a gRNA molecule (i.e., a gRNA-targeting gRNA molecule), or (3) a nucleic acid sequence engineered into the Cas9 components that is designed with minimal homology to other nucleic acid sequences in the cell to minimize off-target cleavage (i.e., an engineered control sequence-targeting gRNA molecule).


The targeting sequence for the governing gRNA can be selected to increase regulation or control of the Cas9 system and/or to reduce or minimize off-target effects of the system. For example, a governing gRNA can minimize undesirable cleavage, e.g., “recleavage” after Cas9 mediated alteration of a target nucleic acid or off-target cutting of Cas9, by inactivating (e.g., cleaving) a nucleic acid that encodes a Cas9 molecule. In an embodiment, a governing gRNA places temporal or other limit(s) on the level of expression or activity of the Cas9 molecule/gRNA molecule complex. In an embodiment, the governing gRNA reduces off-target or other unwanted activity.


A target sequence for the governing gRNA can be disposed in the control or coding region of the Cas9 encoding sequence. This can be a Cas9 sequence or a non-Cas9 sequence, e.g., a sequence which is selected for, or which results in, reduced or minimized off target effect. The silencing, or inactivation, can be effected by cleaving the targeted nucleic acid sequence or by binding a Cas9 molecule/governing gRNA molecule complex to the targeted nucleic acid sequence.


In an aspect, the disclosure features a gRNA molecule that targets, optionally inactivates, a Cas9 molecule. In an embodiment, the gRNA molecule targets a nucleic acid sequence that encodes the Cas9 molecule. For example, a sequence that encodes the Cas9 molecule can comprise one or more of: a sequence encoding the amino acid sequence of the Cas9 molecule, a sequence encoding the amino acid sequence of the Cas9 molecule comprising non-translated sequence, or a sequence encoding the amino acid sequence of the Cas9 molecule comprising non-transcribed sequence.


In an embodiment, the Cas9 molecule is an eaCas9 molecule. In another embodiment, the Cas9 molecule is an eiCas9 molecule.


In an embodiment, the gRNA is configured to provide a Cas9 molecule-mediated cleavage event in the nucleic acid sequence that encodes the Cas9 molecule. In an embodiment, the gRNA molecule comprises a targeting domain configured to provide a Cas9 molecule-mediated cleavage event in the nucleic acid sequence that encodes the Cas9 molecule.


In an embodiment, the gRNA molecule:


targets the Cas9 molecule-amino acid coding sequence of the nucleic acid sequence;


is configured to provide a Cas9 molecule-mediated cleavage event in the Cas 9 molecule-amino acid coding sequence of the nucleic acid sequence; or comprises a targeting domain configured to provide a Cas9 molecule-mediated cleavage event in the Cas9 molecule-amino acid coding sequence of the nucleic acid sequence.


In an embodiment, the gRNA molecule:


targets a non-coding sequence of the nucleic acid sequence;


is configured to provide a Cas9 molecule-mediated cleavage event in a non-coding sequence of the nucleic acid sequence; or comprises a targeting domain configured to provide a Cas9 molecule-mediated cleavage event in a non-coding sequence of the nucleic acid sequence.


In an embodiment, the gRNA molecule:


targets an untranslated sequence of the nucleic acid sequence;


is configured to provide a Cas9 molecule-mediated cleavage event in an untranslated sequence of the nucleic acid sequence; or


comprises a targeting domain configured to provide a Cas9 molecule-mediated cleavage event in an untranslated sequence of the nucleic acid sequence.


In an embodiment, the gRNA molecule:


targets the nucleic acid sequence 5′ of the Cas 9 molecule-amino acid coding region;


is configured to provide a Cas9 molecule-mediated cleavage event in the nucleic acid sequence 5′ of the Cas9 molecule-coding region; or


comprises a targeting domain configured to provide a Cas9 molecule-mediated cleavage event 5′ of the Cas9 molecule-coding region of the nucleic acid sequence.


In an embodiment, the gRNA molecule:


targets the nucleic acid sequence that encodes the Cas9 molecule 3′ of the Cas9 molecule-coding region;


is configured to provide a Cas9 molecule-mediated cleavage event in the nucleic acid sequence 3′ of the Cas9 molecule-coding region; or


comprises a targeting domain configured to provide a Cas9 molecule-mediated cleavage event 3′ of the Cas9 molecule-coding region of the nucleic acid sequence.


In an embodiment, the gRNA molecule:


targets the promoter region of the nucleic acid sequence,


is configured to provide a Cas9 molecule-mediated cleavage event in the promoter region of nucleic acid sequence; or


comprises a targeting domain configured to provide a Cas9 molecule-mediated cleavage event in the promoter region of the nucleic acid sequence,


wherein the promoter region is functionally linked to the Cas9 molecule amino acid coding region.


In an embodiment, the gRNA molecule:


targets Cas9 molecule intronic sequence of the nucleic acid sequence:


is configured to provide a Cas9 molecule-mediated cleavage event in Cas9 molecule intronic sequence of the nucleic acid sequence; or


comprises a targeting domain configured to provide a Cas9 molecule-mediated cleavage event in Cas9 molecule intronic sequence of the nucleic acid sequence.


In an embodiment, the Cas9 molecule is a S. pyogenes Cas9 molecule. In another embodiment, the Cas9 molecule is a S. aureus Cas9 molecule.


In an embodiment, the gRNA molecule is selected from Tables E1-E6. In another embodiment, the gRNA molecule is selected from Tables E7-E12.


In an embodiment, the gRNA is a chimeric gRNA. In another embodiment, the gRNA is a modular gRNA.


In an embodiment, the governing gRNA molecule targets the coding sequence, or a control region, e.g., a promoter, for the Cas9 system component to be negatively regulated. For example, the gRNA can target the coding sequence for Cas9, or a control region, e.g., a promoter, that regulates the expression of the Cas9 coding sequence. In an embodiment, the governing gRNA, e.g., a Cas9-targeting gRNA molecule, or a nucleic acid that encodes it, is introduced separately, e.g., later than the Cas9 molecule or a nucleic acid that encodes it. For example, a first vector, e.g., a viral vector, e.g., an AAV vector, can introduce nucleic acid encoding a Cas9 and one or more gRNAs and a second vector, e.g., a viral vector, e.g., an AAV vector, can introduce a nucleic acid encoding a governing gRNA molecule, e.g., a Cas9-targeting gRNA molecule. The second vector can be introduced after the first. In an embodiment, the governing gRNA. e.g., a Cas9-targeting gRNA molecule, or a nucleic acid that encodes it, can be introduced together, e.g., at the same time or in the same vector, with the Cas9 molecule or a nucleic acid that encodes it, but, e.g., under transcriptional control elements, e.g., a promoter or an enhancer, that are activated at a later time, e.g., such that after a period of time the transcription of Cas9 is silenced. In an embodiment, the transcriptional control element is activated intrinsically. In an embodiment, the transcriptional element is activated via the introduction of an external trigger.


In an aspect, the disclosure features a nucleic acid comprising a sequence that encodes a governing gRNA molecule. In an embodiment, the governing gRNA molecule comprises a Cas9 molecule-targeting gRNA molecule. In an embodiment, the nucleic acid comprises a sequence that encodes a gRNA molecule described herein. In an embodiment, the nucleic acid is purified.


In another aspect, the disclosure features a nucleic acid, e.g., one or more vectors, e.g., one or more viral vectors, e.g., one or more AAV vectors, comprising:


a) a first nucleic acid sequence that encodes a governing gRNA molecule, e.g., a Cas9 molecule-targeting gRNA molecule or a gRNA molecule-targeting gRNA molecule; and


b) a second nucleic acid sequence that encodes a Cas9 molecule, e.g., an eaCas9 or an eiCas9 molecule.


In an embodiment, the governing gRNA molecule comprises a Cas9 molecule-targeting gRNA molecule. In another embodiment, the governing gRNA molecule comprises a gRNA molecule-targeting gRNA molecule.


In an embodiment, the governing gRNA molecule comprises a Cas9 molecule-targeting gRNA molecule and the Cas9 molecule-targeting gRNA molecule targets the second nucleic acid sequence that encodes the Cas9 molecule.


In an embodiment, the Cas9 molecule is an eaCas9 molecule. In another embodiment, the Cas9 molecule is an eiCas9 molecule.


In an embodiment, the gRNA molecule is configured to provide a Cas9 molecule-mediated cleavage event in the second nucleic acid sequence. In an embodiment, the gRNA molecule comprises a targeting domain configured to provide a Cas9 molecule-mediated cleavage event in the second nucleic acid sequence. In an embodiment, the gRNA molecule is a gRNA molecule described herein and targets the second nucleic acid sequence.


In an embodiment, the nucleic acid is purified.


In an embodiment, component a) and component b) are provided on the same nucleic acid, e.g., the same vector, e.g., the same viral vector, e.g., the same AAV vector. In another embodiment, component a) and component b) are provided on different nucleic acids, e.g., different vectors, e.g., different viral vectors, e.g., different AAV vectors.


In an embodiment, the nucleic acid is configured such that a Cas9 molecule-targeting gRNA transcribed from said nucleic acid forms a complex with a Cas9 molecule produced from said nucleic acid.


In an embodiment, said complex is capable of inactivating or silencing, e.g., by cleaving, the nucleic acid sequence that comprises or encodes said Cas9 molecule sequence. In an embodiment, the inactivating comprises cleaving.


In an embodiment, said first nucleic acid sequence is under the control of a first control region, e.g., promoter, and said second nucleic acid sequence is under the control of a second control region, e.g., promoter, and said first and second control regions, e.g., promoters, are different, e.g., one is a constitutive promoter and one is an inducible promoter. In an embodiment, one of the first and second control regions is a constitutive promoter and one is an inducible promoter.


In an embodiment, said first nucleic acid sequence and said second nucleic acid sequence are differentially expressed, e.g., differentially expressed in terms of level of expression or temporally, e.g., the first sequence is expressed later than said second sequence, or the first sequence is expressed at a lower level than said second sequence.


In an embodiment, the nucleic acid further comprises:


c) a third nucleic acid sequence that encodes a gRNA molecule, e.g., a second gRNA molecule, comprising a targeting domain which is complementary with a target nucleic acid, e.g., wherein the second gRNA does not target b).


In an embodiment, the target nucleic acid is disclosed herein, e.g., a sequence from:


a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment, said first nucleic acid sequence is under the control of a first control region, e.g., promoter,


said second nucleic acid sequence is under the control of said second control region, e.g., promoter, or a third control region, e.g., promoter,


said third nucleic acid sequence is under the control of said second control region, e.g., promoter, or said third control region, e.g., promoter, and


said first control region, e.g., promoter, is different from said second and/or said third control region, e.g., promoter.


In an embodiment, said first nucleic acid sequence and said third nucleic acid sequence are differentially expressed, e.g., differentially expressed in terms of level of expression or temporally, e.g., the first sequence is expressed later than said third sequence, or the first sequence is expressed at a lower level than said third sequence.


In an embodiment, the nucleic acid further comprises a template nucleic acid (referred to interchangeably herein as a swap nucleic acid sequence), e.g., having 5′ and 3′ flanking region sequences recognized by one or more governing gRNAs.


In an embodiment, the nucleic acid sequence that comprises or encodes the Cas9 molecule sequence or the gRNA molecule sequence (e.g., targeted by the governing gRNA as described herein) further comprises a nucleic acid sequence that is capable of being used as a template nucleic acid, e.g., after being cleaved or excised (e.g., by the method described herein) from the nucleic acid sequence that comprises or encodes the Cas9 molecule sequence or the gRNA molecule sequence, e.g., as a donor DNA for homologous recombination. In an embodiment, a first governing gRNA molecule targets a region 5′ of a nucleic acid sequence comprising the template nucleic acid sequence and a second governing gRNA molecule targets a region 3′ of the nucleic acid sequence comprising the template nucleic acid sequence. For example, at least two (e.g., two, three, four, five or more) governing gRNAs can be used to produce one or more (e.g., two, three, four or more) template nucleic acids. In another embodiment, a single governing gRNA molecule targets both the regions 5′ and 3′ of the nucleic acid sequence comprising the template nucleic acid sequence. For example, the region (e.g., targeted by the governing gRNA molecule) 5′ of the nucleic acid sequence comprising the template nucleic acid sequence can be the same or substantially the same as the region (e.g., targeted by the governing gRNA molecule) 3′ of the nucleic acid sequence comprising the template nucleic acid sequence. In an embodiment, the nucleic acid sequence comprising the template nucleic acid sequence is in a vector, e.g., a vector described herein. In an embodiment, the vector is a viral vector, e.g., an AAV vector.


In an aspect, the disclosure features a vector comprising a nucleic acid described herein. In an embodiment, the vector is a viral vector. In an embodiment, the viral vector, is an AAV rector.


In an aspect, the disclosure features a composition, e.g., a pharmaceutical composition, comprising:


a) a governing gRNA molecule, e.g., a governing gRNA molecule described herein, or a nucleic acid that encodes a governing gRNA molecule, e.g., a nucleic acid described herein.


In an embodiment, the composition comprises one or more (e.g., 2 or all) of;


b) a Cas9 molecule, e.g., a Cas9 molecule described herein, or a nucleic acid sequence that encodes the Cas 9 molecule, e.g., a nucleic acid sequence described herein;


c) a second gRNA molecule or a nucleic acid encoding the second gRNA molecule; or


d) a template nucleic acid.


In an embodiment, the governing gRNA molecule comprises a Cas9 molecule-targeting gRNA molecule. In an embodiment, the Cas 9 molecule-targeting gRNA comprises a gRNA molecule described herein.


In an embodiment, the gRNA molecule is configured to provide a Cas 9 molecule-mediated cleavage event in the nucleic acid sequence that encodes the Cas 9 molecule.


In an embodiment, the composition comprises a Cas9 molecule-targeting gRNA molecule and a nucleic acid encoding the Cas9 molecule. In another embodiment, the composition comprises a Cas9 molecule-targeting gRNA molecule and the Cas9 molecule.


In an embodiment, the composition further comprises:


c) a second gRNA molecule or a nucleic acid encoding the second gRNA molecule.


In an embodiment, the second gRNA targets a Cas 9 molecule to a target nucleic acid.


In an embodiment, the composition further comprises:


d) a template nucleic acid.


In an embodiment, the composition comprises a second gRNA or a nucleic acid encoding the second gRNA.


In an embodiment, the template nucleic acid is configured to mediate repair of a break positioned by the second gRNA.


In an embodiment, each of a), b), c) and d) is present as a nucleic acid and are encoded on the same nucleic acid molecule. In an embodiment, a first sequence selected from a), b), c) and d) is encoded on a first nucleic acid molecule and a second sequence selected from a), b), c), and d) is encoded on a second nucleic acid molecule.


In another aspect, the disclosure features a composition, e.g., a pharmaceutical composition, comprising the nucleic acid described herein. For example, the nucleic acid, e.g., one or more vectors, e.g., one or more viral vectors, e.g., one or more AAV vectors, can comprise:


a) a first nucleic acid sequence that encodes a governing gRNA molecule, e.g., a Cas9-targeting gRNA molecule or a gRNA-targeting gRNA molecule; and


b) a second nucleic acid sequence that encodes a Cas9 molecule, e.g., an eaCas9 or an eiCas9 molecule.


In an embodiment, said nucleic acid comprises an AAV vector.


In an aspect, the disclosure features a composition, e.g., a pharmaceutical composition, comprising nucleic acid sequence, e.g., a DNA, that encodes a governing gRNA molecule, e.g., a Cas9-targeting gRNA molecule or a gRNA-targeting gRNA molecule, and one or more of


a) a Cas9 molecule,


b) a second Cas9 molecule,


c) a gRNA molecule, and


d) a second gRNA molecule.


In an embodiment, each of a), b), c) and d) present are encoded on the same nucleic acid molecule. In an embodiment, a first sequence selected from a, b, c and d is encoded on a first nucleic acid molecule and a second sequence selected from a, b, c, and d is encoded on a second nucleic acid molecule. In an embodiment, said nucleic acid encodes: a and c; a, c, and d; or a, b, c, and d.


In an aspect, the disclosure features a pharmaceutical preparation comprising:


a gRNA molecule described herein;


a nucleic acid described herein;


a vector described herein; or


a composition described herein.


In an aspect, the disclosure features a cell comprising:


a gRNA molecule described herein;


a nucleic acid described herein;


a vector described herein; or


a composition described herein.


In an embodiment, the cell comprises:


a nucleic acid sequence encoding a Cas 9 molecule, wherein a sequence that encodes the Cas9 molecule can comprise one or more of: a sequence encoding the amino acid sequence of the Cas9 molecule, a sequence encoding the amino acid sequence of the Cas9 molecule comprising non-translated sequence, and a sequence encoding the amino acid sequence of the Cas9 molecule comprising non-transcribed sequence; and


a governing gRNA molecule.


In an embodiment, the governing gRNA molecule comprises a gRNA molecule that targets the nucleic acid sequence that encodes the Cas 9 molecule. In an embodiment, the gRNA molecule is a gRNA molecule described herein.


In an embodiment, the cell further comprises a Cas 9 molecule.


In an embodiment, the cell further comprises a second gRNA molecule or a nucleic acid encoding the second gRNA molecule. In an embodiment, the second gRNA targets a Cas9 molecule to a target nucleic acid.


In an embodiment, the cell further comprises a template nucleic acid. In an embodiment, the template nucleic acid is configured to mediate repair of a break in the target nucleic acid positioned by the second gRNA molecule.


In an embodiment, the cell comprises target nucleic acid cleaved by second gRNA molecule mediated targeting of the Cas9 molecule.


In an embodiment, the cell comprises the target nucleic acid that has been cleaved and repaired. In an embodiment, the repair comprises template nucleic acid mediated repair.


In an embodiment, the nucleic acid sequence encoding the Cas9 molecule has not been cleaved. In an embodiment, the nucleic acid sequence encoding the Cas9 molecule can express Cas 9 molecule.


In an embodiment, the nucleic acid sequence encoding the Cas9 molecule has been cleaved by gRNA mediated targeting of Cas 9 molecule. In an embodiment, the cleaved nucleic acid sequence encoding the Cas9 molecule has reduced ability to express Cas9 molecule, as compared to the same molecule not having been cleaved. In an embodiment, the cleaved nucleic acid sequence encoding the Cas9 molecule is substantially incapable of expressing Cas 9 molecule.


In an embodiment, the cell comprises one or both of:


a cleaved nucleic acid sequence encoding the Cas9 molecule; or


a target nucleic acid having a repaired Cas9 molecule-mediated cleavage event.


In an embodiment, the cell is a vertebrate, mammalian, rodent, goat, pig, bird, chicken, turkey, cow, horse, sheep, fish, primate, or human cell.


In another embodiment, the cell is a plant cell. In an embodiment, the plant cell is a monocot or a dicot.


In an embodiment, the cell is a human cell. In an embodiment, the cell is a somatic cell, germ cell, or prenatal cell. In an embodiment, the cell is a zygotic, blastocyst or embryonic cell, a stem cell, a mitotically competent cell, a meiotically competent cell.


In an aspect, the disclosure features a method of altering a cell. e.g., altering the structure, e.g., sequence, of a target nucleic acid of a cell, comprising contacting said cell with the nucleic acid described herein. For example, the nucleic acid, e.g., one or more vectors, e.g., one or more viral vectors, e.g., one or more AAV vectors, can comprise:


a) a first nucleic acid sequence that encodes a governing gRNA molecule, e.g., a Cas9-targeting gRNA molecule or a gRNA-targeting gRNA molecule; and


b) a second nucleic acid sequence that encodes a Cas9 molecule, e.g., an eaCas9 or an eiCas9 molecule.


In an embodiment, the cell is a mammalian, primate, or human cell. In an embodiment, the cell is a human cell, e.g., a cell described herein, e.g., in Section VIIA. In an embodiment, the cell is: a somatic cell, germ cell, prenatal cell, e.g., zygotic, blastocyst or embryonic cell, a stem cell, a mitotically competent cell, or a meiotically competent cell. In an embodiment, the target nucleic acid is a chromosomal nucleic acid.


In another aspect, the disclosure features a method of altering a cell, e.g., altering the structure, e.g., sequence, of a target nucleic acid of a cell, comprising contacting the cell with an effective amount of:


a gRNA molecule described herein;


a nucleic acid described herein;


a vector described herein; or


a composition described herein.


In an embodiment, the cell is a vertebrate, mammalian, rodent, goat, pig, bird, chicken, turkey, cow, horse, sheep, fish, primate, or human cell.


In another embodiment, the cell is a plant cell. In an embodiment, the plant cell is a monocot or a dicot.


In an embodiment, the cell is a human cell. In an embodiment, the cell is a somatic cell, germ cell, or prenatal cell. In an embodiment, the cell is a zygotic, blastocyst or embryonic cell, a stem cell, a mitotically competent cell, a meiotically competent cell.


In an embodiment, the subject is a mammal, primate, or human.


In an embodiment, the target nucleic acid is a chromosomal nucleic acid.


In another aspect, the disclosure features a method of treating a subject, e.g., by altering the structure, e.g., altering the sequence, of a target nucleic acid, comprising administering to the subject, an effective amount of the nucleic acid described herein. For example, the nucleic acid, e.g., one or more vectors, e.g., one or more viral vectors, e.g., one or more AAV vectors, can comprise:


a) a first nucleic acid sequence that encodes a governing gRNA molecule, e.g., a Cas9-targeting gRNA molecule or a gRNA-targeting gRNA molecule; and


b) a second nucleic acid sequence that encodes a Cas9 molecule, e.g., an eaCas9 or an eiCas9 molecule.


In an embodiment, the subject is a mammalian, primate, or human. In an embodiment, the target nucleic acid is the nucleic acid of a human cell, e.g., a cell described herein, e.g., in Section VIIA. In an embodiment, the target nucleic acid is the nucleic acid of: a somatic cell, germ cell, prenatal cell, e.g., zygotic, blastocyst or embryonic cell, a stem cell, a mitotically competent cell, or a meiotically competent cell. In an embodiment, the target nucleic acid is a chromosomal nucleic acid.


In another aspect, the disclosure features a method of treating a subject, e.g., by altering the structure, e.g., altering the sequence, of a target nucleic acid, in a cell of the subject, comprising contacting the cell or the subject, with an effective amount of the nucleic acid of:


a gRNA molecule described herein:


a nucleic acid described herein:


a vector described herein; or


a composition described herein.


In an embodiment, the cell is a vertebrate, mammalian, rodent, goat, pig, bird, chicken, turkey, cow, horse, sheep, fish, primate, or human cell.


In another embodiment, the cell is a plant cell. In an embodiment, the plant cell is a monocot or a dicot.


In an embodiment, the cell is a human cell. In an embodiment, the cell is a somatic cell, germ cell, or prenatal cell. In an embodiment, the cell is a zygotic, blastocyst or embryonic cell, a stem cell, a mitotically competent cell, a meiotically competent cell.


In an embodiment, the subject is a mammal, primate, or human.


In an embodiment, the target nucleic acid is a chromosomal nucleic acid.


In an aspect, the disclosure features a reaction mixture comprising a cell and:


a gRNA molecule described herein;


a nucleic acid described herein;


a vector described herein; or


a composition described herein.


In another aspect, the disclosure features a reaction mixture comprising a composition described herein and a cell, e.g., a cell described herein.


In an aspect, the disclosure features a kit comprising:


a gRNA molecule described herein:


a nucleic acid described herein;


a vector described herein; or


a composition described herein.


In an embodiment, the kit comprises an instruction for using the gRNA molecule, the nucleic acid, the vector, or the composition, in a method described herein.


In another aspect, the disclosure features a composition, e.g., pharmaceutical composition, comprising a governing gRNA molecule described herein.


In an embodiment, the composition further comprises a Cas9 molecule, e.g., an eaCas9 or an eiCas9 molecule. In an embodiment, said Cas9 molecule is an eaCas9 molecule. In an embodiment, said Cas9 molecule is an eiCas9 molecule.


In an embodiment, the composition further comprises a gRNA molecule comprising a targeting domain which is complementary with a target sequence from a target nucleic acid disclosed herein, e.g., a sequence from: a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In another aspect, the disclosure features a composition, e.g., pharmaceutical composition, comprising a gRNA molecule described herein.


In an embodiment, the composition further comprises a Cas9 molecule, e.g., an eaCas9 or an eiCas9 molecule. In an embodiment, said Cas9 molecule is an eaCas9 molecule. In another embodiment, said Cas9 molecule is an eiCas9 molecule.


In an embodiment, said composition comprises a payload, e.g., a payload described herein, e.g., in Section VI, e.g., in Table VI-1, VI-2, VI-3, VI-4, VI-5, or VI-6.


In an embodiment, the payload comprises: an epigenetic modifier, e.g., a molecule that modifies DNA or chromatin; component, e.g., a molecule that modifies a histone, e.g., an epigenetic modifier described herein, e.g., in Section VI; a transcription factor, e.g., a transcription factor described herein, e.g., in Section VI; a transcriptional activator domain; an inhibitor of a transcription factor, e.g., an anti-transcription factor antibody, or other inhibitors; a small molecule; an antibody; an enzyme; an enzyme that interacts with DNA, e.g., a helicase, restriction enzyme, ligase, or polymerase; and/or a nucleic acid, e.g., an enzymatically active nucleic acid, e.g., a ribozyme, or an mRNA, siRNA, of antisense oligonucleotide. In an embodiment, the composition further comprises a Cas9 molecule. e.g., an eiCas9, molecule.


In an embodiment, said payload is coupled, e.g., covalently or noncovalently, to a Cas9 molecule, e.g., an eiCas9 molecule. In an embodiment, said payload is coupled to said Cas9 molecule by a linker. In an embodiment, said linker is or comprises a bond that is cleavable under physiological, e.g., nuclear, conditions. In an embodiment, said linker is, or comprises, a bond described herein. e.g., in Section XI. In an embodiment, said linker is, or comprises, an ester bond. In an embodiment, said payload comprises a fusion partner fused to a Cas9 molecule, e.g., an eaCas9 molecule or an eiCas9 molecule.


In an embodiment, said payload is coupled, e.g., covalently or noncovalently, to the gRNA molecule. In an embodiment, said payload is coupled to said gRNA molecule by a linker. In an embodiment, said linker is or comprises a bond that is cleavable under physiological, e.g., nuclear, conditions. In an embodiment, said linker is, or comprises, a bond described herein, e.g., in Section XI. In an embodiment, said linker is, or comprises, an ester bond.


In an embodiment, the composition comprises an eaCas9 molecule. In an embodiment, the composition comprises an eaCas9 molecule which forms a double stranded break in the target nucleic acid.


In an embodiment, the composition comprises an eaCas9 molecule which forms a single stranded break in the target nucleic acid. In an embodiment, said single stranded break is formed in the complementary strand of the target nucleic acid. In an embodiment, said single stranded break is formed in the strand which is not the complementary strand of the target nucleic acid.


In an embodiment, the composition comprises HNH-like domain cleavage activity but having no, or no significant, N-terminal RuvC-like domain cleavage activity. In an embodiment, the composition comprises N-terminal RuvC-like domain cleavage activity but having no, or no significant, HNH-like domain cleavage activity.


In an embodiment, said double stranded break is within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position. In an embodiment, said single stranded break is within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.


In an embodiment, the composition further comprises a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV. In an embodiment, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.


In an embodiment, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VI-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment, the composition further comprises a second gRNA molecule, e.g., a second gRNA molecule described herein.


In an embodiment, said gRNA molecule and said second gRNA molecule mediate breaks at different sites in the target nucleic acid, e.g., flanking a target position. In an embodiment, said gRNA molecule and said second gRNA molecule are complementary to the same strand of the target. In an embodiment, said gRNA molecule and said second gRNA molecule are complementary to the different strands of the target.


In an embodiment, said Cas9 molecule mediates a double stranded break.


In an embodiment, said gRNA molecule and said second gRNA molecule are configured such that first and second break made by the Cas9 molecule flank a target position. In an embodiment, said double stranded break is within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.


In an embodiment, the composition further comprises a template nucleic acid. In an embodiment, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.


In an embodiment, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of a target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VI-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment, the template nucleic acid is a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein. e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment, the template nucleic acid is a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment, said Cas9 molecule mediates a single stranded break.


In an embodiment, said gRNA molecule and said second gRNA molecule are configured such that a first and second break are formed in the same strand of the nucleic acid target, e.g., in the case of transcribed sequence, the template strand or the non-template strand.


In an embodiment, said first and second break flank a target position.


In an embodiment, one of said first and second single stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.


In an embodiment, the composition further comprises a template nucleic acid. In an embodiment, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position. In an embodiment, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment, said gRNA molecule and said second gRNA molecule are configured such that a first and a second breaks are formed in different strands of the target. In an embodiment, said first and second break flank a target position. In an embodiment, one of said first and second single stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.


In an embodiment, the composition further comprises a template nucleic acid. In an embodiment, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.


In an embodiment, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VI-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment, the composition comprises a second Cas9 molecule.


In an embodiment, one or both of said Cas9 molecule and said second Cas9 molecule are eiCas9 molecules. In an embodiment, said eiCas9 molecule is coupled to a payload by a linker and said second eiCas9 molecules is coupled to a second payload by a second linker.


In an embodiment, said payload and said second payload are the same. In an embodiment, said payload and said second payload are different. In an embodiment, said linker and said second linker are the same. In an embodiment, said linker and said second linker are different, e.g., have different release properties, e.g., different release rates.


In an embodiment, said payload and said second payload are each described herein, e.g., in Section VI, e.g., in Table VI-1, VI-2, VI-3, VI-4, VI-5, or VI-6. In an embodiment, said payload and said second payload can interact, e.g., they are subunits of a protein.


In an embodiment, one of both of said Cas9 molecule and said second Cas9 molecule are eaCas9 molecules.


In an embodiment, said eaCas9 molecule comprises a first cleavage activity and said second eaCas9 molecule comprises a second cleavage activity. In an embodiment, said cleavage activity and said second cleavage activity are the same, e.g., both are N-terminal RuvC-like domain activity or are both HNH-like domain activity. In an embodiment, said cleavage activity and said second cleavage activity are different, e.g., one is N-terminal RuvC-like domain activity and one is HNH-like domain activity.


In an embodiment, said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for, e.g., NGGNG, NNAGAAW (W=A or T), or NAAR (R=A or G). In an embodiment, said Cas9 molecule of S. aureus recognizes the sequence motif NNGRR (R=A or G) and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. In an embodiment, one of said Cas 9 molecule and said second Cas 9 molecule recognizes an S. aureus PAM. In an embodiment, said Cas9 molecule of N. meningitidis recognizes the sequence motif NNNNGATT and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. In an embodiment, one of said Cas 9 molecule and said second Cas 9 molecule recognizes an N. meningitidis PAM.


In an embodiment, said Cas9 molecule and said second Cas9 molecule both mediate double stranded breaks.


In an embodiment, said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for another PAM, e.g., another PAM described herein. In an embodiment, said gRNA molecule and said second gRNA molecule are configured such that first and second break flank a target position. In an embodiment, one of said first and second double stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.


In an embodiment, the composition further comprises a template nucleic acid. In an embodiment, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.


In an embodiment, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VI-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, XII-1, or Section VIII.


In an embodiment, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment, one of said Cas9 molecule and said second Cas9 molecule mediates a double stranded break and the other mediates a single stranded break.


In an embodiment, said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for another PAM, e.g., another PAM described herein. In an embodiment, said gRNA molecule and said second gRNA molecule are configured such that a first and second break flank a target position. In an embodiment, said first and second break flank a target position. In an embodiment, one of said first and second breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.


In an embodiment, the composition further comprises a template nucleic acid. In an embodiment, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.


In an embodiment, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VI-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment, said Cas9 molecule and said second Cas9 molecule both mediate single stranded breaks.


In an embodiment, said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for another PAM, e.g., another PAM described herein. In an embodiment, said first and second break flank a target position.


In an embodiment, one of said first and second single stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.


In an embodiment, the composition further comprises a template nucleic acid. In an embodiment, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.


In an embodiment, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VI-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment, said gRNA molecule, said second gRNA molecule are configured such that a first and second break are in the same strand.


In an embodiment, said Cas9 molecule and said second Cas9 molecule are specific for different PAMs. e.g., one is specific for NGG and the other is specific for another PAM, e.g., another PAM described herein. In an embodiment, said gRNA molecule, said second gRNA molecule are configured such that a first and second break flank a target position. In an embodiment, one of said first and second single stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.


In an embodiment, the composition further comprises a template nucleic acid. In an embodiment, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.


In an embodiment, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment, said first and second break are on the different strands.


In an embodiment, said Cas9 molecule and said second Cas9 molecule are specific for different PAMs. e.g., one is specific for NGG and the other is specific for another PAM, e.g., another PAM described herein. In an embodiment, said gRNA molecule, said second gRNA molecule are configured such that a first and second break are on different strands.


In an embodiment, said gRNA molecule, said second gRNA molecule are configured such that a first and second break flank a target position. In an embodiment, said first and second break flank a target position.


In an embodiment, one of said first and second single stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.


In an embodiment, the composition further comprises a template nucleic acid. In an embodiment, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.


In an embodiment, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or Section VIII.


In yet another aspect, the disclosure features a composition, e.g., a pharmaceutical composition, comprising governing gRNA molecule, e.g., a Cas9-targeting gRNA molecule or a gRNA-targeting gRNA molecule, a gRNA molecule and a second gRNA molecule described herein.


In an embodiment, the composition further comprises a nucleic acid, e.g., a DNA or mRNA, that encodes a Cas9 molecule described herein. In an embodiment, the composition further comprises a nucleic acid, e.g., a DNA or RNA, that encodes a second Cas9 molecule described herein. In an embodiment, the composition further comprises a template nucleic acid described herein.


In one aspect, the disclosure features a composition, e.g., a pharmaceutical composition, comprising, nucleic acid sequence, e.g., a DNA, that encodes a governing gRNA molecule, e.g., a Cas9-targeting gRNA molecule or a gRNA-targeting gRNA molecule, and one or more gRNA molecules described herein.


In an embodiment, said nucleic acid comprises a promoter operably linked to the sequence that encodes a gRNA molecule, e.g., a promoter described herein.


In an embodiment, said nucleic acid comprises a second promoter operably linked to the sequence that encodes a second gRNA molecule, e.g., a promoter described herein. In an embodiment, the promoter and second promoter are different promoters. In an embodiment, the promoter and second promoter are the same.


In an embodiment, the nucleic acid further encodes a Cas9 molecule described herein. In an embodiment, the nucleic acid further encodes a second Cas9 molecule described herein.


In an embodiment, said nucleic acid comprises a promoter operably linked to the sequence that encodes a Cas9 molecule, e.g., a promoter described herein.


In an embodiment, said nucleic acid comprises a second promoter operably linked to the sequence that encodes a second Cas9 molecule, e.g., a promoter described herein. In an embodiment, the promoter and second promoter are different promoters. In an embodiment, the promoter and second promoter are the same.


In an embodiment, the composition further comprises a template nucleic acid e.g., a template nucleic acid described herein, e.g., in Section IV.


In another aspect, the disclosure features a composition, e.g., a pharmaceutical composition, comprising nucleic acid sequence that encodes one or more of: a) a Cas9 molecule, b) a second Cas9 molecule, c) a gRNA molecule, d) a second gRNA molecule, and e) a governing gRNA molecule, e.g., a Cas9-targeting gRNA molecule or a gRNA-targeting gRNA molecule.


In an embodiment, each of a), b), c) d) and e) present are encoded on the same nucleic acid molecule.


In an embodiment, a first sequence selected from of a), b), c), d) and e) is encoded on a first nucleic acid molecule and a second sequence selected from a), b), c), d) and e) is encoded on a second nucleic acid molecule.


In an embodiment, said nucleic acid encodes: a), c) and e); a), c), d) and e); or a), b), c), d) and e).


In an embodiment, the composition further comprises a Cas9 molecule, e.g., comprising one or more of the Cas9 molecules wherein said nucleic acid does not encode a Cas9 molecule.


In an embodiment, the composition further comprises an mRNA encoding Cas9 molecule, e.g., comprising one or more mRNAs encoding one or more of the Cas9 molecules wherein said nucleic acid does not encode a Cas9 molecule.


In an embodiment, the composition further comprises a template nucleic acid e.g., a template nucleic acid described herein, e.g., in Section IV.


In yet another aspect, the disclosure features a nucleic acid described herein.


In one aspect, the disclosure features a composition comprising: a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule; and a second eaCas9 molecule); and c) optionally, a template nucleic acid e.g., a template nucleic acid described herein, e.g., in Section IV.


In another aspect, the disclosure features a composition comprising: a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a nucleic acid, e.g. a DNA or mRNA encoding an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV; and d) a governing gRNA molecule, e.g., a Cas9-targeting gRNA molecule.


In yet another aspect, the disclosure features a composition comprising: a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV; and d) a governing gRNA molecule, e.g., a gRNA-targeting gRNA molecule.


In still another aspect, the disclosure features a composition comprising: a) nucleic acid, e.g., a DNA, which encodes a gRNA molecule or (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) nucleic acid, e.g. a DNA or mRNA encoding eaCas9 molecule or (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule) (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); c) optionally, a template nucleic acid. e.g., a template nucleic acid described herein, e.g., in Section IV; and d) a governing gRNA molecule, e.g., a Cas9-targeting gRNA molecule or a gRNA-targeting gRNA molecule.


In one aspect, the disclosure features a method of altering a cell, e.g., altering the structure, e.g., sequence, of a target nucleic acid of a cell, comprising contacting said cell with:


1) a composition comprising:

    • a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
    • b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule; and a second eaCas9 molecule); and
    • c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV;


2) a composition comprising:

    • a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
    • b) a nucleic acid, e.g. a DNA or mRNA encoding an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); and
    • c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV; and
    • d) a governing gRNA molecule, e.g., a Cas9-targeting gRNA molecule; 3) a composition comprising:
    • a) a nucleic acid. e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); and
    • c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV; and
    • d) a governing gRNA molecule, e.g., a Cas9-targeting gRNA molecule or a gRNA-targeting gRNA molecule; and/or


4) a composition comprising:

    • a) nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) nucleic acid, e.g. a DNA or mRNA encoding eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule), (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and
    • c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV; and
    • d) a governing gRNA molecule, e.g., a Cas9-targeting gRNA molecule or a gRNA-targeting gRNA molecule.


In an embodiment, a gRNA molecule or nucleic acid encoding a gRNA molecule, and an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, are delivered in or by, one dosage form, mode of delivery, or formulation.


In an embodiment, a) a gRNA molecule or nucleic acid encoding a gRNA molecule is delivered in or by, a first dosage form, a first mode of delivery, or a first formulation; and b) an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, is delivered in or by a second dosage form, second mode of delivery, or second formulation. In an embodiment, a governing gRNA molecule (or a nucleic acid that encodes it), e.g., a Cas9-targeting gRNA molecule or a gRNA-targeting gRNA molecule, is provided in the dosage form that contains the component it inactivates, or in another dosage form, mode of delivery, or formulation.


In an embodiment, the cell is an animal or plant cell. In an embodiment, the cell is a mammalian, primate, or human cell. In an embodiment, the cell is a human cell, e.g., a cell from described herein, e.g., in Section VIIA. In an embodiment, the cell is: a somatic cell, germ cell, prenatal cell, e.g., zygotic, blastocyst or embryonic, blastocyst cell, a stem cell, a mitotically competent cell, a meiotically competent cell. In an embodiment, the cell is a human cell, e.g., a cancer cell or other cell characterized by a disease or disorder.


In an embodiment, the target nucleic acid is a chromosomal nucleic acid. In an embodiment, the target nucleic acid is an organellar nucleic acid. In an embodiment, the target nucleic acid is a mitochondrial nucleic acid. In an embodiment, the target nucleic acid is a chloroplast nucleic acid.


In an embodiment, the cell is a cell of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite.


In an embodiment, the target nucleic acid is the nucleic acid of a disease causing organism, e.g., of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite.


In an embodiment, said method comprises: modulating the expression of a gene or inactivating a disease organism.


In an embodiment, said cell is a cell characterized by unwanted proliferation, e.g., a cancer cell. In an embodiment, said cell is a cell characterized by an unwanted genomic component, e.g., a viral genomic component. In an embodiment, the cell is a cell described herein, e.g., in Section IIA. In an embodiment, a control or structural sequence of at least, 2 3, 4, 5 or 6 or more genes is altered.


In an embodiment, the target nucleic acid is a rearrangement, a rearrangement that comprises a kinase gene, or a rearrangement that comprises a tumor suppressor gene. In an embodiment, the targent nucleic acid comprises a kinase gene or a tumor suppressor gene.


In an embodiment, the method comprises cleaving a target nucleic acid within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position. In an embodiment, said composition comprises a template nucleic acid.


In an embodiment, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.


In an embodiment, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VI-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment,


a) a control region, e.g., a cis-acting or tans-acting control region, of a gene is cleaved;


b) the sequence of a control region, e.g., a cis-acting or trans-acting control region, of a gene is altered, e.g., by an alteration that modulates, e.g., increases or decreases, expression a gene under control of the control region, e.g., a control sequence is disrupted or a new control sequence is inserted:


c) the coding sequence of a gene is cleaved;


d) the sequence of a transcribed region, e.g., a coding sequence of a gene is altered, e.g., a mutation is corrected or introduced, an alteration that increases expression of or activity of the gene product is effected, e.g., a mutation is corrected; and/or


e) the sequence of a transcribed region, e.g., the coding sequence of a gene is altered. e.g., a mutation is corrected or introduced, an alteration that decreases expression of or activity of the gene product is effected, e.g., a mutation is inserted, e.g., the sequence of one or more nucleotides is altered so as to insert a stop codon.


In an embodiment, a control region or transcribed region, e.g., a coding sequence, of at least 2, 3, 4, 5, or 6 or more genes are altered.


In another aspect, the disclosure features a method of treating a subject, e.g., by altering the structure, e.g., altering the sequence, of a target nucleic acid, comprising administering to the subject, an effective amount of:


1) a composition comprising:

    • a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
    • b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule; and a second eaCas9 molecule); and
    • c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV;


2) a composition comprising:

    • a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
    • b) a nucleic acid, e.g. a DNA or mRNA encoding an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); and
    • c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV; and
    • d) a governing gRNA molecule, e.g., a Cas9-targeting gRNA molecule;


3) a composition comprising:

    • a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
    • b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); and
    • c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV; and
    • d) a governing gRNA molecule, e.g., a gRNA-targeting gRNA molecule;
    • and/or


4) a composition comprising:

    • a) nucleic acid, e.g., a DNA, which encodes a gRNA molecule or (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
    • b) nucleic acid, e.g. a DNA or mRNA encoding eaCas9 molecule or (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule), (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and
    • c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV; and
    • d) a governing gRNA molecule, e.g., a Cas9-targeting gRNA molecule or a gRNA-targeting gRNA molecule.


In an embodiment, a gRNA molecule or nucleic acid encoding a gRNA molecule, and an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, are delivered in or by one dosage form, mode of delivery, or formulation. In an embodiment, a governing gRNA molecule (or a nucleic acide that encodes it), e.g., a Cas9-targeting gRNA molecule or a gRNA-targeting gRNA molecule, is provided in the dosage form that contains the component it inactivates, or in another dosage form, mode of delivery, or formulation.


In an embodiment, a gRNA molecule or nucleic acid encoding a gRNA molecule is delivered in or by a first dosage form, in a first mode of delivery, or first formulation; and an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, is delivered in or by a second dosage form, second mode of delivery, or second formulation. In an embodiment a governing gRNA molecule (or a nucleic acide that encodes it), e.g., a Cas9-targeting gRNA molecule or a gRNA-targeting gRNA molecule, can provided in the dosage form that contains the component it inactivates, or in another dosage form, mode of delivery, or formulation.


In an embodiment, the subject is an animal or plant. In an embodiment, the subject is a mammalian, primate, or human.


In an embodiment, the target nucleic acid is the nucleic acid of a human cell, e.g., a cell described herein, e.g., in Section VIIA. In an embodiment, the target nucleic acid is the nucleic acid of: a somatic cell, germ cell, prenatal cell, e.g., zygotic, blastocyst or embryonic, blastocyst cell, a stem cell, a mitotically competent cell, a meiotically competent cell.


In an embodiment, the target nucleic acid is a chromosomal nucleic acid. In an embodiment, the target nucleic acid is an organellar nucleic acid. In an embodiment, the nucleic acid is a mitochondrial nucleic acid. In an embodiment, the nucleic acid is a chloroplast nucleic acid.


In an embodiment, the target nucleic acid is the nucleic acid of a disease causing organism, e.g., of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite. In an embodiment, said method comprises modulating expression of a gene or inactivating a disease organism.


In an embodiment, the target nucleic acid is the nucleic acid of a cell characterized by unwanted proliferation, e.g., a cancer cell. In an embodiment, said target nucleic acid comprises an unwanted genomic component, e.g., a viral genomic component. In an embodiment, a control or structural sequence of at least, 2 3, 4, 5 or 6 or more genes is altered. In an embodiment, the target nucleic acid is a rearrangement, a rearrangement that comprises a kinase gene, or a rearrangement that comprises a tumor suppressor gene. In an embodiment, the targent nucleic acid comprises a kinase gene or a tumor suppressor gene.


In an embodiment, the method comprises cleaving a target nucleic acid within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.


In an embodiment, said composition comprises a template nucleic acid. In an embodiment, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.


In an embodiment, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


In an embodiment, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in:


In an embodiment,


a) a control region, e.g., a cis-acting or trans-acting control region, of a gene is cleaved;


b) the sequence of a control region, e.g., a cis-acting or trans-acting control region, of a gene is altered, e.g., by an alteration that modulates, e.g., increases or decreases, expression a gene under control of the control region, e.g., a control sequence is disrupted or a new control sequence is inserted;


c) the coding sequence of a gene is cleaved;


d) the sequence of a transcribed region, e.g., a coding sequence of a gene is altered, e.g., a mutation is corrected or introduced, an alteration that increases expression of or activity of the gene product is effected, e.g., a mutation is corrected:


e) the non-coding sequence of a gene or an intergenic region between genes is cleaved; and/or


f) the sequence of a transcribed region, e.g., the coding sequence of a gene is altered, e.g., a mutation is corrected or introduced, an alteration that decreases expression of or activity of the gene product is effected, e.g., a mutation is inserted, e.g., the sequence of one or more nucleotides is altered so as to insert a stop codon.


In an embodiment, a control region or transcribed region, e.g., a coding sequence, of at least 2, 3, 4, 5, or 6 or more genes are altered.


In one aspect, the disclosure features a composition comprising: a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and c) a payload coupled, covalently or non-covalently, to a complex of the gRNA molecule and the Cas9 molecule, e.g., coupled to the Cas9 molecule or the gRNA molecule.


In another aspect, the disclosure features a composition comprising: a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a nucleic acid, e.g. a DNA or mRNA encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and c) a payload which is: coupled, covalently or non-covalently, the gRNA molecule; or a fusion partner with the Cas9 molecule.


In yet another aspect, the disclosure features a composition comprising: a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); c) a payload which is coupled, covalently or non-covalently, to the Cas9 molecule; and d) a governing gRNA molecule, e.g., a gRNA-targeting gRNA molecule.


In still another aspect, the disclosure features a composition comprising: a) nucleic acid, e.g., a DNA, which encodes a gRNA molecule or (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) nucleic acid, e.g. a DNA or mRNA, encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule), wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules; c) a payload which is a fusion partner with the Cas9 molecule; and d) a governing gRNA molecule, e.g., a Cas9-targeting gRNA molecule or a gRNA-targeting gRNA molecule.


In one aspect, the disclosure features a method of delivering a payload to a cell, e.g., by targeting a payload to target nucleic acid, comprising contacting said cell with:


1) a composition comprising:

    • a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
    • b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and
    • c) a payload coupled, covalently or non-covalently, to a complex of the gRNA molecule and the Cas9 molecule, e.g., coupled to the Cas9 molecule or the gRNA molecule;


2) a composition comprising:

    • a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
    • b) a nucleic acid, e.g. a DNA or mRNA encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule);
    • c) a payload which is: coupled, covalently or non-covalently, the gRNA molecule; or a fusion partner with the Cas9 molecule;


3) a composition comprising:

    • a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
    • b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule);
    • c) a payload which is coupled, covalently or non-covalently, to the Cas9 molecule; and
    • d) a governing gRNA molecule, e.g., a gRNA-targeting gRNA molecule; and/or


4) a composition comprising:

    • a) nucleic acid, e.g., a DNA, which encodes a gRNA molecule or (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
    • b) nucleic acid, e.g. a DNA or mRNA, encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule), wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules;
    • c) a payload which is a fusion partner with the Cas9 molecule; and
    • d) a governing gRNA molecule, e.g., a Cas9-targeting gRNA molecule or a gRNA-targeting gRNA molecule.


In an embodiment, a gRNA molecule or nucleic acid encoding a gRNA molecule, and an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, are delivered in or by one dosage form, mode of delivery, or formulation. In an embodiment, a governing gRNA molecule (or a nucleic acide that encodes it). e.g., a Cas9-targeting gRNA molecule or a gRNA-targeting gRNA molecule, is provided in the dosage form that contains the component it inactivates, or in another dosage form, mode of delivery, or formulation.


In an embodiment, a gRNA molecule or nucleic acid encoding a gRNA molecule is delivered in or by a first dosage form, first mode of delivery, or first formulation; and a Cas9 molecule, or nucleic acid encoding a Cas9 molecule, is delivered in or by a second dosage form, second mode of delivery, or second formulation. In an embodiment, a governing gRNA molecule (or a nucleic acide that encodes it), e.g., a Cas9-targeting gRNA molecule or a gRNA-targeting gRNA molecule, is provided in the dosage form that contains the component it inactivates, or in another dosage form, mode of delivery, or formulation.


In an embodiment, the cell is an animal or plant cell. In an embodiment, the cell is a mammalian, primate, or human cell. In an embodiment, the cell is a human cell, e.g., a human cell described herein, e.g., in Section VIIA. In an embodiment, the cell is: a somatic cell, germ cell, prenatal cell, e.g., zygotic, blastocyst or embryonic, blastocyst cell, a stem cell, a mitotically competent cell, a meiotically competent cell. In an embodiment, the cell is a human cell, e.g., a cancer cell, a cell comprising an unwanted genetic element, e.g., all or part of a viral genome.


In an embodiment, the gRNA mediates targeting of a chromosomal nucleic acid. In an embodiment, the gRNA mediates targeting of a selected genomic signature. In an embodiment, the gRNA mediates targeting of an organellar nucleic acid. In an embodiment, the gRNA mediates targeting of a mitochondrial nucleic acid. In an embodiment, the gRNA mediates targeting of a chloroplast nucleic acid.


In an embodiment, the cell is a cell of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite.


In an embodiment, the gRNA mediates targeting of the nucleic acid of a disease causing organism, e.g., of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite.


In an embodiment, the payload comprises a payload described herein, e.g., in Section VI.


In an embodiment, said cell is a cell characterized by unwanted proliferation, e.g., a cancer cell. In an embodiment, said cell is characterized by an unwanted genomic component, e.g., a viral genomic component.


In an embodiment, a control or structural sequence of at least 2 3, 4, 5, or 6 or more genes is altered.


In an embodiment, the gRNA targets a selected genomic signature, e.g., a mutation, e.g., a germline or acquired somatic mutation. In an embodiment, the target nucleic acid is a rearrangement, a rearrangement that comprises a kinase gene, or a rearrangement that comprises a tumor suppressor gene. In an embodiment, the targent nucleic acid comprises a kinase gene or a tumor suppressor gene. In an embodiment, the gRNA targets a cancer cell, e.g., a cancer cell disclosed herein, e.g., in Section VIIA. In an embodiment, the gRNA targets a cell which has been infected with a virus.


In another aspect, the disclosure features a method of treating a subject, e.g., by targeting a payload to target nucleic acid, comprising administering to the subject, an effective amount of:


1) a composition comprising:

    • a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
    • b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and
    • c) a payload coupled, covalently or non-covalently, to a complex of the gRNA molecule and the Cas9 molecule, e.g., coupled to the Cas9 molecule;


2) a composition comprising:

    • a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
    • b) a nucleic acid, e.g. a DNA or mRNA encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule);
    • c) a payload which is:
      • coupled, covalently or non-covalently, the gRNA molecule; or is a fusion partner with the Cas9 molecule; and
    • d) a governing gRNA molecule, e.g., a Cas9-targeting gRNA molecule;


3) a composition comprising:

    • a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
    • b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and
    • c) a payload which is coupled, covalently or non-covalently, to the Cas9 molecule; and
    • d) a governing gRNA molecule, e.g., a gRNA-targeting gRNA molecule;


and/or


4) a composition comprising:

    • a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule or (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
    • b) a nucleic acid, e.g. a DNA or mRNA, encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule), (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules);
    • c) a payload which is a fusion partner with the Cas9 molecule; and
    • d) a governing gRNA molecule, e.g., a Cas9-targeting gRNA molecule or a gRNA-targeting gRNA molecule.


In an embodiment, a gRNA molecule or nucleic acid encoding a gRNA molecule, and an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, are delivered in or by one dosage form, mode of delivery, or formulation. In an embodiment a governing gRNA molecule (or a nucleic acide that encodes it), e.g., a Cas9-targeting gRNA molecule or a gRNA-targeting gRNA molecule, can provided in the dosage form that contains the component it inactivates, or in another dosage form, mode of delivery, or formulation.


In an embodiment, a gRNA molecule or nucleic acid encoding a gRNA molecule is delivered in or by a first dosage, mode of delivery form or formulation; and a Cas9 molecule, or nucleic acid encoding a Cas9 molecule, is delivered in or by a second dosage form, mode of delivery, or formulation. In an embodiment a governing gRNA molecule (or a nucleic acide that encodes it), e.g., a Cas9-targeting gRNA molecule or a gRNA-targeting gRNA molecule, can provided in the dosage form that contains the component it inactivates, or in another dosage form, mode of delivery, or formulation.


In an embodiment, the subject is an animal or plant cell. In an embodiment, the subject is a mammalian, primate, or human cell.


In an embodiment, the gRNA mediates targeting of a human cell, e.g., a human cell described herein, e.g., in Section VIIA. In an embodiment, the gRNA mediates targeting of: a somatic cell, germ cell, prenatal cell, e.g., zygotic, blastocyst or embryonic, blastocyst cell, a stem cell, a mitotically competent cell, a meiotically competent cell. In an embodiment, the gRNA mediates targeting of a cancer cell or a cell comprising an unwanted genomic element, e.g., all or part of a viral genome. In an embodiment, the gRNA mediates targeting of a chromosomal nucleic acid. In an embodiment, the gRNA mediates targeting of a selected genomic signature. In an embodiment, the gRNA mediates targeting of an organellar nucleic acid. In an embodiment, the gRNA mediates targeting of a mitochondrial nucleic acid. In an embodiment, the gRNA mediates targeting of a chloroplast nucleic acid. In an embodiment, the gRNA mediates targeting of the nucleic acid of a disease causing organism, e.g., of a disease causing organism. e.g., a virus, bacterium, fungus, protozoan, or parasite. In an embodiment, the gRNA targets a cell characterized by unwanted proliferation, e.g., a cancer cell, e.g., a cancer cell from Section VIIA, e.g., from Table VII-11. In an embodiment, the gRNA targets a cell characterized by an unwanted genomic component, e.g., a viral genomic component.


In an embodiment, a control element, e.g., a promoter or enhancer, is targeted. In an embodiment, the target nucleic acid is a rearrangement, a rearrangement that comprises a kinase gene, or a rearrangement that comprises a tumor suppressor gene. In an embodiment, the targent nucleic acid comprises a kinase gene or a tumor suppressor gene. In an embodiment, the gRNA targets a selected genomic signature, e.g., a mutation, e.g., a germline or acquired somatic mutation.


In an embodiment, the gRNA targets a cancer cell. In an embodiment, the gRNA targets a cell which has been infected with a virus.


In an embodiment, at least one eaCas9 molecule and a payload are administered. In an embodiment, the payload comprises a payload described herein, e.g., in Section VI.


Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.


Headings, including numeric and alphabetical headings and subheadings, are for organization and presentation and are not intended to be limiting.


Other features and advantages of the invention will be apparent from the detailed description, drawings, and from the claims.





BRIEF DESCRIPTION OF THE DRAWING

The Figures described below, that together make up the Drawing, are for illustration purposes only, not for limitation.



FIG. 1A-G are representations of several exemplary gRNAs.



FIG. 1A depicts a modular gRNA molecule derived in part (or modeled on a sequence in part) from Streptococcus pyogenes (S. pyogenes) as a duplexed structure (SEQ ID NOS 42 and 43, respectively, in order of appearance);



FIG. 1B depicts a unimolecular (or chimeric) gRNA molecule derived in part from S. pyogenes as a duplexed structure (SEQ ID NO: 44):



FIG. 1C depicts a unimolecular gRNA molecule derived in part from S. pyogenes as a duplexed structure (SEQ ID NO: 45);



FIG. 1D depicts a unimolecular gRNA molecule derived in part from S. pyogenes as a duplexed structure (SEQ ID NO: 46);



FIG. 1E depicts a unimolecular gRNA molecule derived in part from S. pyogenes as a duplexed structure (SEQ ID NO: 47);



FIG. 1F depicts a modular gRNA molecule derived in part from Streptococcus thermophilus (S. thermophilus) as a duplexed structure (SEQ ID NOS 48 and 49, respectively, in order of appearance);



FIG. 1G depicts an alignment of modular gRNA molecules of S. pyogenes and S. thermophilus (SEQ ID NOS 50-53, respectively, in order of appearance).



FIG. 2 depicts an alignment of Cas9 sequences from Chylinski et al., RNA BIOL. 2013; 10(5); 726-737. The N-terminal RuvC-like domain is boxed and indicated with a “Y”. The other two RuvC-like domains are boxed and indicated with a “B”. The HNH-like domain is boxed and indicated by a “G”. Sm; S. mutans (SEQ ID NO: 1); Sp: S. pyogenes (SEQ ID NO: 2); St: S. thermophilus (SEQ ID NO: 3); Li: L. innocua (SEQ ID NO: 4). Motif: this is a motif based on the four sequences: residues conserved in all four sequences are indicated by single letter amino acid abbreviation; “*” indicates any amino acid found in the corresponding position of any of the four sequences; and“−” indicates any amino acid, e.g., any of the 20 naturally occurring amino acids.



FIG. 3A shows an alignment of the N-terminal RuvC-like domain from the Cas9 molecules disclosed in Chylinski et al. (SEQ ID NOS 54-103, respectively, in order of appearance). The last line of FIG. 3A identifies 3 highly conserved residues.



FIG. 3B shows an alignment of the N-terminal RuvC-like domain from the Cas9 molecules disclosed in Chylinski et al. with sequence outliers removed (SEQ ID NOS 104-177, respectively, in order of appearance). The last line of FIG. 3B identifies 4 highly conserved residues.



FIG. 4A shows an alignment of the HNH-like domain from the Cas9 molecules disclosed in Chylinski et al. (SEQ ID NOS 178-252, respectively, in order of appearance). The last line of FIG. 4A identifies conserved residues.



FIG. 4B shows an alignment of the HNH-like domain from the Cas9 molecules disclosed in Chylinski et al. with sequence outliers removed (SEQ ID NOS 253-302, respectively, in order of appearance). The last line of FIG. 4B identifies 3 highly conserved residues.



FIG. 5 depicts an alignment of Cas9 sequences from S. pyogenes and Neisseria meningitidis (N. meningitidis). The N-terminal RuvC-like domain is boxed and indicated with a “Y”. The other two RuvC-like domains are boxed and indicated with a “B”. The HNH-like domain is boxed and indicated with a “G”. Sp: S. pyogenes; Nm: N. meningitidis. Motif: this is a motif based on the two sequences: residues conserved in both sequences are indicated by a single amino acid designation; “*” indicates any amino acid found in the corresponding position of any of the two sequences: “-” indicates any amino acid, e.g., any of the 20 naturally occurring amino acids, and“−” indicates any amino acid, e.g., any of the 20 naturally occurring amino acids, or absent.



FIG. 6 shows a nucleic acid sequence encoding Cas9 of N. meningitidis (SEQ ID NO: 303). Sequence indicated by an “R” is an SV40 NLS; sequence indicated as “G” is an HA tag; sequence indicated by an “O” is a synthetic NLS sequence. The remaining (unmarked) sequence is the open reading frame (ORF).



FIG. 7 depicts the levels of Cas9 protein expression in cells transfected with each of the Cas9-targeted governing gRNAs at 1, 2, 3, 6 and 9 days following transfection.





DEFINITIONS

“Governing gRNA molecule”, as used herein, refers to a gRNA molecule that can complex with a Cas9 molecule to inactivate or silence a component of the Cas9 system. In an embodiment, the governing gRNA molecule inactivates or silences a nucleic acid that comprises the sequence encoding the Cas9 molecule. In an embodiment, it inactivates or silences the nucleic acid that comprises the sequence encoding the gRNA molecule. In an embodiment, the governing gRNA, e.g., a Cas9-targeting gRNA molecule, or a gRNA targeting gRNA molecule, limits the effect of the Cas9 molecule/gRNA molecule complex-mediated gene targeting. In an embodiment, it places temporal, level of expression, or other limits, on activity of the Cas9 molecule/gRNA molecule complex. In an embodiment, it reduces off-target or other unwanted activity. Governing gRNA molecules can act as to inhibit, e.g., entirely or substantially inhibit, the production of a component of the Cas9 system, e.g., the Cas9 molecule, and thereby limit, or govern, its activity.


The governing gRNA molecule can target any region of the nucleic acid that comprises the sequence encoding the component to be negatively regulated, within or outside the transcribed or translated region of the component, as long as production of the component is reduced.


In an embodiment, a governing gRNA molecule comprises a targeting sequence that is complementary with a target sequence on the nucleic acid on which the sequence encoding the component to be negatively regulated resides.


In an embodiment, a governing gRNA molecule comprises a targeting sequence that is complementary with a sequence of the component to be negatively regulated.


In an embodiment, a Cas9-targeting gRNA molecule can include a targeting sequence that targets the nucleic acid on which the sequence that encodes the Cas9 molecule resides.


In an embodiment, a Cas9-targeting gRNA molecule can include a targeting sequence that targets the Cas9 molecule sequence.


In an embodiment, a gRNA-targeting gRNA molecule can include a targeting sequence that targets the nucleic acid on which the sequence that encodes the gRNA molecule resides.


In an embodiment, a gRNA-targeting gRNA molecule can include a targeting sequence that targets the gRNA molecule sequence.


“Domain”, as used herein, is used to describe segments of a protein or nucleic acid. Unless otherwise indicated, a domain is not required to have any specific functional property.


Calculations of “homology” or “sequence identity” between two sequences (the terms are used interchangeably herein) are performed as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The optimal alignment is determined as the best score using the GAP program in the GCG software package with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frame shift gap penalty of 5. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein, In an embodiment, amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences.


“Modulator”, as used herein, refers to an entity, e.g., a drug, that can alter the activity (e.g., enzymatic activity, transcriptional activity, or translational activity), amount, distribution, or structure of a subject molecule or genetic sequence. In an embodiment, modulation comprises cleavage, e.g., breaking of a covalent or non-covalent bond, or the forming of a covalent or non-covalent bond, e.g., the attachment of a moiety, to the subject molecule. In an embodiment, a modulator alters the, three dimensional, secondary, tertiary, or quaternary structure, of a subject molecule. A modulator can increase, decrease, initiate, or eliminate a subject activity.


“Large molecule”, as used herein, refers to a molecule having a molecular weight of at least 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kD. Large molecules include proteins, polypeptides, nucleic acids, biologics, and carbohydrates.


“Polypeptide”, as used herein, refers to a polymer of amino acids having less than 100 amino acid residues. In an embodiment, it has less than 50, 20, or 10 amino acid residues.


“Reference molecule”, e.g., a reference Cas9 molecule or reference gRNA, as used herein, refers to a molecule to which a subject molecule, e.g., a subject Cas9 molecule of subject gRNA molecule, e.g., a modified or candidate Cas9 molecule is compared. For example, a Cas9 molecule can be characterized as having no more than 10% of the nuclease activity of a reference Cas9 molecule. Examples of reference Cas9 molecules include naturally occurring unmodified Cas9 molecules, e.g., a naturally occurring Cas9 molecule such as a Cas9 molecule of S. pyogenes, or S. thermophilus. In an embodiment, the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology with the Cas9 molecule to which it is being compared. In an embodiment, the reference Cas9 molecule is a sequence, e.g., a naturally occurring or known sequence, which is the parental form on which a change, e.g., a mutation has been made.


“Replacement”, or “replaced”, as used herein with reference to a modification of a molecule does not require a process limitation but merely indicates that the replacement entity is present.


“Small molecule”, as used herein, refers to a compound having a molecular weight less than about 2 kD, e.g., less than about 2 kD, less than about 1.5 kD, less than about 1 kD, or less than about 0.75 kD.


“Subject”, as used herein, may mean either a human or non-human animal. The term includes, but is not limited to, mammals (e.g., humans, other primates, pigs, rodents (e.g., mice and rats or hamsters), rabbits, guinea pigs, cows, horses, cats, dogs, sheep, and goats). In an embodiment, the subject is a human. In an embodiment, the subject is poultry.


“Treat”, “treating” and “treatment”, as used herein, mean the treatment of a disease in a mammal, e.g., in a human, including (a) inhibiting the disease, i.e., arresting or preventing its development; (b) relieving the disease, i.e., causing regression of the disease state; or (c) curing the disease.


“X” as used herein in the context of an amino acid sequence, refers to any amino acid (e.g., any of the twenty natural amino acids) unless otherwise specified.


DETAILED DESCRIPTION

I. gRNA Molecules


A gRNA molecule, as that term is used herein, refers to a nucleic acid that promotes the specific targeting or homing of a gRNA molecule/Cas9 molecule complex to a target nucleic acid, gRNA molecules can be unimolecular (having a single RNA molecule), sometimes referred to herein as “chimeric” gRNAs, or modular (comprising more than one, and typically two, separate RNA molecules). A gRNA molecule comprises a number of domains. The gRNA molecule domains are described in more detail below. Typically, gRNA will incorporate the functions or structure of both crRNA and tracrRNA, e.g., the functions of processed or mature crRNA and of processed or mature tracrRNA. Chimeric or unimolecular gRNA molecules can have a single RNA molecule, e.g., which incorporates both crRNA function or structure and the tracrRNA function or structure. A modular gRNA molecule can comprise a RNA molecule that incorporates the crRNA function or structure another that incorporates the tracrRNA function or structure. Several exemplary gRNA structures, with domains indicated thereon, are provided in FIG. 1. While not wishing to be bound by theory with regard to the three dimensional form, or intra- or inter-strand interactions of an active form of a gRNA, regions of high complementarity are sometimes shown as duplexes in FIG. 1 and other depictions provided herein.


In an embodiment, a unimolecular, or chimeric, gRNA comprises, preferably from 5′ to 3′:

    • a targeting domain, e.g., comprising 15, 16, 17, 18, 19, or 20 nucleotides (which is complementary to a target nucleic acid);
    • a first complementarity domain;
    • a linking domain;
    • a second complementarity domain (which is complementary to the first complementarity domain);
    • a proximal domain; and
    • optionally, a tail domain.


In an embodiment, a modular gRNA comprises:

    • a first strand comprising, preferably from 5′ to 3′;
      • a targeting domain (which is complementary with a target sequence from a target nucleic acid disclosed herein, e.g., a sequence from: a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII); and
      • a first complementarity domain; and
        • a second strand, comprising, preferably from 5′ to 3′:
      • optionally, a 5′ extension domain;
      • a second complementarity domain; and
      • a proximal domain; and
      • optionally, a tail domain.


The domains are discussed briefly below:


1) The Targeting Domain:



FIGS. 1A-1G provide examples of the placement of targeting domains.


The targeting domain comprises a nucleotide sequence that is complementary, e.g., at least 80, 85, 90, or 95% complementary, e.g., fully complementary, to the target sequence on the target nucleic acid. The targeting domain is part of an RNA molecule and will therefore comprise the base uracil (U), while any DNA encoding the gRNA molecule will comprise the base thymine (T). While not wishing to be bound by theory, it is believed that the complementarity of the targeting domain with the target sequence contributes to specificity of the interaction of the gRNA molecule/Cas9 molecule complex with a target nucleic acid. It is understood that in a targeting domain and target sequence pair, the uracil bases in the targeting domain will pair with the adenine bases in the target sequence. In an embodiment, the target domain itself comprises, in the 5′ to 3′ direction, an optional secondary domain, and a core domain. In an embodiment, the core domain is fully complementary with the target sequence. In an embodiment, the targeting domain is 5 to 50, e.g., 10 to 40, e.g., 10 to 30, e.g., 15 to 30, e.g., 15 to 25 nucleotides in length. In an embodiment, the targeting domain is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length. The strand of the target nucleic acid with which the targeting domain is complementary is referred to herein as the complementary strand. Some or all of the nucleotides of the domain can have a modification, e.g., modification found in Section X herein.


In an embodiment, the targeting domain is 15 nucleotides in length.


In an embodiment, the targeting domain is 16 nucleotides in length.


In an embodiment, the targeting domain is 17 nucleotides in length.


In an embodiment, the targeting domain is 18 nucleotides in length.


In an embodiment, the targeting domain is 19 nucleotides in length.


In an embodiment, the targeting domain is 20 nucleotides in length.


In an embodiment, the targeting domain is 21 nucleotides in length.


In an embodiment, the targeting domain is 22 nucleotides in length.


In an embodiment, the targeting domain is 23 nucleotides in length.


In an embodiment, the targeting domain is 24 nucleotides in length.


In an embodiment, the targeting domain is 25 nucleotides in length.


In an embodiment, the targeting domain is 26 nucleotides in length.


In an embodiment, the targeting domain comprises 15 nucleotides.


In an embodiment, the targeting domain comprises 16 nucleotides.


In an embodiment, the targeting domain comprises 17 nucleotides.


In an embodiment, the targeting domain comprises 18 nucleotides.


In an embodiment, the targeting domain comprises 19 nucleotides.


In an embodiment, the targeting domain comprises 20 nucleotides.


In an embodiment, the targeting domain comprises 21 nucleotides.


In an embodiment, the targeting domain comprises 22 nucleotides.


In an embodiment, the targeting domain comprises 23 nucleotides.


In an embodiment, the targeting domain comprises 24 nucleotides.


In an embodiment, the targeting domain comprises 25 nucleotides.


In an embodiment, the targeting domain comprises 26 nucleotides.


Targeting domains are discussed in more detail below.


2) The First Complementarity Domain:



FIGS. 1A-1G provide examples of first complementarity domains.


The first complementarity domain is complementary with the second complementarity domain, and in an embodiment, has sufficient complementarity to the second complementarity domain to form a duplexed region under at least some physiological conditions. In an embodiment, the first complementarity domain is 5 to 30 nucleotides in length. In an embodiment, the first complementarity domain is 5 to 25 nucleotides in length. In an embodiment, the first complementary domain is 7 to 25 nucleotides in length. In an embodiment, the first complementary domain is 7 to 22 nucleotides in length. In an embodiment, the first complementary domain is 7 to 18 nucleotides in length. In an embodiment, the first complementary domain is 7 to 15 nucleotides in length. In an embodiment, the first complementary domain is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.


In an embodiment, the first complementarity domain comprises 3 subdomains, which, in the 5′ to 3′ direction are: a 5′ subdomain, a central subdomain, and a 3′ subdomain. In an embodiment, the 5′ subdomain is 4-9, e.g., 4, 5, 6, 7, 8 or 9 nucleotides in length. In an embodiment, the central subdomain is 1, 2, or 3, e.g., 1, nucleotide in length. In an embodiment, the 3′ subdomain is 3 to 25, e.g., 4-22, 4-18, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, nucleotides in length.


The first complementarity domain can share homology with, or be derived from, a naturally occurring first complementarity domain. In an embodiment, it has at least 50% homology with a first complementarity domain disclosed herein, e.g., an S. pyogenes, or S. thermophilus, first complementarity domain.


Some or all of the nucleotides of the domain can have a modification, e.g., modification found in Section X herein.


First complementarity domains are discussed in more detail below.


3) The Linking Domain



FIGS. 1B-1E provide examples of linking domains.


A linking domain serves to link the first complementarity domain with the second complementarity domain of a unimolecular gRNA. The linking domain can link the first and second complementarity domains covalently or non-covalently. In an embodiment, the linkage is covalent. In an embodiment, the linking domain covalently couples the first and second complementarity domains, see, e.g., FIGS. 1B-1E. In an embodiment, the linking domain is, or comprises, a covalent bond interposed between the first complementarity domain and the second complementarity domain. Typically, the linking domain comprises one or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.


In modular gRNA molecules the two molecules can be associated by virtue of the hybridization of the complementarity domains, see e.g., FIG. 1A.


A wide variety of linking domains are suitable for use in unimolecular gRNA molecules. Linking domains can consist of a covalent bond, or be as short as one or a few nucleotides, e.g., 1, 2, 3, 4, or 5 nucleotides in length.


In an embodiment, a linking domain is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in length. In an embodiment, a linking domain is 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, or 2 to 5 nucleotides in length. In an embodiment, a linking domain shares homology with, or is derived from, a naturally occurring sequence, e.g., the sequence of a tracrRNA that is 5′ to the second complementarity domain. In an embodiment, the linking domain has at least 50% homology with a linking domain disclosed herein.


Some or all of the nucleotides of the domain can have a modification, e.g., modification found in Section X herein.


Linking domains are discussed in more detail below.


4) The 5′ Extension Domain


In an embodiment, a modular gRNA can comprise additional sequence, 5′ to the second complementarity domain, referred to herein as the 5′ extension domain, see, e.g., FIG. 1A. In an embodiment, the 5′ extension domain is, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4 nucleotides in length. In an embodiment, the 5′ extension domain is 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length.


5) The Second Complementarity Domain:



FIGS. 1A-1F provide examples of second complementarity domains.


The second complementarity domain is complementary with the first complementarity domain, and in an embodiment, has sufficient complementarity to the second complementarity domain to form a duplexed region under at least some physiological conditions. In an embodiment, e.g., as shown in FIG. 1A or FIG. 1B, the second complementarity domain can include sequence that lacks complementarity with the first complementarity domain, e.g., sequence that loops out from the duplexed region.


In an embodiment, the second complementarity domain is 5 to 27 nucleotides in length. In an embodiment, it is longer than the first complementarity region.


In an embodiment, the second complementary domain is 7 to 27 nucleotides in length. In an embodiment, the second complementary domain is 7 to 25 nucleotides in length. In an embodiment, the second complementary domain is 7 to 20 nucleotides in length. In an embodiment, the second complementary domain is 7 to 17 nucleotides in length. In an embodiment, the complementary domain is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.


In an embodiment, the second complementarity domain comprises 3 subdomains, which, in the 5′ to 3′ direction are: a 5′ subdomain, a central subdomain, and a 3′ subdomain. In an embodiment, the 5′ subdomain is 3 to 25, e.g., 4 to 22, 4 to 18, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In an embodiment, the central subdomain is 1, 2, 3, 4 or 5, e.g., 3, nucleotides in length. In an embodiment, the 3′ subdomain is 4 to 9, e.g., 4, 5, 6, 7, 8 or 9 nucleotides in length.


In an embodiment, the 5′ subdomain and the 3′ subdomain of the first complementarity domain, are respectively, complementary, e.g., fully complementary, with the 3′ subdomain and the 5′ subdomain of the second complementarity domain.


The second complementarity domain can share homology with or be derived from a naturally occurring second complementarity domain. In an embodiment, it has at least 50% homology with a second complementarity domain disclosed herein, e.g., an S. pyogenes, or S. thermophilus, first complementarity domain.


Some or all of the nucleotides of the domain can have a modification, e.g., modification found in Section X herein.


6) A Proximal Domain:



FIGS. 1A-1F provide examples of proximal domains.


In an embodiment, the proximal domain is 5 to 20 nucleotides in length. In an embodiment, the proximal domain can share homology with or be derived from a naturally occurring proximal domain. In an embodiment, it has at least 50% homology with a proximal domain disclosed herein, e.g., an S. pyogenes, or S. thermophilus, proximal domain.


Some or all of the nucleotides of the domain can have a modification, e.g., modification found in Section X herein.


7) A Tail Domain:



FIG. 1A and FIGS. 1C-1F provide examples of tail domains.


As can be seen by inspection of the tail domains in FIG. 1A and FIGS. 1C-1F, a broad spectrum of tail domains are suitable for use in gRNA molecules. In an embodiment, the tail domain is O (absent), 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In an embodiment, the tail domain nucleotides are from or share homology with sequence from the 5′ end of a naturally occurring tail domain, see e.g., FIG. 1D or FIG. 1E. In an embodiment, the tail domain includes sequences that are complementary to each other and which, under at least some physiological conditions, form a duplexed region.


In an embodiment, the tail domain is absent or is 1 to 50 nucleotides in length. In an embodiment, the tail domain can share homology with or be derived from a naturally occurring proximal tail domain. In an embodiment, it has at least 50% homology with a tail domain disclosed herein, e.g., an S. pyogenes, or S. thermophilus, tail domain.


Some or all of the nucleotides of the domain can have a modification, e.g., modification found in Section X herein.


In an embodiment, the tail domain includes nucleotides at the 3′ end that are related to the method of in vitro or in vivo transcription. When a T7 promoter is used for in vitro transcription of the gRNA, these nucleotides may be any nucleotides present before the 3′ end of the DNA template. When a U6 promoter is used for in vivo transcription, these nucleotides may be the sequence UUUUUU. When alternate pol-III promoters are used, these nucleotides may be various numbers or uracil bases or may include alternate bases.


The domains of gRNA molecules are described in more detail below.


The Targeting Domain


The “targeting domain” of the gRNA is complementary to the “target domain” on the target nucleic acid. The strand of the target nucleic acid comprising the nucleotide sequence complementary to the core domain of the gRNA is referred to herein as the “complementary strand” of the target nucleic acid. Guidance on the selection of targeting domains can be found, e.g., in Fu Y et al., NAT BIOTECHNOL 2014 (doi: 10.1038/nbt.2808) and Sternberg S H et al., NATURE 2014 (doi: 10.1038/nature13011).


In an embodiment, the targeting domain is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.


In an embodiment, the targeting domain comprises 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.


In an embodiment, the targeting domain is 15 nucleotides in length.


In an embodiment, the targeting domain is 16 nucleotides in length.


In an embodiment, the targeting domain is 17 nucleotides in length.


In an embodiment, the targeting domain is 18 nucleotides in length.


In an embodiment, the targeting domain is 19 nucleotides in length.


In an embodiment, the targeting domain is 20 nucleotides in length.


In an embodiment, the targeting domain is 21 nucleotides in length.


In an embodiment, the targeting domain is 22 nucleotides in length.


In an embodiment, the targeting domain is 23 nucleotides in length.


In an embodiment, the targeting domain is 24 nucleotides in length.


In an embodiment, the targeting domain is 25 nucleotides in length.


In an embodiment, the targeting domain is 26 nucleotides in length.


In an embodiment, the targeting domain comprises 15 nucleotides.


In an embodiment, the targeting domain comprises 16 nucleotides.


In an embodiment, the targeting domain comprises 17 nucleotides.


In an embodiment, the targeting domain comprises 18 nucleotides.


In an embodiment, the targeting domain comprises 19 nucleotides.


In an embodiment, the targeting domain comprises 20 nucleotides.


In an embodiment, the targeting domain comprises 21 nucleotides.


In an embodiment, the targeting domain comprises 22 nucleotides.


In an embodiment, the targeting domain comprises 23 nucleotides.


In an embodiment, the targeting domain comprises 24 nucleotides.


In an embodiment, the targeting domain comprises 25 nucleotides.


In an embodiment, the targeting domain comprises 26 nucleotides.


In an embodiment, the targeting domain is 10+/−5, 20+/−5, 30+/−5, 40+/−5, 50+/−5, 60+/−5, 70+/−5, 80+/−5, 90+/−5, or 100+/−5 nucleotides, in length.


In an embodiment, the targeting domain is 20+/−5 nucleotides in length.


In an embodiment, the targeting domain is 20+/−10, 30+/−10, 40+/−10, 50+/−10, 60+/−10, 70+/−10, 80+/−10, 90+/−10, or 100+/−10 nucleotides, in length.


In an embodiment, the targeting domain is 30+/−10 nucleotides in length.


In an embodiment, the targeting domain is 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20 or 10 to 15 nucleotides in length. In an embodiment, the targeting domain is 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, or 20 to 25 nucleotides in length.


Typically the targeting domain has full complementarity with the target sequence. In an embodiment the targeting domain has or includes 1, 2, 3, 4, 5, 6, 7 or 8 nucleotides that are not complementary with the corresponding nucleotide of the targeting domain.


In an embodiment, the target domain includes 1, 2, 3, 4 or 5 nucleotides that are complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 5′ end. In an embodiment, the target domain includes 1, 2, 3, 4 or 5 nucleotides that are complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 3′ end.


In an embodiment, the target domain includes 1, 2, 3, or 4 nucleotides that are not complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 5′ end. In an embodiment, the target domain includes 1, 2, 3, or 4 nucleotides that are not complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 3′ end.


In an embodiment, the degree of complementarity, together with other properties of the gRNA, is sufficient to allow targeting of a Cas9 molecule to the target nucleic acid.


In an embodiment, the targeting domain comprises two consecutive nucleotides that are not complementary to the target domain (“non-complementary nucleotides”), e.g., two consecutive noncomplementary nucleotides that are within 5 nucleotides of the 5′ end of the targeting domain, within 5 nucleotides of the 3′ end of the targeting domain, or more than 5 nucleotides away from one or both ends of the targeting domain.


In an embodiment, no two consecutive nucleotides within 5 nucleotides of the 5′ end of the targeting domain, within 5 nucleotides of the 3′ end of the targeting domain, or within a region that is more than 5 nucleotides away from one or both ends of the targeting domain, are not complementary to the targeting domain.


In an embodiment, there are no noncomplementary nucleotides within 5 nucleotides of the 5′ end of the targeting domain, within 5 nucleotides of the 3′ end of the targeting domain, or within a region that is more than 5 nucleotides away from one or both ends of the targeting domain.


In an embodiment, the targeting domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X. However, in an embodiment, the targeting domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the targeting domain can be modified with a phosphorothioate, or other modification from Section X. In an embodiment, a nucleotide of the targeting domain can comprise a 2′ modification (e.g., a modification at the 2′ position on ribose), e.g., a 2′ acetylation, e.g., a 2′ methylation, or other modification from Section X.


In an embodiment, the targeting domain includes 1, 2, 3, 4, 5, 6, 7 or 8 or more modifications. In an embodiment, the targeting domain includes 1, 2, 3, or 4 modifications within 5 nucleotides of its 5′ end. In an embodiment, the targeting domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3′ end.


In an embodiment, the targeting domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5′ end of the targeting domain, within 5 nucleotides of the 3′ end of the targeting domain, or more than 5 nucleotides away from one or both ends of the targeting domain.


In an embodiment, no two consecutive nucleotides are modified within 5 nucleotides of the 5′ end of the targeting domain, within 5 nucleotides of the 3′ end of the targeting domain, or within a region that is more than 5 nucleotides away from one or both ends of the targeting domain. In an embodiment, no nucleotide is modified within 5 nucleotides of the 5′ end of the targeting domain, within 5 nucleotides of the 3′ end of the targeting domain, or within a region that is more than 5 nucleotides away from one or both ends of the targeting domain.


Modifications in the targeting domain can be selected so as to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section III, gRNA's having a candidate targeting domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in a system in Section III. The candidate targeting domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.


In an embodiment, all of the modified nucleotides are complementary to and capable of hybridizing to corresponding nucleotides present in the target domain. In an embodiment, 1, 2, 3, 4, 5, 6, 7 or 8 or more modified nucleotides are not complementary to or capable of hybridizing to corresponding nucleotides present in the target domain.


In an embodiment, the targeting domain comprises, preferably in the 5′ 3′ direction: a secondary domain and a core domain. These domains are discussed in more detail below.


The Core Domain and Secondary Domain of the Targeting Domain


The “core domain” of the targeting domain is complementary to the “core domain target” on the target nucleic acid. In an embodiment, the core domain comprises about 8 to about 13 nucleotides from the 3′ end of the targeting domain (e.g., the most 3′ 8 to 13 nucleotides of the targeting domain).


In an embodiment, the core domain is 6+/−2, 7+/−2, 8+/−2, 9+/−2, 10+/−2, 11+/−2, 12+/−2, 13+/−2, 14+/−2, 15+/−2, or 16+/−2 nucleotides in length.


In an embodiment, the core domain is 10+/−2 nucleotides in length.


In an embodiment, the core domain is 10+/−4 nucleotides in length.


In an embodiment, the core domain is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleotides in length.


In an embodiment, the core domain is 8 to 13, e.g., 8 to 12, 8 to 11, 8 to 10, 8 to 9, 9 to 13, 9 to 12, 9 to 11, or 9 to 10 nucleotides in length.


In an embodiment, the core domain is 6 to 16, e.g., 6 to 15, 6 to 14, 6 to 13, 7 to 14, 7 to 13, 7 to 12, 7 to 11, 7 to 10, 8 to 14, 8 to 13, 8 to 12, 8 to 11, 8 to 10, or 8 to 9 nucleotides in length.


The core domain is complementary with the core domain target. Typically the core domain has exact complementarity with the core domain target. In an embodiment, the core domain can have 1, 2, 3, 4 or 5 nucleotides that are not complementary with the corresponding nucleotide of the core domain. In an embodiment, the degree of complementarity, together with other properties of the gRNA, is sufficient to allow targeting of a Cas9 molecule to the target nucleic acid.


The “secondary domain” of the targeting domain of the gRNA is complementary to the “secondary domain target” of the target nucleic acid.


In an embodiment, the secondary domain is positioned 5′ to the core domain.


In an embodiment, the secondary domain is absent or optional.


In an embodiment, if the targeting domain is, or is at least, 26 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 12 to 17 nucleotides in length.


In an embodiment, if the targeting domain is, or is at least, 25 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 12 to 17 nucleotides in length.


In an embodiment, if the targeting domain is, or is at least, 24 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 11 to 16 nucleotides in length.


In an embodiment, if the targeting domain is, or is at least, 23 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 10 to 15 nucleotides in length.


In an embodiment, if the targeting domain is, or is at least, 22 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 9 to 14 nucleotides in length.


In an embodiment, if the targeting domain is, or is at least, 21 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 8 to 13 nucleotides in length.


In an embodiment, if the targeting domain is, or is at least, 20 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 7 to 12 nucleotides in length.


In an embodiment, if the targeting domain is, or is at least, 19 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 6 to 11 nucleotides in length.


In an embodiment, if the targeting domain is, or is at least, 18 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 5 to 10 nucleotides in length.


In an embodiment, if the targeting domain is, or is at least, 17 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 4 to 9 nucleotides in length.


In an embodiment, if the targeting domain is, or is at least, 16 nucleotides in length and the core domain (counted from the 3′ end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 3 to 8 nucleotides in length.


In an embodiment, the secondary domain is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or nucleotides in length.


The secondary domain is complementary with the secondary domain target. Typically the secondary domain has exact complementarity with the secondary domain target. In an embodiment the secondary domain can have 1, 2, 3, 4 or 5 nucleotides that are not complementary with the corresponding nucleotide of the secondary domain. In an embodiment, the degree of complementarity, together with other properties of the gRNA, is sufficient to allow targeting of a Cas9 molecule to the target nucleic acid.


In an embodiment, the core domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X. However, in an embodiment, the core domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the core domain can be modified with a phosphorothioate, or other modification from Section X. In an embodiment, a nucleotide of the core domain can comprise a 2′ modification (e.g., a modification at the 2′ position on ribose), e.g., a 2′-acetylation, e.g., a 2′ methylation, or other modification from Section X. Typically, a core domain will contain no more than 1, 2, or 3 modifications.


Modifications in the core domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section III, gRNA's having a candidate core domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described at Section III. The candidate core domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.


In an embodiment, the secondary domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X. However, in an embodiment, the secondary domain comprises one or more modifications, e.g., modifications that render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the secondary domain can be modified with a phosphorothioate, or other modification from Section X. In an embodiment, a nucleotide of the secondary domain can comprise a 2′ modification (e.g., a modification at the 2′ position on ribose), e.g., a 2′-acetylation, e.g., a 2′ methylation, or other modification from Section X. Typically, a secondary domain will contain no more than 1, 2, or 3 modifications.


Modifications in the secondary domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section III, gRNA's having a candidate secondary domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described at Section III. The candidate secondary domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.


In an embodiment, (1) the degree of complementarity between the core domain and its target, and (2) the degree of complementarity between the secondary domain and its target, may differ. In an embodiment, (1) may be greater than (2). In an embodiment, (1) may be less than (2). In an embodiment, (1) and (2) may be the same. e.g., each may be completely complementary with its target.


In an embodiment, (1) the number of modifications (e.g., modifications from Section X) of the nucleotides of the core domain and (2) the number of modification (e.g., modifications from Section X) of the nucleotides of the secondary domain, may differ. In an embodiment, (1) may be less than (2). In an embodiment, (1) may be greater than (2). In an embodiment, (1) and (2) may be the same, e.g., each may be free of modifications.


The First and Second Complementarity Domains


The first complementarity domain is complementary with the second complementarity domain.


Typically the first domain does not have exact complementarity with the second complementarity domain target. In an embodiment, the first complementarity domain can have 1, 2, 3, 4 or 5 nucleotides that are not complementary with the corresponding nucleotide of the second complementarity domain. In an embodiment, 1, 2, 3, 4, 5 or 6, e.g., 3 nucleotides, will not pair in the duplex, and, e.g., form a non-duplexed or looped-out region. In an embodiment, an unpaired, or loop-out, region, e.g., a loop-out of 3 nucleotides, is present on the second complementarity domain. In an embodiment, the unpaired region begins 1, 2, 3, 4, 5, or 6, e.g., 4, nucleotides from the 5′ end of the second complementarity domain.


In an embodiment, the degree of complementarity, together with other properties of the gRNA, is sufficient to allow targeting of a Cas9 molecule to the target nucleic acid.


In an embodiment, the first and second complementarity domains are:


independently, 6+/−2, 7+/−2, 8+/−2, 9+/−2, 10+/−2, 11+/−2, 12+/−2, 13+/−2, 14+/−2, 15+/−2, 16+/−2, 17+/−2, 18+/−2, 19+/−2, or 20+/−2, 21+/−2, 22+/−2, 23+/−2, or 24+/−2 nucleotides in length;


independently, 6, 7, 8, 9, 10, 11, 12, 13, 14, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length; or


independently, 5 to 24, 5 to 23, 5 to 22, 5 to 21, 5 to 20, 7 to 18, 9 to 16, or 10 to 14 nucleotides in length.


In an embodiment, the second complementarity domain is longer than the first complementarity domain, e.g., 2, 3, 4, 5, or 6, e.g., 6, nucleotides longer.


In an embodiment, the first and second complementary domains, independently, do not comprise modifications, e.g., modifications of the type provided in Section X.


In an embodiment, the first and second complementary domains, independently, comprise one or more modifications, e.g., modifications that the render the domain less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the domain can be modified with a phosphorothioate, or other modification from Section X. In an embodiment, a nucleotide of the domain can comprise a 2′ modification (e.g., a modification at the 2′ position on ribose), e.g., a 2′-acetylation, e.g., a 2′ methylation, or other modification from Section X.


In an embodiment, the first and second complementary domains, independently, include 1, 2, 3, 4, 5, 6, 7 or 8 or more modifications. In an embodiment, the first and second complementary domains, independently, include 1, 2, 3, or 4 modifications within 5 nucleotides of its 5′ end. In an embodiment, the first and second complementary domains, independently, include as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3′ end.


In an embodiment, the first and second complementary domains, independently, include modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5′ end of the domain, within 5 nucleotides of the 3′ end of the domain, or more than 5 nucleotides away from one or both ends of the domain. In an embodiment, the first and second complementary domains, independently, include no two consecutive nucleotides that are modified, within 5 nucleotides of the 5′ end of the domain, within 5 nucleotides of the 3′ end of the domain, or within a region that is more than 5 nucleotides away from one or both ends of the domain. In an embodiment, the first and second complementary domains, independently, include no nucleotide that is modified within 5 nucleotides of the 5′ end of the domain, within 5 nucleotides of the 3′ end of the domain, or within a region that is more than 5 nucleotides away from one or both ends of the domain.


Modifications in a complementarity domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section III, gRNA's having a candidate complementarity domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described in Section III. The candidate complementarity domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.


In an embodiment, the first complementarity domain has at least 60, 70, 80, 85%, 90%, or 95% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference first complementarity domain, e.g., a naturally occurring, e.g., an S. pyogenes, or S. thermophilus, first complementarity domain, or a first complementarity domain described herein, e.g., from FIGS. 1A-1F.


In an embodiment, the second complementarity domain has at least 60, 70, 80, 85%, 90%, or 95% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference second complementarity domain, e.g., a naturally occurring, e.g., an S. pyogenes, or S. thermophilus, second complementarity domain, or a second complementarity domain described herein. e.g., from FIGS. 1A-1F.


The duplexed region formed by first and second complementarity domains is typically 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 base pairs in length (excluding any looped out or unpaired nucleotides).


In an embodiment, the first and second complementarity domains, when duplexed, comprise 11 paired nucleotides, for example, in the gRNA sequence (one paired strand underlined, one bolded):









(SEQ ID NO: 5)


NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU





AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC.






In an embodiment, the first and second complementarity domains, when duplexed, comprise 15 paired nucleotides, for example in the gRNA sequence (one paired strand underlined, one bolded):









(SEQ ID NO: 27)


NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGAAAAGCAUAGCAA





GUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCG





GUGC.






In an embodiment the first and second complementarity domains, when duplexed, comprise 16 paired nucleotides, for example in the gRNA sequence (one paired strand underlined, one bolded):









(SEQ ID NO: 28)


NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGGAAACAGCAUAGC





AAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU





CGGUGC.






In an embodiment the first and second complementarity domains, when duplexed, comprise 21 paired nucleotides, for example in the gRNA sequence (one paired strand underlined, one bolded):









(SEQ ID NO: 29)


NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGUUUUGGAAACAAA





ACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU





GGCACCGAGUCGGUGC.






In an embodiment, nucleotides are exchanged to remove poly-U tracts, for example in the gRNA sequences (exchanged nucleotides underlined):









(SEQ ID NO: 30)


NNNNNNNNNNNNNNNNNNNNGUAUUAGAGCUAGAAAUAGCAAGUUAAUAU





AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC;





(SEQ ID NO: 31)


NNNNNNNNNNNNNNNNNNNNGUUUAAGAGCUAGAAAUAGCAAGUUUAAAU





AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC;


and





(SEQ ID NO: 32)


NNNNNNNNNNNNNNNNNNNNGUAUUAGAGCUAUGCUGUAUUGGAAACAAU





ACAGCAUAGCAAGUUAAUAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU





GGCACCGAGUCGGUGC.






The 5′ Extension Domain


In an embodiment, a modular gRNA can comprise additional sequence, 5′ to the second complementarity domain. In an embodiment, the 5′ extension domain is 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, or 2 to 4 nucleotides in length. In an embodiment, the 5′ extension domain is 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length.


In an embodiment, the 5′ extension domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X. However, in an embodiment, the 5′ extension domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the 5′ extension domain can be modified with a phosphorothioate, or other modification from Section X. In an embodiment, a nucleotide of the 5′ extension domain can comprise a 2′ modification (e.g., a modification at the 2′ position on ribose), e.g., a 2′-acetylation, e.g., a 2′ methylation, or other modification from Section X.


In an embodiment, the 5′ extension domain can comprise as many as 1, 2, 3, 4, 5, 6, 7 or 8 modifications. In an embodiment, the 5′ extension domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 5′ end, e.g., in a modular gRNA molecule. In an embodiment, the 5′ extension domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3′ end, e.g., in a modular gRNA molecule.


In an embodiment, the 5′ extension domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5′ end of the 5′ extension domain, within 5 nucleotides of the 3′ end of the 5′ extension domain, or more than 5 nucleotides away from one or both ends of the 5′ extension domain. In an embodiment, no two consecutive nucleotides are modified within 5 nucleotides of the 5′ end of the 5′ extension domain, within 5 nucleotides of the 3′ end of the 5′ extension domain, or within a region that is more than 5 nucleotides away from one or both ends of the 5′ extension domain. In an embodiment, no nucleotide is modified within 5 nucleotides of the 5′ end of the 5′ extension domain, within 5 nucleotides of the 3′ end of the 5′ extension domain, or within a region that is more than 5 nucleotides away from one or both ends of the 5′ extension domain.


Modifications in the 5′ extension domain can be selected to not interfere with gRNA molecule efficacy, which can be evaluated by testing a candidate modification in the system described in Section III, gRNAs having a candidate 5′ extension domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described at Section III. The candidate 5′ extension domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.


In an embodiment, the 5′ extension domain has at least 60, 70, 80, 85, 90 or 95% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference 5′ extension domain, e.g., a naturally occurring, e.g., an S. pyogenes, or S. thermophilus, 5′ extension domain, or a 5′ extension domain described herein, e.g., from FIG. 1A and FIG. 1F.


The Linking Domain


In a unimolecular gRNA molecule the linking domain is disposed between the first and second complementarity domains. In a modular gRNA molecule, the two molecules are associated with one another by the complementarity domains.


In an embodiment, the linking domain is 10+/−5, 20+/−5, 30+/−5, 40+/−5, 50+/−5, 60+/−5, 70+/−5, 80+/−5, 90+/−5, or 100+/−5 nucleotides, in length.


In an embodiment, the linking domain is 20+/−10, 30+/−10, 40+/−10, 50+/−10, 60+/−10, 70+/−10, 80+/−10, 90+/−10, or 100+/−10 nucleotides, in length.


In an embodiment, the linking domain is 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20 or 10 to 15 nucleotides in length. In an embodiment, the targeting domain is 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, or 20 to 25 nucleotides in length.


In an embodiment, the linking domain 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 an embodiment, the linking domain is a covalent bond.


In an embodiment, the linking domain comprises a duplexed region, typically adjacent to or within 1, 2, or 3 nucleotides of the 3′ end of the first complementarity domain and/or the 5-end of the second complementarity domain. In an embodiment, the duplexed region can be 20+/−10, 30+/−10, 40, +/−10 or 50+/−10 base pairs in length. In an embodiment, the duplexed region can be 10+/−5, 15+/−5, 20+/−5, or 30+/−5 base pairs in length. In an embodiment, the duplexed region can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 base pairs in length.


Typically the sequences forming the duplexed region have exact complementarity with one another, though in an embodiment as many as 1, 2, 3, 4, 5, 6, 7 or 8 nucleotides are not complementary with the corresponding nucleotides.


In an embodiment, the linking domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X. However, in an embodiment the linking domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the linking domain can be modified with a phosphorothioate, or other modification from Section X. In an embodiment, a nucleotide of the linking domain can comprise a 2′ modification (e.g., a modification at the 2′ position on ribose), e.g., a 2′-acetylation, e.g., a 2′ methylation, or other modification from Section X.


In an embodiment, the linking domain can comprise as many as 1, 2, 3, 4, 5, 6, 7 or 8 modifications.


Modifications in a linking domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section III, gRNA's having a candidate linking domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated a system described in Section III. A candidate linking domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.


In an embodiment, the linking domain has at least 60, 70, 80, 85, 90 or 95% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference linking domain, e.g., a linking domain described herein, e.g., from FIG. 1B-1E.


The Proximal Domain


In an embodiment, the proximal domain is 6+/−2, 7+/−2, 8+/−2, 9+/−2, 10+/−2, 11+/−2, 12+/−2, 13+/−2, 14+/−2, 14+/−2, 16+/−2, 17+/−2, 18+/−2, 19+/−2, or 20+/−2 nucleotides in length.


In an embodiment, the proximal domain is 6, 7, 8, 9, 10, 11, 12, 13, 14, 14, 16, 17, 18, 19, or 20 nucleotides in length.


In an embodiment, the proximal domain is 5 to 20, 7, to 18, 9 to 16, or 10 to 14 nucleotides in length.


In an embodiment, the proximal domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X. However, in an embodiment, the proximal domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the proximal domain can be modified with a phosphorothioate, or other modification from Section X. In an embodiment, a nucleotide of the proximal domain can comprise a 2′ modification (e.g., a modification at the 2′ position on ribose), e.g., a 2′-acetylation, e.g., a 2′ methylation, or other modification from Section X.


In an embodiment, the proximal domain can comprise as many as 1, 2, 3, 4, 5, 6, 7 or 8 modifications. In an embodiment, the proximal domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 5′ end, e.g., in a modular gRNA molecule. In an embodiment, the target domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3′ end, e.g., in a modular gRNA molecule.


In an embodiment, the proximal domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5′ end of the proximal domain, within 5 nucleotides of the 3′ end of the proximal domain, or more than 5 nucleotides away from one or both ends of the proximal domain. In an embodiment, no two consecutive nucleotides are modified within 5 nucleotides of the 5′ end of the proximal domain, within 5 nucleotides of the 3′ end of the proximal domain, or within a region that is more than 5 nucleotides away from one or both ends of the proximal domain. In an embodiment, no nucleotide is modified within 5 nucleotides of the 5′ end of the proximal domain, within 5 nucleotides of the 3′ end of the proximal domain, or within a region that is more than 5 nucleotides away from one or both ends of the proximal domain.


Modifications in the proximal domain can be selected to not interfere with gRNA molecule efficacy, which can be evaluated by testing a candidate modification in the system described in Section III, gRNA's having a candidate proximal domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described at Section III. The candidate proximal domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.


In an embodiment, the proximal domain has at least 60%, 70%, 80%, 85%, 90%, or 95% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference proximal domain, e.g., a naturally occurring, e.g., an S. pyogenes, or S. thermophilus, proximal domain, or a proximal domain described herein, e.g., from FIG. 1A-1F.


The Tail Domain


In an embodiment, the tail domain is 10+/−5, 20+/−5, 30+/−5, 40+/−5, 50+/−5, 60+/−5, 70+/−5, 80+/−5, 90+/−5, or 100+/−5 nucleotides, in length.


In an embodiment, the tail domain is 20+/−5 nucleotides in length.


In an embodiment, the tail domain is 20+/−10, 30+/−10, 40+/−10, 50+/−10, 60+/−10, 70+/−10, 80+/−10, 90+/−10, or 100+/−10 nucleotides, in length.


In an embodiment, the tail domain is 25+/−10 nucleotides in length.


In an embodiment, the tail domain is 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20 or 10 to 15 nucleotides in length.


In an embodiment, the tail domain is 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, or 20 to 25 nucleotides in length.


In an embodiment, the tail domain is 1 to 20, 1 to 1, 1 to 10, or 1 to 5 nucleotides in length.


In an embodiment, the tail domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X. However, in an embodiment, the tail domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the tail domain can be modified with a phosphorothioate, or other modification from Section X. In an embodiment, a nucleotide of the tail domain can comprise a 2′ modification (e.g., a modification at the 2′ position on ribose), e.g., a 2′-acetylation, e.g., a 2′ methylation, or other modification from Section X.


In an embodiment, the tail domain can have as many as 1, 2, 3, 4, 5, 6, 7 or 8 modifications. In an embodiment, the target domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 5′ end. In an embodiment, the target domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3′ end.


In an embodiment, the tail domain comprises a tail duplex domain, which can form a tail duplexed region. In an embodiment, the tail duplexed region can be 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 base pairs in length. In an embodiment, a further single stranded domain exists 3′ to the tail duplexed domain. In an embodiment, this domain is 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In an embodiment, it is 4 to 6 nucleotides in length.


In an embodiment, the tail domain has at least 60, 70, 80, or 90% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference tail domain, e.g., a naturally occurring, e.g., an S. pyogenes, or S. thermophilus, tail domain, or a tail domain described herein, e.g., from FIG. 1A and FIGS. 1C-1F.


In an embodiment, the proximal and tail domain, taken together comprise the following sequences:









(SEQ ID NO: 33)


AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU;





(SEQ ID NO: 34)


AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGGUGC;





(SEQ ID NO: 35)


AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGGAU





C;





(SEQ ID NO: 36)


AAGGCUAGUCCGUUAUCAACUUGAAAAAGUG;





(SEQ ID NO: 37)


AAGGCUAGUCCGUUAUCA;


or





(SEQ ID NO: 38)


AAGGCUAGUCCG.






In an embodiment, the tail domain comprises the 3′ sequence UUUUUU, e.g., if a U6 promoter is used for transcription.


In an embodiment, the tail domain comprises the 3′ sequence UUUU, e.g., if an H1 promoter is used for transcription.


In an embodiment, tail domain comprises variable numbers of 3′ Us depending, e.g., on the termination signal of the pol-III promoter used.


In an embodiment, the tail domain comprises variable 3′ sequence derived from the DNA template if a T7 promoter is used.


In an embodiment, the tail domain comprises variable 3′ sequence derived from the DNA template, e.g., if in vitro transcription is used to generate the RNA molecule.


In an embodiment, the tail domain comprises variable 3′ sequence derived from the DNA template, e.g., if a pol-II promoter is used to drive transcription.


Modifications in the tail domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section III, gRNA's having a candidate tail domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described in Section III. The candidate tail domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.


In an embodiment, the tail domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5′ end of the tail domain, within 5 nucleotides of the 3′ end of the tail domain, or more than 5 nucleotides away from one or both ends of the tail domain. In an embodiment, no two consecutive nucleotides are modified within 5 nucleotides of the 5′ end of the tail domain, within 5 nucleotides of the 3′ end of the tail domain, or within a region that is more than 5 nucleotides away from one or both ends of the tail domain. In an embodiment, no nucleotide is modified within 5 nucleotides of the 5′ end of the tail domain, within 5 nucleotides of the 3′ end of the tail domain, or within a region that is more than 5 nucleotides away from one or both ends of the tail domain.


In an embodiment a gRNA has the following structure:


5′ [targeting domain]-[first complementarity domain]-[linking domain]-[second complementarity domain]-[proximal domain]-[tail domain]-3′


wherein,

    • the targeting domain comprises a core domain and optionally a secondary domain, and is 10 to 50 nucleotides in length;


the first complementarity domain is 5 to 25 nucleotides in length and, in an embodiment has

    • at least 50, 60, 70, 80, 85, 90, or 95% homology with a reference first
    • complementarity domain disclosed herein;
    • the linking domain is 1 to 5 nucleotides in length;


      the proximal domain is 5 to 20 nucleotides in length and, in an embodiment has at least 50, 60, 70, 80, 85, 90 or 95% homology with a reference proximal domain disclosed herein;


and


the tail domain is absent or a nucleotide sequence is 1 to 50 nucleotides in length and, in an embodiment has at least 50, 60, 70, 80, 85, 90 or 95% homology with a reference tail domain disclosed herein.


Exemplary Chimeric gRNAs


In an embodiment, a unimolecular, or chimeric, gRNA comprises, preferably from 5′ to 3′:

    • a targeting domain, e.g., comprising 15, 16, 17, 18, 19 or 20 nucleotides (which is complementary to a target nucleic acid);
    • a first complementarity domain;
    • a linking domain;
    • a second complementarity domain (which is complementary to the first complementarity domain);
    • a proximal domain; and
    • a tail domain,
    • wherein.
    • (a) the proximal and tail domain, when taken together, comprise
    • at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides;
    • (b) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain; or
    • (c) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.


In an embodiment, the sequence from (a), (b), or (c), has at least 60, 75, 80, 85, 90, 95, or 99% homology with the corresponding sequence of a naturally occurring gRNA, or with a gRNA described herein.


In an embodiment, the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.


In an embodiment, there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.


In an embodiment, there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.


In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length.


In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length.


In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length.


In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length.


In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length.


In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length.


In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length.


In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length.


In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length.


In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length.


In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length. In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.


In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.


In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.


In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.


In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.


In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.


In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.


In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.


In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.


In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.


In an embodiment, the targeting domain has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.


In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.


Exemplary Modular gRNAs


In an embodiment, a modular gRNA comprises:

    • a first strand comprising, preferably from 5′ to 3′;
      • a targeting domain, e.g., comprising 15, 16, 17, 18, 19, or 20 nucleotides;
      • a first complementarity domain; and
      • a second strand, comprising, preferably from 5′ to 3′;
      • optionally a 5′ extension domain:
      • a second complementarity domain;
      • a proximal domain; and
      • a tail domain,
    • wherein:


(a) the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides;


(b) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain; or


(c) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.


In an embodiment, the sequence from (a), (b), or (c), has at least 60, 75, 80, 85, 90, 95, or 99% homology with the corresponding sequence of a naturally occurring gRNA, or with a gRNA described herein.


In an embodiment, the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.


In an embodiment, there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.


In an embodiment, there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length.


In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length.


In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length.


In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length.


In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length.


In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length.


In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length.


In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length.


In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length.


In an embodiment, the targeting domain has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length.


In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 5 nucleotides in length.


In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 5 nucleotides in length.


In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.


In an embodiment, the targeting domain comprises, has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.


In an embodiment, the targeting domain comprises, n has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.


In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.


In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.


In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.


In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.


In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.


In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.


In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.


In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.


In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.


In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain.


In an embodiment, the targeting domain comprises, has, or consists of, 26 nucleotides (e.g., 26 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 26 nucleotides in length; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.


Methods for Designing gRNAs


Methods for designing gRNAs are described herein, including methods for selecting, designing and validating target domains. Exemplary targeting domains are also provided herein.


Targeting Domains discussed herein can be incorporated into the gRNAs described herein.


Methods for selection and validation of target sequences as well as off-target analyses are described. e.g., in Mali et al., 2013 SCIENCE 339(6121); 823-826; Hsu et al., 2013 NAT BIOTECHNOL, 31(9): 827-32; Fu et al., 2014 NAT BIOTECHNOL, doi: 10.1038/nbt.2808. PubMed PMID: 24463574: Heigwer et al., 2014 NAT METHODS 11(2): 122-3. doi: 10.1038/nmeth.2812. PubMed PMID: 24481216; Bae et al., 2014 BIOINFORMATICS PubMed PMID: 24463181; Xiao A et al., 2014 BIOINFORMATICS PubMed PMID: 24389662.


For example, a software tool can be used to optimize the choice of gRNA within a user's target sequence, e.g., to minimize total off-target activity across the genome. Off target activity may be other than cleavage. For each possible gRNA choice e.g., using S. pyogenes Cas9, the tool can identify all off-target sequences (e.g., preceding either NAG or NGG PAMs) across the genome that contain up to certain number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of mismatched base-pairs. The cleavage efficiency at each off-target sequence can be predicted, e.g., using an experimentally-derived weighting scheme. Each possible gRNA is then ranked according to its total predicted off-target cleavage; the top-ranked gRNAs represent those that are likely to have the greatest on-target and the least off-target cleavage. Other functions, e.g., automated reagent design for CRISPR construction, primer design for the on-target Surveyor assay, and primer design for high-throughput detection and quantification of off-target cleavage via next-gen sequencing, can also be included in the tool. Candidate gRNA molecules can be evaluated by art-known methods or as described in Section IV herein.


II. Cas9 Molecules


Cas9 molecules of a variety of species can be used in the methods and compositions described herein. While the S. pyogenes and S. thermophilus Cas9 molecules are the subject of much of the disclosure herein, Cas9 molecules of, derived from, or based on the Cas9 proteins of other species listed herein can be used as well. In other words, while the much of the description herein uses S. pyogenes and S. thermophilus Cas9 molecules, Cas9 molecules from the other species can replace them, e.g., Staphylococcus aureus and Neisseria meningitidis Cas9 molecules. Additional Cas9 species include: Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp., cycliphilus denitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroides sp., Blastopirellula marina, Bradyrhizobium sp., Brevibacillus laterosporus, Campylobacter coli, Campylobacter jejuni, Campylobacter Zari, Candidatus Puniceispirillum, Clostridium cellulolyticum, Clostridium pefringens, Corynebacterium accolens, Corynebacterium diphtheria, Corynebacterium matruchotii, Dinoroseobacter shibae, Eubacterium dolichum, gamma proteobacterium, Gluconacetobacter diazoirophicus, Haemophilus parainfluenzae, Haemophilus sputorum, Helicobacter canadensis, Helicobacter cinaedi, Helicobacter mustelae, Ilyobacter polvtropus, Kingella kingae, Lactobacillus crispatus, Listeria ivanovii, Listeria monocytogenes, Listeriaceae bacterium, Methylocystis sp., Methylosinus trichosporium, Mobiluncus mulieris, Neisseria bacilliformis, Neisseria cinerea, Neisseriaflavescens, Neisseria lactamica, Neisseria sp., Neisseria wadsworthii, Nitrosomonas sp., Parvibaculum lavamentivorans, Pasteurella multocida, Phascolarctobacterium succinatutens, Ralstonia syzygii, Rhodopseudomonaspalustris, Rhodovulum sp., Simonsiella muelleri, Sphingomnonas sp., Sporolactobacillus vineae, Staphylococcus lugdunensis, Streplococcus sp., Subdoligranulum sp., Tistrella mobilis, Treponema sp., or Verminephrobacter eisenmae.


A Cas9 molecule, as that term is used herein, refers to a molecule that can interact with a gRNA molecule and, in concert with the gRNA molecule, localize (e.g., target or home) to a site which comprises a target domain and PAM sequence.


In an embodiment, the Cas9 molecule is capable of cleaving a target nucleic acid molecule. A Cas9 molecule that is capable of cleaving a target nucleic acid molecule is referred to herein as an eaCas9 (an enzymatically active Cas9) molecule. In an embodiment, an eaCas9 molecule, comprises one or more of the following activities:


a nickase activity, i.e., the ability to cleave a single strand, e.g., the non-complementary strand or the complementary strand, of a nucleic acid molecule;


a double stranded nuclease activity, i.e., the ability to cleave both strands of a double stranded nucleic acid and create a double stranded break, which in an embodiment is the presence of two nickase activities:


an endonuclease activity;


an exonuclease activity; and


a helicase activity, i.e., the ability to unwind the helical structure of a double stranded nucleic acid.


In an embodiment, an enzymatically active Cas9 or an eaCas9 molecule cleaves both DNA strands and results in a double stranded break. In an embodiment, an eaCas9 molecule cleaves only one strand, e.g., the strand to which the gRNA hybridizes to, or the strand complementary to the strand the gRNA hybridizes with. In an embodiment, an eaCas9 molecule comprises cleavage activity associated with an HNH-like domain. In an embodiment, an eaCas9 molecule comprises cleavage activity associated with an N-terminal RuvC-like domain. In an embodiment, an eaCas9 molecule comprises cleavage activity associated with an HNH-like domain and cleavage activity associated with an N-terminal RuvC-like domain. In an embodiment, an eaCas9 molecule comprises an active, or cleavage competent, HNH-like domain and an inactive, or cleavage incompetent, N-terminal RuvC-like domain. In an embodiment, an eaCas9 molecule comprises an inactive, or cleavage incompetent, HNH-like domain and an active, or cleavage competent, N-terminal RuvC-like domain.


In an embodiment, the ability of an eaCas9 molecule to interact with and cleave a target nucleic acid is PAM sequence dependent. A PAM sequence is a sequence in the target nucleic acid. In an embodiment, cleavage of the target nucleic acid occurs upstream from the PAM sequence. EaCas9 molecules from different bacterial species can recognize different sequence motifs (e.g., PAM sequences). In an embodiment, an eaCas9 molecule of S. pyogenes recognizes the sequence motif NGG and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. See, e.g., Mali et al., SCIENCE 2013; 339(6121): 823-826. In an embodiment, an eaCas9 molecule of S. thermophilus recognizes the sequence motif NGGNG and NNAGAAW (W=A or T) and directs cleavage of a core target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from these sequences. See, e.g., Horvath et al., SCIENCE 2010; 327(5962): 167-170, and Deveau et al., J BACTERIOL 2008; 190(4): 1390-1400. In an embodiment, an eaCas9 molecule of S. Imutans recognizes the sequence motif NGG or NA AR (R=A or G) and directs cleavage of a core target nucleic acid sequence 1 to 10, e.g., 3 to 5 base pairs, upstream from this sequence. See, e.g., Deveau et al., J BACTERIOL 2008; 190(4): 1390-1400. In an embodiment, an eaCas9 molecule of S. aureus recognizes the sequence motif NNGRR (R=A or G) and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. In an embodiment, an eaCas9 molecule of N. meningitidis recognizes the sequence motif NNNNGATT and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. See, e.g., Hou et al., PNAS EARLY EDITION 2013, 1-6. The ability of a Cas9 molecule to recognize a PAM sequence can be determined, e.g., using a transformation assay described in Jinek et al., SCIENCE 2012, 337:816.


Some Cas9 molecules have the ability to interact with a gRNA molecule, and in conjunction with the gRNA molecule home (e.g., targeted or localized) to a core target domain, but are incapable of cleaving the target nucleic acid, or incapable of cleaving at efficient rates. Cas9 molecules having no, or no substantial, cleavage activity are referred to herein as an eiCas9 (an enzymatically inactive Cas9) molecule. For example, an eiCas9 molecule can lack cleavage activity or have substantially less, e.g., less than 20, 10, 5, 1 or 0.1% of the cleavage activity of a reference Cas9 molecule, as measured by an assay described herein.


Exemplary naturally occurring Cas9 molecules are described in Chylinski et al., RNA Biology 2013; 10:5, 727-737. Such Cas9 molecules include Cas9 molecules of a cluster 1 bacterial family, cluster 2 bacterial family, cluster 3 bacterial family, cluster 4 bacterial family, cluster 5 bacterial family, cluster 6 bacterial family, a cluster 7 bacterial family, a cluster 8 bacterial family, a cluster 9 bacterial family, a cluster 10 bacterial family, a cluster 11 bacterial family, a cluster 12 bacterial family, a cluster 13 bacterial family, a cluster 14 bacterial family, a cluster 15 bacterial family, a cluster 16 bacterial family, a cluster 17 bacterial family, a cluster 18 bacterial family, a cluster 19 bacterial family, a cluster 20 bacterial family, a cluster 21 bacterial family, a cluster 22 bacterial family, a cluster 23 bacterial family, a cluster 24 bacterial family, a cluster 25 bacterial family, a cluster 26 bacterial family, a cluster 27 bacterial family, a cluster 28 bacterial family, a cluster 29 bacterial family, a cluster 30 bacterial family, a cluster 31 bacterial family, a cluster 32 bacterial family, a cluster 33 bacterial family, a cluster 34 bacterial family, a cluster 35 bacterial family, a cluster 36 bacterial family, a cluster 37 bacterial family, a cluster 38 bacterial family, a cluster 39 bacterial family, a cluster 40 bacterial family, a cluster 41 bacterial family, a cluster 42 bacterial family, a cluster 43 bacterial family, a cluster 44 bacterial family, a cluster 45 bacterial family, a cluster 46 bacterial family, a cluster 47 bacterial family, a cluster 48 bacterial family, a cluster 49 bacterial family, a cluster 50 bacterial family, a cluster 51 bacterial family, a cluster 52 bacterial family, a cluster 53 bacterial family, a cluster 54 bacterial family, a cluster 55 bacterial family, a cluster 56 bacterial family, a cluster 57 bacterial family, a cluster 58 bacterial family, a cluster 59 bacterial family, a cluster 60 bacterial family, a cluster 61 bacterial family, a cluster 62 bacterial family, a cluster 63 bacterial family, a cluster 64 bacterial family, a cluster 65 bacterial family, a cluster 66 bacterial family, a cluster 67 bacterial family, a cluster 68 bacterial family, a cluster 69 bacterial family, a cluster 70 bacterial family, a cluster 71 bacterial family, a cluster 72 bacterial family, a cluster 73 bacterial family, a cluster 74 bacterial family, a cluster 75 bacterial family, a cluster 76 bacterial family, a cluster 77 bacterial family, or a cluster 78 bacterial family.


Exemplary naturally occurring Cas9 molecules include a Cas9 molecule of a cluster 1 bacterial family. Examples include a Cas9 molecule of: S. pyogenes (e.g., strain SF370, MGAS10270, MGAS10750, MGAS2096, MGAS315, MGAS5005, MGAS6180, MGAS9429, NZ131 and SSI-1), S. thermophilus (e.g., strain LMD-9), S. pseudoporcinus (e.g., strain SPIN 20026), S. mutans (e.g., strain UA159, NN2025), S. macacae (e.g., strain NCTC11558), S. gallolyticus (e.g., strain UCN34, ATCC BAA-2069), S. equines (e.g., strain ATCC 9812, MGCS 124), S. dysdalactiae (e.g., strain GGS 124), S. bovis (e.g., strain ATCC 700338), S. anginosus (e.g., strain F0211), S. agalactiae (e.g., strain NEM316, A909), Listeria monocytogenes (e.g., strain F6854), Listeria innocua (L. innocua, e.g., strain Clip11262), Enterococcus italicus (e.g., strain DSM 15952), or Enterococcus faecium (e.g., strain 1,231,408). Additional exemplary Cas9 molecules are a Cas9 molecule of Neisseria meningitidis (Hou et al. PNAS Early Edition 2013, 1-6) and a S. aureus Cas9 molecule.


In an embodiment, a Cas9 molecule, e.g., an eaCas9 molecule or eiCas9 molecule, comprises an amino acid sequence:


having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with;


differs at no more than, 2, 5, 10, 15, 20, 30, or 40% of the amino acid residues when compared with;


differs by at least 1, 2, 5, 10 or 20 amino acids but by no more than 100, 80, 70, 60, 50, 40 or 30 amino acids from: or


is identical to:


any Cas9 molecule sequence described herein or a naturally occurring Cas9 molecule sequence, e.g., a Cas9 molecule from a species listed herein or described in Chylinski et al., RNA Biology 2013, 10:5, 727-737; Hou et al. PNAS Early Edition 2013, 1-6. In an embodiment, the Cas9 molecule comprises one or more of the following activities: a nickase activity; a double stranded cleavage activity (e.g., an endonuclease and/or exonuclease activity); a helicase activity; or the ability, together with a gRNA molecule, to localize to a target nucleic acid.


In an embodiment, a Cas9 molecule comprises the amino acid sequence of the consensus sequence of FIG. 2, wherein “*” indicates any amino acid found in the corresponding position in the amino acid sequence of a Cas9 molecule of S. pyogenes, S. thermophilus, S. mutans and L. innocua, and “-” indicates any amino acid. In an embodiment, a Cas9 molecule differs from the sequence of the consensus sequence disclosed in FIG. 2 by at least 1, but no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues. In an embodiment, a Cas9 molecule comprises the amino acid sequence of SEQ ID NO:7 of FIG. 5, wherein “*” indicates any amino acid found in the corresponding position in the amino acid sequence of a Cas9 molecule of S. pyogenes, or N. meningitidis. “−” indicates any amino acid, and “−” indicates any amino acid or absent. In an embodiment, a Cas9 molecule differs from the sequence of SEQ ID NO:6 or 7 by at least 1, but no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues.


A comparison of the sequence of a number of Cas9 molecules indicate that certain regions are conserved. These are identified below as:


region 1 (residues 1 to 180, or in the case of region 1′ residues 120 to 180)


region 2 (residues 360 to 480):


region 3 (residues 660 to 720):


region 4 (residues 817 to 900); and


region 5 (residues 900 to 960).


In an embodiment, a Cas9 molecule comprises regions 1-5, together with sufficient additional Cas9 molecule sequence to provide a biologically active molecule, e.g., a Cas9 molecule having at least one activity described herein. In an embodiment, each of regions 1-6, independently, have, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with the corresponding residues of a Cas9 molecule described herein, e.g., a sequence from FIG. 2 or from FIG. 5.


In an embodiment, a Cas9 molecule, e.g., an eaCas9 molecule or eiCas9 molecule, comprises an amino acid sequence referred to as region 1:


having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with amino acids 1-180 (the numbering is according to the motif sequence in FIG. 2; 52% of residues in the four Cas9 sequences in FIG. 2 are conserved) of the amino acid sequence of Cas9 of S. pyogenes;


differs by at least 1, 2, 5, 10 or 20 amino acids but by no more than 90, 80, 70, 60, 50, 40 or 30 amino acids from amino acids 1-180 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans. L. innocua, N. meningitidis, or S. aureus; or


is identical to 1-180 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans, L. innocua, N. meningitidis, or S. aureus.


In an embodiment, a Cas9 molecule, e.g., an eaCas9 molecule or eiCas9 molecule, comprises an amino acid sequence referred to as region 1′:


having 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with amino acids 120-180 (55% of residues in the four Cas9 sequences in FIG. 2 are conserved) of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or, L. innocua. N. meningitidis, or S. aureus;


differs by at least 1, 2, or 5 amino acids but by no more than 35, 30, 25, 20 or 10 amino acids from amino acids 120-180 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus; or


is identical to 120-180 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus. S. mutans or, L. innocua. N. meningitidis, or S. aureus.


In an embodiment, a Cas9 molecule, e.g., an eaCas9 molecule or eiCas9 molecule, comprises an amino acid sequence referred to as region 2:


having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with amino acids 360-480 (52% of residues in the four Cas9 sequences in FIG. 2 are conserved) of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus;


differs by at least 1, 2, or 5 amino acids but by no more than 35, 30, 25, 20 or 10 amino acids from amino acids 360-480 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus; or is identical to 360-480 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus. S. mutans or, L. innocua, N. meningitidis, or S. aureus.


In an embodiment, a Cas9 molecule, e.g., an eaCas9 molecule or eiCas9 molecule, comprises an amino acid sequence referred to as region 3:


having 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with amino acids 660-720 (56% of residues in the four Cas9 sequences in FIG. 2 are conserved) of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus;


differs by at least 1, 2, or 5 amino acids but by no more than 35, 30, 25, 20 or 10 amino acids from amino acids 660-720 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus. S. mutans or, L. innocua. N. meningitidis, or S. aureus; or


is identical to 660-720 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus.


In an embodiment, a Cas9 molecule. e.g., an eaCas9 molecule or eiCas9 molecule, comprises an amino acid sequence referred to as region 4:


having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with amino acids 817-900 (55% of residues in the four Cas9 sequences in FIG. 2 are conserved) of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus;


differs by at least 1, 2, or 5 amino acids but by no more than 35, 30, 25, 20 or 10 amino acids from amino acids 817-900 of the amino acid sequence of Cas9 of S. pyogenes. S. thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus; or


is identical to 817-900 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus.


In an embodiment, a Cas9 molecule. e.g., an eaCas9 molecule or eiCas9 molecule, comprises an amino acid sequence referred to as region 5:


having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with amino acids 900-960 (60% of residues in the four Cas9 sequences in FIG. 2 are conserved) of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus;


differs by at least 1, 2, or 5 amino acids but by no more than 35, 30, 25, 20 or 10 amino acids from amino acids 900-960 of the amino acid sequence of Cas9 of S. pyogenes. S. thermophilus. S. mutans or, L. innocua. N. meningitidis, or S. aureus; or


is identical to 900-960 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus.


A RuvC-Like Domain and an HNH-Like Domain


In an embodiment, a Cas9 molecule comprises an HNH-like domain and an RuvC-like domain. In an embodiment, cleavage activity is dependent on a RuvC-like domain and an HNH-like domain. A Cas9 molecule, e.g., an eaCas9 or eiCas9 molecule, can comprise one or more of the following domains: a RuvC-like domain and an HNH-like domain. In an embodiment, a cas9 molecule is an eaCas9 molecule and the eaCas9 molecule comprises a RuvC-like domain, e.g., a RuvC-like domain described below, and/or an HNH-like domain, e.g., an HNH-like domain described below. In an embodiment, a Cas9 molecule is an eiCas9 molecule comprising one or more difference in an RuvC-like domain and/or in an HNH-like domain as compared to a reference Cas9 molecule, and the eiCas9 molecule does not cleave a nucleic acid, or cleaves with significantly less efficiency than does wildtype. e.g., when compared with wild type in a cleavage assay, e.g., as described herein, cuts with less than 50, 25, 10, or 1% of the a reference Cas9 molecule, as measured by an assay described herein.


RuvC-Like Domains


In an embodiment, a RuvC-like domain cleaves, a single strand, e.g., the non-complementary strand of the target nucleic acid molecule. A Cas9 molecule can include more than one RuvC-like domain (e.g., one, two, three or more RuvC-like domains). In an embodiment, an RuvC-like domain is at least 5, 6, 7, 8 amino acids in length but not more than 20, 19, 18, 17, 16 or 15 amino acids in length. In an embodiment, the cas9 molecule comprises an N-terminal RuvC-like domain of about 10 to 20 amino acids, e.g., about 15 amino acids in length.


N-Terminal RuvC-Like Domains


Some naturally occurring Cas9 molecules comprise more than one RuvC-like domain, with cleavage being dependent on the N-terminal RuvC-like domain. Accordingly, Cas9 molecules can comprise an N-terminal RuvC-like domain. Exemplary N-terminal RuvC-like domains are described below.


In an embodiment, an eaCas9 molecule comprises an N-terminal RuvC-like domain comprising an amino acid sequence of formula I:










D-X1-G-X2-X3-X4-X5-G-X6-X7-X8-X9,
(SEQ ID NO: 8)






wherein,


X1 is selected from I, V, M, L and T (e.g., selected from I, V, and L);


X2 is selected from T, I, V, S, N, Y, E and L (e.g., selected from T, V, and I);


X3 is selected from N, S, G, A, D, T, R, M and F (e.g., A or N);


X4 is selected from S, Y, N and F (e.g., S);


X5 is selected from V, I, L, C, T and F (e.g., selected from V, I and L);


X6 is selected from W, F, V, Y, S and L (e.g., W);


X7 is selected from A, S, C, V and G (e.g., selected from A and S);


X8 is selected from V, I, L, A, M and H (e.g., selected from V, I, M and L); and


X9 is selected from any amino acid or is absent (e.g., selected from T, V, I, L, ‘F, S, A, Y, M and R, or, e.g., selected from T, V, I, L and).


In an embodiment, the N-terminal RuvC-like domain differs from a sequence of SEQ ID N0:8, by as many as 1 but no more than 2, 3, 4, or 5 residues.


In embodiment the N-terminal RuvC-like domain is cleavage competent.


In embodiment the N-terminal RuvC-like domain is cleavage incompetent.


In an embodiment, an eaCas9 molecule comprises an N-terminal RuvC-like domain comprising an amino acid sequence of formula II:










D-X1-G-X2-X3-S-X5-G-X6-X7-X8-X9,
(SEQ ID NO: 9)






wherein


X1 is selected from 1, V, M, L and T (e.g., selected from I, V, and L);


X2 is selected from T, I, V, S, N, Y, E and L (e.g., selected from T, V, and I);


X3 is selected from N, S, G, A, D, T, R, M and F (e.g., A or N);


X5 is selected from V, I, L, C, T and F (e.g., selected from V, I and L);


X6 is selected from W, F, V, Y, S and L (e.g., W);


X7 is selected from A, S, C, V and G (e.g., selected from A and S);


X8 is selected from V, I, L, A, M and H (e.g., selected from V, I, M and L); and


X9 is selected from any amino acid or is absent (e.g., selected from T, V, I, L, ‘F, S, A, Y, M and R or selected from e.g., T, V, I, L and).


In an embodiment, the N-terminal RuvC-like domain differs from a sequence of SEQ ID NO:9 by as many as 1, but no more than 2, 3, 4, or 5 residues.


In an embodiment, the N-terminal RuvC-like domain comprises an amino acid sequence of formula III:










D-I-G-X2-X3-S-V-G-W-A-X8-X9,
(SEQ ID NO: 10)






wherein


X2 is selected from T, I, V, S, N, Y, E and L (e.g., selected from T, V, and I);


X3 is selected from N, S, G, A, D, T, R, M and F (e.g., A or N);


X8 is selected from V, I, L, A, M and H (e.g., selected from V, I, M and L); and


X9 is selected from any amino acid or is absent (e.g., selected from T, V, I, L, ‘F, S, A, Y, M and R or selected from e.g., T, V, I, L and).


In an embodiment, the N-terminal RuvC-like domain differs from a sequence of SEQ ID NO: 10 by as many as 1, but no more than, 2, 3, 4, or 5 residues.


In an embodiment, the N-terminal RuvC-like domain comprises an amino acid sequence of formula III:












D-I-G-T-N-S-V-G-W-A-V-X,
(SEQ ID NO: 11)






wherein


X is a non-polar alkyl amino acid or a hydroxyl amino acid, e.g., X is selected from V, I, L and T (e.g., the eaCas9 molecule can comprise an N-terminal RuvC-like domain shown in FIG. 2 (depicted as “Y”)).


In an embodiment, the N-terminal RuvC-like domain differs from a sequence of SEQ ID NO: 11 by as many as 1 but no more than, 2, 3, 4, or 5 residues.


In an embodiment, the N-terminal RuvC-like domain differs from a sequence of an N-terminal RuvC-like domain disclosed herein, e.g., in FIG. 3A or FIG. 5, as many as 1, but no more than 2, 3, 4, or 5 residues. In an embodiment, 1, 2, or all 3 of the highly conserved residues identified in FIG. 3A or FIG. 5 are present.


In an embodiment, the N-terminal RuvC-like domain differs from a sequence of an N-terminal RuvC-like domain disclosed herein, e.g., in FIG. 3B, as many as 1, but no more than 2, 3, 4, or 5 residues. In an embodiment, 1, 2, 3 or all 4 of the highly conserved residues identified in FIG. 3B are present.


Additional RuvC-Like Domains


In addition to the N-terminal RuvC-like domain, a Cas9 molecule, e.g., an eaCas9 molecule, can comprise one or more additional RuvC-like domains. In an embodiment, a Cas9 molecule can comprise two additional RuvC-like domains. Preferably, the additional RuvC-like domain is at least 5 amino acids in length and, e.g., less than 15 amino acids in length, e.g., 5 to 10 amino acids in length, e.g., 8 amino acids in length.


An additional RuvC-like domain can comprise an amino acid sequence:












I-X1-X2-E-X3-A-R-E,
(SEQ ID NO: 12)







wherein


X1 is V or H,


X2 is I, L or V (e.g., I or V); and


X3 is M or T.


In an embodiment, the additional RuvC-like domain comprises the amino acid sequence:












I-V-X2-E-M-A-R-E,
(SEQ ID NO: 13)







wherein


X2 is I, L or V (e.g., I or V) (e.g., the eaCas9 molecule can comprise an additional RuvC-like domain shown in FIG. 2 or FIG. 5 (depicted as “B”)).


An additional RuvC-like domain can comprise an amino acid sequence:












H-H-A-X1-D-A-X2-X3,
(SEQ ID NO: 14)







wherein


X1 is H or L;


X2 is R or V; and


X3 is E or V.


In an embodiment, the additional RuvC-like domain comprises the amino acid sequence:












H-H-A-H-D-A-Y-L.
(SEQ ID NO: 15)






In an embodiment, the additional RuvC-like domain differs from a sequence of SEQ ID NO:13, 15, 12 or 14 by as many as 1, but no more than 2, 3, 4, or 5 residues.


In an embodiment, the sequence flanking the N-terminal RuvC-like domain is a sequences of formula V:










K-X1′-Y-X2′-X3′-X4′-Z-T-D-X9′-Y,
(SEQ ID NO: 16)






wherein


X1′ is selected from K and P,


X2′ is selected from V, L, I, and F (e.g., V, I and L);


X3′ is selected from G, A and S (e.g., G),


X4′ is selected from L, I, V and F (e.g., L);


X9′ is selected from D, E. N and Q; and


Z is an N-terminal RuvC-like domain, e.g., as described above.


HNH-Like Domains


In an embodiment, an HNH-like domain cleaves a single stranded complementary domain, e.g., a complementary strand of a double stranded nucleic acid molecule. In an embodiment, an HNH-like domain is at least 15, 20, 25 amino acids in length but not more than 40, 35 or 30 amino acids in length, e.g., 20 to 35 amino acids in length, e.g., 25 to 30 amino acids in length. Exemplary HNH-like domains are described below.


In an embodiment, an eaCas9 molecule comprises an HNH-like domain having an amino acid sequence of formula VI:









(SEQ ID NO: 17)


X1-X2-X3-H-X4-X5-P-X6-X-7-X8-X9-X10-X11-X12-X13-





X14-X15-N-X16-X17-X18-X19-X20-X21-X22-X23-N,







wherein


X1 is selected from D, E, Q and N (e.g., D and E);


X2 is selected from L, I, R, Q, V, M and K;


X3 is selected from D and E;


X4 is selected from I, V, T, A and L (e.g., A, I and V);


X5 is selected from V, Y, I, L, F and W (e.g., V, I and L);


X6 is selected from Q, H, R, K, Y, I, L, F and W;


X7 is selected from S, A, D, T and K (e.g., S and A);


X8 is selected from F, L, V, K, Y, M, I, R, A, E, D and Q (e.g., F);


X9 is selected from L, R, T, I, V, S, C, Y, K, F and G;


X10 is selected from K, Q, Y, T, F, L, W, M, A, E, G, and S;


X11 is selected from D, S, N, R, L and T (e.g., D):


X12 is selected from D. N and S;


X13 is selected from S, A. T. G and R (e.g., S);


X14 is selected from I, L, F, S, R, Y, Q, W, D, K and H (e.g., I, L and F);


X15 is selected from D, S, I, N, E, A, H, F, L, Q, M, G, Y and V;


X16 is selected from K, L, R, M, T and F (e.g., L, R and K):


X17 is selected from V, L, I, A and T:


X18 is selected from L, I, V and A (e.g., L and I);


X19 is selected from T, V, C, E, S and A (e.g., T and V):


X20 is selected from R, F, T, W, E, L, N, C, K, V, S, Q, I, Y, H and A;


X21 is selected from S, P, R, K, N, A, H, Q, G and L;


X22 is selected from D, G, T, N, S, K, A, I, E, L, Q, R and Y; and


X23 is selected from K, V, A, E, Y, I, C, L, S, T, G, K, M, D and F.


In an embodiment, a HNH-like domain differs from a sequence of SEQ ID NO: 17 by at least 1, but no more than, 2, 3, 4, or 5 residues.


In an embodiment, the HNH-like domain is cleavage competent.


In an embodiment, the HNH-like domain is cleavage incompetent.


In an embodiment, an eaCas9 molecule comprises an HNH-like domain comprising an amino acid sequence of formula VII:









(SEQ ID NO: 18)


X1-X2-X3-H-X4-X5-P-X6-S-X8-X9-X10-D-D-S-X14-X15-





N-K-V-L-X19-X20-X21-X22-X23-N,






wherein


X1 is selected from D and E;


X2 is selected from L, I, R, Q, V, M and K;


X3 is selected from D and E;


X4 is selected from I, V. T. A and L (e.g., A, I and V);


X5 is selected from V, Y, I, L, F and W (e.g., V. I and L);


X6 is selected from Q, H, R, K, Y, I, L, F and W:


X8 is selected from F, L, V, K, Y, M, I, R, A, E, D and Q (e.g., F);


X9 is selected from L, R, T, I, V, S, C, Y, K, F and G;


X10 is selected from K, Q, Y, T, F, L, W, M, A, E, G, and S;


X14 is selected from I, L, F, S, R, Y, Q, W, D, K and H (e.g., I, L and F);


X15 is selected from D, S, I, N, E, A, H, F, L, Q, M, G, Y and V;


X19 is selected from T, V, C, E, S and A (e.g., T and V);


X20 is selected from R, F, T, W, E, L, N, C, K, V, S, Q, I, Y, H and A;


X21 is selected from S, P, R, K, N, A, H, Q, G and L:


X22 is selected from D, G, T, N, S, K, A, I, E, L, Q, R and Y; and


X23 is selected from K, V, A, E, Y, I, C, L, S, T, G, K, M, D and F.


In an embodiment, the HNH-like domain differs from a sequence of SEQ ID NO: 18 by 1, 2, 3, 4, or 5 residues.


In an embodiment, an eaCas9 molecule comprises an HNH-like domain comprising an amino acid sequence of formula VII:









(SEQ ID NO: 19)


X1-V-X3-H-I-V-P-X6-S-X8-X9-X10-D-D-S-X14-X15-N-K-





V-L-T-X20-X21-X22-X23-N,






wherein


X1 is selected from D and E:


X3 is selected from D and E:


X6 is selected from Q, H, R, K, Y, I, L and W;


X8 is selected from F, L, V, K, Y, M, I, R, A, E, D and Q (e.g., F);


X9 is selected from L, R, T, I, V, S, C, Y, K, F and G:


X10 is selected from K, Q, Y, T, F, L, W, M, A, E, G, and S;


X14 is selected from I, L, F, S, R, Y, Q, W, D, K and H (e.g., I, L and F):


X15 is selected from D, S, I, N, E, A, H, F, L, Q, M, G, Y and V;


X20 is selected from R, F, T, W, E, L, N, C, K, V, S, Q, I, Y, H and A:


X21 is selected from S, P, R, K, N, A, H, Q, G and L:


X22 is selected from D, G, T, N, S, K, A, I, E, L, Q, R and Y; and


X23 is selected from K, V, A, E, Y, I, C, L, S, T, G, K, M, D and F.


In an embodiment, the HNH-like domain differs from a sequence of SEQ ID NO: 19 by 1, 2, 3, 4, or 5 residues.


In an embodiment, an eaCas9 molecule comprises an HNH-like domain having an amino acid sequence of formula VIII:









(SEQ ID NO: 20)


D-X2-D-H-I-X5-P-Q-X7-F-X9-X10-D-X12-S-I-D-N-X16-V-





L-X19-X20-S-X22-X23-N,






wherein


X2 is selected from I and V:


X5 is selected from 1 and V;


X7 is selected from A and S;


X9 is selected from I and L;


X10 is selected from K and T;


X12 is selected from D and N;


X16 is selected from R, K and L; X19 is selected from T and V:


X20 is selected from S and R;


X22 is selected from K, D and A; and


X23 is selected from E, K, G and N (e.g., the eaCas9 molecule can comprise an HNH-like domain as described herein).


In an embodiment, the HNH-like domain differs from a sequence of SEQ ID NO:20 by as many as 1, but no more than 2, 3, 4, or 5 residues.


In an embodiment, an eaCas9 molecule comprises the amino acid sequence of formula IX:









(SEQ ID NO: 21)


L-Y-Y-L-Q-N-G-X1′-D-M-Y-X2′-X3′-X4′-X5′-L-D-I-X6′-





X7′-L-S-X8′-Y-Z-N-R-X9′-K-X10′-D-X11′-V-P,






wherein


X1′ is selected from K and R;


X2′ is selected from V and T;


X3′ is selected from G and D;


X4′ is selected from E, Q and D;


X5′ is selected from E and D;


X6′ is selected from D. N and H;


X7′ is selected from Y, R and N;


X8′ is selected from Q, D and N; X9′ is selected from G and E;


X10′ is selected from Sand G;


X11′ is selected from D and N; and


Z is an HNH-like domain, e.g., as described above.


In an embodiment, the eaCas9 molecule comprises an amino acid sequence that differs from a sequence of SEQ ID NO:21 by as many as 1, but no more than 2, 3, 4, or 5 residues.


In an embodiment, the HNH-like domain differs from a sequence of an HNH-like domain disclosed herein, e.g., in FIG. 4A or FIG. 5, as many as 1, but no more than 2, 3, 4, or 5 residues.


In an embodiment, the HNH-like domain differs from a sequence of an HNH-like domain disclosed herein. e.g., in FIG. 4B, by as many as 1, but no more than 2, 3, 4, or 5 residues. In an embodiment, 1, 2, all 3 of the highly conserved residues identified in FIG. 4B are present.


Altered Cas9 Molecules


Naturally occurring Cas9 molecules possess a number of properties, including: nickase activity, nuclease activity (e.g., endonuclease and/or exonuclease activity); helicase activity; the ability to associate functionally with a gRNA molecule; and the ability to target (or localize to) a site on a nucleic acid (e.g., PAM recognition and specificity). In an embodiment, a Cas9 molecules can include all or a subset of these properties. In a typical embodiment, Cas9 molecules have the ability to interact with a gRNA molecule and, in concert with the gRNA molecule, localize to a site in a nucleic acid. Other activities, e.g., PAM specificity, cleavage activity, or helicase activity can vary more widely in Cas9 molecules.


Cas9 molecules with desired properties can be made in a number of ways, e.g., by alteration of a parental, e.g., naturally occurring Cas9 molecules to provide an altered Cas9 molecule having a desired property. For example, one or more mutations or differences relative to a parental Cas9 molecule can be introduced. Such mutations and differences comprise: substitutions (e.g., conservative substitutions or substitutions of non-essential amino acids); insertions; or deletions. In an embodiment, a Cas9 molecule can comprises one or more mutations or differences, e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40 or 50 mutations but less than 200, 100, or 80 mutations relative to a reference Cas9 molecule.


In an embodiment, a mutation or mutations do not have a substantial effect on a Cas9 activity, e.g. a Cas9 activity described herein. In an embodiment, a mutation or mutations have a substantial effect on a Cas9 activity. e.g. a Cas9 activity described herein. In an embodiment, exemplary activities comprise one or more of PAM specificity, cleavage activity, and helicase activity. A mutation(s) can be present, e.g., in: one or more RuvC-like domain, e.g., an N-terminal RuvC-like domain; an HNH-like domain; a region outside the RuvC-like domains and the HNH-like domain. In an embodiment, a mutation(s) is present in an N-terminal RuvC-like domain. In an embodiment, a mutation(s) is present in an HNH-like domain. In an embodiment, mutations are present in both an N-terminal RuvC-like domain and an HNH-like domain.


Whether or not a particular sequence, e.g., a substitution, may affect one or more activity, such as targeting activity, cleavage activity, etc, can be evaluated or predicted, e.g., by evaluating whether the mutation is conservative or by the method described in Section III. In an embodiment, a “non-essential” amino acid residue, as used in the context of a Cas9 molecule, is a residue that can be altered from the wild-type sequence of a Cas9 molecule, e.g., a naturally occurring Cas9 molecule, e.g., an eaCas9 molecule, without abolishing or more preferably, without substantially altering a Cas9 activity (e.g., cleavage activity), whereas changing an “essential” amino acid residue results in a substantial loss of activity (e.g., cleavage activity).


In an embodiment, the altered Cas9 molecule is an eaCas9 molecule comprising the fixed amino acid residues of S. pyogenes shown in the consensus sequence disclosed in FIG. 2, and has one or more amino acids that differ from the amino acid sequence of S. pyogenes (e.g., has a substitution) at one or more residue (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) represented by an“−” in the consensus sequence disclosed in FIG. 2 or SEQ ID N0:7. In an embodiment, the altered Cas9 molecule is an eiCas9 molecule wherein one or more of the fixed amino acid residues of S. pyogenes shown in the consensus sequence disclosed in FIG. 2 (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100.200 amino acid residues) is mutated.


In an embodiment, the altered Cas9 molecule comprises a sequence in which:


the sequence corresponding to the fixed sequence of the consensus sequence disclosed in FIG. 2 differs at no more than 1, 2, 3, 4, 5, 10, 15, or 20% of the fixed residues in the consensus sequence disclosed in FIG. 2;


the sequence corresponding to the residues identified by “*” in the consensus sequence disclosed in FIG. 2 differ at no more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40% of the “*” residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. pyogenes Cas9 molecule; and,


the sequence corresponding to the residues identified by “-” in the consensus sequence disclosed in FIG. 2 differ at no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 55, or 60% of the “−” residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. pyogenes Cas9 molecule.


In an embodiment, the altered Cas9 molecule is an eaCas9 molecule comprising the fixed amino acid residues of S. thermophilus shown in the consensus sequence disclosed in FIG. 2, and has one or more amino acids that differ from the amino acid sequence of S. thermophilus (e.g., has a substitution) at one or more residue (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) represented by an“−” in the consensus sequence disclosed in FIG. 2.


In an embodiment, the altered Cas9 molecule is an eiCas9 molecule wherein one or more of the fixed amino acid residues of S. thermophilus shown in the consensus sequence disclosed in FIG. 2 (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100.200 amino acid residues) is mutated.


In an embodiment the altered Cas9 molecule comprises a sequence in which:


the sequence corresponding to the fixed sequence of the consensus sequence disclosed in FIG. 2 differs at no more than 1, 2, 3, 4, 5, 10, 15, or 20% of the fixed residues in the consensus sequence disclosed in FIG. 2;


the sequence corresponding to the residues identified by “*” in the consensus sequence disclosed in FIG. 2 differ at no more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40% of the “*” residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. thermophilus Cas9 molecule; and,


the sequence corresponding to the residues identified by “-” in the consensus sequence disclosed in FIG. 2 differ at no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 55, or 60% of the “-” residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. thermophilus Cas9 molecule.


In an embodiment, the altered Cas9 molecule is an eaCas9 molecule comprising the fixed amino acid residues of S. mutans shown in the consensus sequence disclosed in FIG. 2, and has one or more amino acids that differ from the amino acid sequence of S. mutans (e.g., has a substitution) at one or more residue (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) represented by an“−” in the consensus sequence disclosed in FIG. 2. In an embodiment, the altered Cas9 molecule is an eiCas9 molecule wherein one or more of the fixed amino acid residues of S. mutans shown in the consensus sequence disclosed in FIG. 2 (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) is mutated.


In an embodiment the altered Cas9 molecule comprises a sequence in which:


the sequence corresponding to the fixed sequence of the consensus sequence disclosed in FIG. 2 differs at no more than 1, 2, 3, 4, 5, 10, 15, or 20% of the fixed residues in the consensus sequence disclosed in FIG. 2;


the sequence corresponding to the residues identified by “*” in the consensus sequence disclosed in FIG. 2 differ at no more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40% of the “*” residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. mutans Cas9 molecule; and,


the sequence corresponding to the residues identified by “-” in the consensus sequence disclosed in FIG. 2 differ at no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 55, or 60% of the “−” residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S, mutans Cas9 molecule.


In an embodiment, the altered Cas9 molecule is an eaCas9 molecule comprising the fixed amino acid residues of L. innocula shown in the consensus sequence disclosed in FIG. 2, and has one or more amino acids that differ from the amino acid sequence of L. innocula (e.g., has a substitution) at one or more residue (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) represented by an“−” in the consensus sequence disclosed in FIG. 2. In an embodiment, the altered Cas9 molecule is an eiCas9 molecule wherein one or more of the fixed amino acid residues of L. innocula shown in the consensus sequence disclosed in FIG. 2 (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) is mutated.


In an embodiment the altered Cas9 molecule comprises a sequence in which:


the sequence corresponding to the fixed sequence of the consensus sequence disclosed in FIG. 2 differs at no more than 1, 2, 3, 4, 5, 10, 15, or 20% of the fixed residues in the consensus sequence disclosed in FIG. 2;


the sequence corresponding to the residues identified by “*” in the consensus sequence disclosed in FIG. 2 differ at no more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40% of the “*” residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an L. innocula Cas9 molecule; and,


the sequence corresponding to the residues identified by “-” in the consensus sequence disclosed in FIG. 2 differ at no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 55, or 60% of the“−” residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an L. innocula Cas9 molecule.


In an embodiment, the altered Cas9 molecule, e.g., an eaCas9 molecule or an eiCas9 molecule, can be a fusion, e.g., of two of more different Cas9 molecules, e.g., of two or more naturally occurring Cas9 molecules of different species. For example, a fragment of a naturally occurring Cas9 molecule of one species can be fused to a fragment of a Cas9 molecule of a second species. As an example, a fragment of Cas9 of S. pyogenes comprising an N-terminal RuvC-like domain can be fused to a fragment of Cas9 of a species other than S. pyogenes (e.g., S. thermophilus) comprising an HNH-like domain.


Cas9 Molecules with altered PAM recognition or no PAM recognition


Naturally occurring Cas9 molecules can recognize specific PAM sequences, for example the PAM recognition sequences described above for S. pyogenes, S. thermophilus, S. mutans, S. aureus and N. meningitidis.


In an embodiment, a Cas9 molecule has the same PAM specificities as a naturally occurring Cas9 molecule. In an embodiment, a Cas9 molecule has a PAM specificity not associated with a naturally occurring Cas9 molecule, or a PAM specificity not associated with the naturally occurring Cas9 molecule to which it has the closest sequence homology. For example, a naturally occurring Cas9 molecule can be altered, e.g., to alter PAM recognition, e.g., to alter the PAM sequence that the Cas9 molecule recognizes to decrease off target sites and/or improve specificity; or eliminate a PAM recognition requirement. In an embodiment, a Cas9 molecule can be altered, e.g., to increase length of PAM recognition sequence and/or improve Cas9 specificity to high level of identity to decrease off target sites and increase specificity. In an embodiment, the length of the PAM recognition sequence is at least 4, 5, 6, 7, 8, 9, 10 or 15 amino acids in length. Cas9 molecules that recognize different PAM sequences and/or have reduced off-target activity can be generated using directed evolution. Exemplary methods and systems that can be used for directed evolution of Cas9 molecules are described, e.g., in Esvelt et al., NATURE 2011, 472(7344): 499-503. Candidate Cas9 molecules can be evaluated, e.g., by methods described in Section III.


Non-Cleaving and Modified-Cleavage Cas9 Molecules


In an embodiment, a Cas9 molecule comprises a cleavage property that differs from naturally occurring Cas9 molecules, e.g., that differs from the naturally occurring Cas9 molecule having the closest homology. For example, a Cas9 molecule can differ from naturally occurring Cas9 molecules, e.g., a Cas9 molecule of S. pyogenes, as follows: its ability to modulate, e.g., decreased or increased, cleavage of a double stranded break (endonuclease and/or exonuclease activity), e.g., as compared to a naturally occurring Cas9 molecule (e.g., a Cas9 molecule of S. pyogenes); its ability to modulate, e.g., decreased or increased, cleavage of a single strand of a nucleic acid, e.g., a non-complimentary strand of a nucleic acid molecule or a complementary strand of a nucleic acid molecule (nickase activity), e.g., as compared to a naturally occurring Cas9 molecule (e.g., a Cas9 molecule of S. pyogenes); or the ability to cleave a nucleic acid molecule, e.g., a double stranded or single stranded nucleic acid molecule, can be eliminated.


Modified Cleavage eaCas9 Molecules


In an embodiment, an eaCas9 molecule comprises one or more of the following activities; cleavage activity associated with an N-terminal RuvC-like domain; cleavage activity associated with an HNH-like domain; cleavage activity associated with an HNH domain and cleavage activity associated with an N-terminal RuvC-like domain.


In an embodiment an eaCas9 molecule comprises an active, or cleavage competent, HNH-like domain (e.g., an HNH-like domain described herein, e.g., SEQ ID NO:17, SEQ ID N0:18. SEQ ID NO:19, SEQ ID N0:20 or SEQ ID NO:21) and an inactive, or cleavage incompetent, N-terminal RuvC-like domain. An exemplary inactive, or cleavage incompetent N-terminal RuvC-like domain can have a mutation of an aspartic acid in an N-terminal RuvC-like domain, e.g., an aspartic acid at position 9 of the consensus sequence disclosed in FIG. 2 or an aspartic acid at position 10 of SEQ ID N0:7, e.g., can be substituted with an alanine. In an embodiment, the eaCas9 differs from wild type in the N-terminal RuvC-like domain and does not cleave the target nucleic acid, or cleaves with significantly less efficiency, e.g., less than 20, 10, 5, 1 or 0.1% of the cleavage activity of a reference Cas9 molecule, e.g., as measured by an assay described herein. The reference Cas9 molecule can by a naturally occurring unmodified Cas9 molecule, e.g., a naturally occurring Cas9 molecule such as a Cas9 molecule of S. pyogenes, or S. thermophilus. In an embodiment, the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology.


In an embodiment, an eaCas9 molecule comprises an inactive, or cleavage incompetent, HNH domain and an active, or cleavage competent, N-terminal RuvC-like domain (e.g., an HNH-like domain described herein, e.g., SEQ ID N0:8. SEQ ID N0:9, SEQ ID NO:10, SEQ ID N0:11, SEQ ID N0:12, SEQ ID N0:13, SEQ ID N0:14 or SEQ ID N0:15). Exemplary inactive, or cleavage incompetent HNH-like domains can have a mutation at one or more of: a histidine in an HNH-like domain, e.g., a histidine at position 856 of the consensus sequence disclosed in FIG. 2, e.g., can be substituted with an alanine; and one or more asparagines in an HNH-like domain, e.g., an asparagine at position 870 of the consensus sequence disclosed in FIG. 2 and/or at position 879 of the consensus sequence disclosed in FIG. 2, e.g., can be substituted with an alanine. In an embodiment, the eaCas9 differs from wild type in the HNH-like domain and does not cleave the target nucleic acid, or cleaves with significantly less efficiency, e.g., less than 20, 10, 5, 1 or 0.1% of the cleavage activity of a reference Cas9 molecule, e.g., as measured by an assay described herein. The reference Cas9 molecule can by a naturally occurring unmodified Cas9 molecule, e.g., a naturally occurring Cas9 molecule such as a Cas9 molecule of S. pyogenes, or S. thermophilus. In an embodiment, the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology.


Non-Cleaving eiCas9 Molecules


In an embodiment, the altered Cas9 molecule is an eiCas9 molecule which does not cleave a nucleic acid molecule (either double stranded or single stranded nucleic acid molecules) or cleaves a nucleic acid molecule with significantly less efficiency, e.g., less than 20, 10, 5, 1 or 0.1% of the cleavage activity of a reference Cas9 molecule, e.g., as measured by an assay described herein. The reference Cas9 molecule can by a naturally occurring unmodified Cas9 molecule, e.g., a naturally occurring Cas9 molecule such as a Cas9 molecule of S. pyogenes, S. thermophilus. S. aureus or N. meningitidis. In an embodiment, the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology. In an embodiment, the eiCas9 molecule lacks substantial cleavage activity associated with an N-terminal RuvC-like domain and cleavage activity associated with an HNH-like domain.


In an embodiment, an eiCas9 molecule comprises an inactive, or cleavage incompetent, N-terminal RuvC-like domain. An exemplary inactive, or cleavage incompetent N-terminal RuvC-like domain can have a mutation of an aspartic acid in an N-terminal RuvC-like domain, e.g., an aspartic acid at position 9 of the consensus sequence disclosed in FIG. 2 or an aspartic acid at position 10 of SEQ ID NO:7, e.g., can be substituted with an alanine.


In an embodiment an eiCas9 molecule comprises an inactive, or cleavage incompetent, HNH domain (e.g., an HNH-like domain described herein, e.g., SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 or SEQ ID NO: 15). Exemplary inactive, or cleavage incompetent HNH-like domains can have a mutation at one or more of: a histidine in an HNH-like domain, e.g., a histidine at position 856 of the consensus sequence disclosed in FIG. 2, e.g., can be substituted with an alanine; and one or more asparagines in an HNH-like domain, e.g., an asparagine at position 870 of the consensus sequence disclosed in FIG. 2 and/or at position 879 of the consensus sequence disclosed in FIG. 2, e.g., can be substituted with an alanine.


A catalytically inactive Cas9 molecule may be fused with a transcription repressor. An eiCas9 fusion protein complexes with a gRNA and localizes to a DNA sequence specified by gRNA's targeting domain, but, unlike an eaCas9, it will not cleave the target DNA. Fusion of an effector domain, such as a transcriptional repression domain, to an eiCas9 enables recruitment of the effector to any DNA site specified by the gRNA. Site specific targeting of an eiCas9 or an eiCas9 fusion protein to a promoter region of a gene can block RNA polymerase binding to the promoter region, a transcription factor (e.g., a transcription activator) and/or a transcriptional enhancer to inhibit transcription activation. Alternatively, site specific targeting of an eiCas9-fusion to a transcription repressor to a promoter region of a gene can be used to decrease transcription activation.


Transcription repressors or transcription repressor domains that may be fused to an eiCas9 molecule can include Knippel associated box (KRAB or SKD), the Mad mSIN3 interaction domain (SID) or the ERP repressor domain (ERD).


In another embodiment, an eiCas9 molecule may be fused with a protein that modifies chromatin. For example, an eiCas9 molecule may be fused to heterochromatin protein 1 (HP1), a histone lysine methyltransferase (e.g., SUV39H1, SUV39H2, G9A, ESET/SETDBl, Pr-SET7/8, SUV4-20H1, RIZ1), a histone lysine demethylates (e.g., LSDl/BHCl 10, SpLsdl/Sw, 1/Safl10, Su(var)3-3, JMJD2A/JHDM3A, JMJD2B, JMJD2C/GASC1, JMJD2D, Rph1, JARID1A/RBP2, JAR1D1B/PLU-1, JAR1D1C/SMCX, JAR1D1D/SMCY, Lid, Jhn2, Jmj2), a histone lysine deacetylases (e.g., HDAC1, HDAC2, HDAC3, HDAC8, Rpd3, Hos1, Cir6, HDAC4, HDAC5, HDAC7, HDAC9, Hda1, Cir3, SIRT1, SIRT2, Sir2, Hst1, Hst2, Hst3, Hst4, HDAC11) and a DNA methylases (DNMT1, DNMT2a/DMNT3b, MET1). An eiCas9-chromatin modifying molecule fusion protein can be used to alter chromatin status to reduce expression a target gene.


The heterologous sequence (e.g., the transcription repressor domain) may be fused to the N- or C-terminus of the eiCas9 protein. In an alternative embodiment, the heterologous sequence (e.g., the transcription repressor domain) may be fused to an internal portion (i.e., a portion other than the N-terminus or C-terminus) of the eiCas9 protein.


The ability of a Cas9 molecule/gRNA molecule complex to bind to and cleave a target nucleic acid can be evaluated, e.g., by the methods described herein in Section III. The activity of a Cas9 molecule, either an eaCas9 or a eiCas9, alone or in a complex with a gRNA molecule may also be evaluated by methods well-known in the art, including, gene expression assays and chromatin-based assays, e.g., chromatin immunoprecipitation (ChiP) and chromatin in vivo assay (CiA).


Nucleic Acids Encoding Cas9 Molecules


Nucleic acids encoding the Cas9 molecules, e.g., an eaCas9 molecule or an eiCas9 molecule are provided herein.


Exemplary nucleic acids encoding Cas9 molecules are described in Cong et al., SCIENCE 2013, 399(6121):819-823; Wang et al., CELL 2013, 153(4):910-918; Mali et al., SCIENCE 2013, 399(6121):823-826; Jinek et al., SCIENCE 2012, 337(6096):816-821. Another exemplary nucleic acid encoding a Cas9 molecule of N. meningitidis is shown in FIG. 6.


In an embodiment, a nucleic acid encoding a Cas9 molecule can be a synthetic nucleic acid sequence. For example, the synthetic nucleic acid molecule can be chemically modified, e.g., as described in Section X. In an embodiment, the Cas9 mRNA has one or more of, e.g., all of the following properties; it is capped, polyadenylated, substituted with 5-methylcytidine and/or pseudouridine.


In addition or alternatively, the synthetic nucleic acid sequence can be codon optimized, e.g., at least one non-common codon or less-common codon has been replaced by a common codon. For example, the synthetic nucleic acid can direct the synthesis of an optimized messenger mRNA, e.g., optimized for expression in a mammalian expression system, e.g., described herein.


In addition, or alternatively, a nucleic acid encoding a Cas9 molecule may comprise a nuclear localization sequence (NLS). Nuclear localization sequences are known in the art.


Provided below is an exemplary codon optimized nucleic acid sequence encoding a Cas9 molecule of S. pyogenes.











(SEQ ID NO: 22)



ATGGATAAAA AGTACAGCAT CGGGCTGGAC ATCGGTACAA







ACTCAGTGGG GTGGGCCGTG ATTACGGACG AGTACAAGGT







ACCCTCCAAA AAATTTAAAG TGCTGGGTAA CACGGACAGA







CACTCTATAA AGAAAAATCT TATTGGAGCC TTGCTGTTCG







ACTCAGGCGA GACAGCCGAA GCCACAAGGT TGAAGCGGAC







CGCCAGGAGG CGGTATACCA GGAGAAAGAA CCGCATATGC







TACCTGCAAG AAATCTTCAG TAACGAGATG GCAAAGGTTG







ACGATAGCTT TTTCCATCGC CTGGAAGAAT CCTTTCTTGT







TGAGGAAGAC AAGAAGCACG AACGGCACCC CATCTTTGGC







AATATTGTCG ACGAAGTGGC ATATCACGAA AAGTACCCGA







CTATCTACCA CCTCAGGAAG AAGCTGGTGG ACTCTACCGA







TAAGGCGGAC CTCAGACTTA TTTATTTGGC ACTCGCCCAC







ATGATTAAAT TTAGAGGACA TTTCTTGATC GAGGGCGACC







TGAACCCGGA CAACAGTGAC GTCGATAAGC TGTTCATCCA







ACTTGTGCAG ACCTACAATC AACTGTTCGA AGAAAACCCT







ATAAATGCTT CAGGAGTCGA CGCTAAAGCA ATCCTGTCCG







CGCGCCTCTC AAAATCTAGA AGACTTGAGA ATCTGATTGC







TCAGTTGCCC GGGGAAAAGA AAAATGGATT GTTTGGCAAC







CTGATCGCCC TCAGTCTCGG ACTGACCCCA AATTTCAAAA







GTAACTTCGA CCTGGCCGAA GACGCTAAGC TCCAGCTGTC







CAAGGACACA TACGATGACG ACCTCGACAA TCTGCTGGCC







CAGATTGGGG ATCAGTACGC CGATCTCTTT TTGGCAGCAA







AGAACCTGTC CGACGCCATC CTGTTGAGCG ATATCTTGAG







AGTGAACACC GAAATTACTA AAGCACCCCT TAGCGCATCT







ATGATCAAGC GGTACGACGA GCATCATCAG GATCTGACCC







TGCTGAAGGC TCTTGTGAGG CAACAGCTCC CCGAAAAATA







CAAGGAAATC TTCTTTGACC AGAGCAAAAA CGGCTAGGCT







GGCTATATAG ATGGTGGGGC CAGTCAGGAG GAATTCTATA







AATTCATCAA GCCCATTCTC GAGAAAATGG ACGGCACAGA







GGAGTTGCTG GTCAAACTTA ACAGGGAGGA CCTGCTGCGG







AAGCAGCGGA CCTTTGACAA CGGGTCTATC CCCCACCAGA







TTCATCTGGG CGAACTGCAC GCAATCCTGA GGAGGCAGGA







GGATTTTTAT CCTTTTCTTA AAGATAACCG CGAGAAAATA







GAAAAGATTC TTACATTCAG GATCCCGTAC TACGTGGGAC







CTCTCGCCCG GGGCAATTCA CGGTTTGCCT GGATGACAAG







GAAGTCAGAG GAGACTATTA CACCTTGGAA CTTCGAAGAA







GTGGTGGACA AGGGTGCATC TGCCCAGTCT TTCATCGAGC







GGATGACAAA TTTTGACAAG AACCTCCCTA ATGAGAAGGT







GCTGCCCAAA CATTCTCTGC TCTACGAGTA CTTTACCGTC







TACAATGAAC TGACTAAAGT CAAGTACGTC ACCGAGGGAA







TGAGGAAGCC GGCATTCCTT AGTGGAGAAC AGAAGAAGGC







GATTGTAGAC CTGTTGTTCA AGACCAACAG GAAGGTGACT







GTGAAGCAAC TTAAAGAAGA CTACTTTAAG AAGATCGAAT







GTTTTGACAG TGTGGAAATT TCAGGGGTTG AAGACCGCTT







CAATGCGTCA TTGGGGACTT ACCATGATCT TCTCAAGATC







ATAAAGGACA AAGACTTCCT GGACAACGAA GAAAATGAGG







ATATTCTCGA AGACATCGTC CTCACCCTGA CCCTGTTCGA







AGACAGGGAA ATGATAGAAG AGCGCTTGAA AACCTATGCC







CACCTCTTCG ACGATAAAGT TATGAAGCAG CTGAAGCGCA







GGAGATACAC AGGATGGGGA AGATTGTCAA GGAAGCTGAT







CAATGGAATT AGGGATAAAC AGAGTGGCAA GACCATACTG







GATTTCCTCA AATCTGATGG CTTCGCCAAT AGGAACTTCA







TGCAACTGAT TCACGATGAC TCTCTTACCT TCAAGGAGGA







CATTCAAAAG GCTCAGGTGA GCGGGCAGGG AGACTCCCTT







CATGAACACA TCGCGAATTT GGCAGGTTCC CCCGCTATTA







AAAAGGGCAT CCTTCAAACT GTCAAGGTGG TGGATGAATT







GGTCAAGGTA ATGGGCAGAC ATAAGCCAGA AAATATTGTG







ATCGAGATGG CCCGCGAAAA GCAGAGCACA CAGAAGGGCC







AGAAAAATAG TAGAGAGCGG ATGAAGAGGA TCGAGGAGGG







CATCAAAGAG CTGGGATCTC AGATTCTCAA AGAAGACCCC







GTAGAAAACA CACAGCTGCA GAACGAAAAA TTGTACTTGT







ACTATCTGCA GAACGGCAGA GACATGTACG TCGACCAAGA







ACTTGATATT AATAGACTGT CCGACTATGA CGTAGACCAT







ATCGTGCCCC AGTCCTTCCT GAAGGACGAC TCCATTGATA







ACAAAGTCTT GACAAGAAGC GACAAGAACA GGGGTAAAAG







TGATAATGTG CCTAGCGAGG AGGTGGTGAA AAAAATGAAG







AACTACTGGC GACAGCTGCT TAATGCAAAG CTCATTACAC







AACGGAAGTT CGATAATCTG ACGAAAGCAG AGAGAGGTGG







CTTGTCTGAG TTGGACAAGG CAGGGTTTAT TAAGCGGCAG







CTGGTGGAAA CTAGGCAGAT CACAAAGCAC GTGGCGCAGA







TTTTGGACAG CCGGATGAAC ACAAAATACG ACGAAAATGA







TAAACTGATA CGAGAGGTCA AAGTTATCAC GCTGAAAAGC







AAGCTGGTGT CCGATTTTCG GAAAGACTTC CAGTTCTACA







AAGTTCGCGA GATTAATAAC TACCATCATG CTCACGATGC







GTACCTGAAC GCTGTTGTCG GGACCGCCTT GATAAAGAAG







TACCCAAAGC TGGAATCCGA GTTCGTATAC GGGGATTACA







AAGTGTACGA TGTGAGGAAA ATGATAGCCA AGTCCGAGCA







GGAGATTGGA AAGGCCACAG CTAAGTACTT CTTTTATTCT







AACATCATGA ATTTTTTTAA GACGGAAATT ACCCTGGCCA







ACGGAGAGAT CAGAAAGCGG CCCCTTATAG AGACAAATGG







TGAAACAGGT GAAATCGTCT GGGATAAGGG CAGGGATTTC







GCTACTGTGA GGAAGGTGCT GAGTATGCCA CAGGTAAATA







TCGTGAAAAA AACCGAAGTA CAGACCGGAG GATTTTCCAA







GGAAAGCATT TTGCCTAAAA GAAACTCAGA CAAGCTCATC







GCCCGCAAGA AAGATTGGGA CCCTAAGAAA TACGGGGGAT







TTGACTCACC CACCGTAGCC TATTCTGTGC TGGTGGTAGC







TAAGGTGGAA AAAGGAAAGT CTAAGAAGCT GAAGTCCGTG







AAGGAACTCT TGGGAATCAC TATCATGGAA AGATCATCCT







TTGAAAAGAA CCCTATCGAT TTCCTGGAGG CTAAGGGTTA







CAAGGAGGTC AAGAAAGACC TCATCATTAA ACTGCCAAAA







TACTCTCTCT TCGAGCTGGA AAATGGCAGG AAGAGAATGT







TGGCCAGCGC CGGAGAGCTG CAAAAGGGAA ACGAGCTTGC







TCTGCCCTCC AAATATGTTA ATTTTCTCTA TCTCGCTTCC







CACTATGAAA AGCTGAAAGG GTCTCCCGAA GATAACGAGC







AGAAGCAGCT GTTCGTCGAA CAGCACAAGC ACTATCTGGA







TGAAATAATC GAACAAATAA GCGAGTTCAG CAAAAGGGTT







ATCCTGGCGG ATGCTAATTT GGACAAAGTA CTGTCTGCTT







ATAACAAGCA CCGGGATAAG CCTATTAGGG AACAAGCCGA







GAATATAATT CACCTCTTTA CACTCACGAA TCTCGGAGCC







CCCGCCGCCT TCAAATACTT TGATACGACT ATCGACCGGA







AACGGTATAC CAGTACCAAA GAGGTCCTCG ATGCCACCCT







CATCCACCAG TCAATTACTG GCCTGTACGA AACACGGATC







GACCTCTCTC AACTGGGCGG CGACTAG






Provided below is the corresponding amino acid sequence of a S. pyogenes Cas9 molecule.









(SEQ ID NO: 23)


MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA





LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR





LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD





LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP





INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP





NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI





LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI





FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR





KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY





YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK





NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD





LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI





IKDKDFLDNEEDEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ





LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD





SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV





MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP





VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD





SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL





TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDLKI





REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK





YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI





TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV





QTGGFSKESILPKRNSDKLIARKKDWDPKYYGGFDSPTVAYSVLVVAKVE





KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK





YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE





DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK





PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ





SITGLYETRIDLSQLGGD*






Provided below is an exemplary codon optimized nucleic acid sequence encoding a Cas9 molecule of N. meningitidis.









(SEQ ID NO: 24)


ATGGCCGCCTTCAAGCCCAACCCCATCAACTACATCCTGGGCCTGGACAT





CGGCATCGCCAGCGTGGGCTGGGCCATGGTGGAGATCGACGAGGACGAGA





ACCCCATCTGCCTGATCGACCTGGGTGTGCGCGTGTTCGAGCGCGCTGAG





GTGCCCAAGACTGGTGACAGTCTGGCTATGGCTCGCCGGCTTGCTCGCTC





TGTTCGGCGCCTTACTCGCCGGCGCGCTCACCGCCTTCTGCGCGCTCGCC





GCCTGCTGAAGCGCGAGGGTGTGCTGCAGGCTGCCGACTTCGACGAGAAC





GGCCTGATCAAGAGCCTGCCCAACACTCCTTGGCAGCTGCGCGCTGCCGC





TCTGGACCGCAAGCTGACTCCTCTGGAGTGGAGCGCCGTGCTGCTGCACC





TGATCAAGCACCGCGGCTACCTGAGCCAGCGCAAGAACGAGGGCGAGACC





GCCGACAAGGAGCTGGGTGCTCTGCTGAAGGGCGTGGCCGACAACGCCCA





CGCCCTGCAGACTGGTGACTTCCGCACTCCTGCTGAGCTGGCCCTGAACA





AGTTCGAGAAGGAGAGCGGCCACATCCGCAACCAGCGCGGCGACTACAGC





CACACCTTCAGCCGCAAGGACCTGCAGGCCGAGCTGATCCTGCTGTTCGA





GAAGCAGAAGGAGTTCGGCAACCCCCACGTGAGCGGCGGCCTGAAGGAGG





GCATCGAGACCCTGCTGATGACCCAGCGCCCCGCCCTGAGCGGCGACGCC





GTGCAGAAGATGCTGGGCCACTGCACCTTCGAGCCAGCCGAGCCCAAGGC





CGCCAAGAACACCTACACCGCCGAGCGCTTCATCTGGCTGACCAAGCTGA





ACAACCTGCGCATCCTGGAGCAGGGCAGCGAGCGCCCCCTGACCGACACC





GAGCGCGCCACCCTGATGGACGAGCCCTACCGCAAGAGCAAGCTGACCTA





CGCCCAGGCCCGCAAGCTGCTGGGTCTGGAGGACACCGCCTTCTTCAAGG





GCCTGCGCTACGGCAAGGACAACGCCGAGGCCAGCACCCTGATGGAGATG





AAGGCCTACCACGCCATCAGCCGCGCCCTGGAGAAGGAGGGCCTGAAGGA





CAAGAAGAGTCCTCTGAACCTGAGCCCCGAGCTGCAGGACGAGATCGGCA





CCGCCTTCAGCCTGTTCAAGACCGACGAGGACATCACCGGCCGCCTGAAG





GACCGCATCCAGCCCGAGATCCTGGAGGCCCTGCTGAAGCACATCAGCTT





CGACAAGTTCGTGCAGATCAGCCTGAAGGCCCTGCGCCGCATCGTGCCCC





TGATGGAGCAGGGCAAGCGCTACGACGAGGCCTGCGCCGAGATCTACGGC





GACCACTACGGCAAGAAGAACACCGAGGAGAAGATCTACCTGCCTCCTAT





CCCCGCCGACGAGATCCGCAACCCCGTGGTGCTGCGCGCCCTGAGCCAGG





CCCGCAAGGTGATCAACGGCGTGGTGCGCCGCTACGGCAGCCCCGCCCGC





ATCCACATCGAGACCGCCCGCGAGGTGGGCAAGAGCTTCAAGGACCGCAA





GGAGATCGAGAAGCGCCAGGAGGAGAACCGCAAGGACCGCGAGAAGGCCG





CCGCCAAGTTCCGCGAGTACTTCCCCAACTTCGTGGGCGAGCCCAAGAGC





AAGGACATCCTGAAGCTGCGCCTGTACGAGCAGCAGCACGGCAAGTGCCT





GTACAGCGGCAAGGAGATCAACCTGGGCCGCCTGAACGAGAAGGGCTACG





TGGAGATCGACCACGCCCTGCCCTTCAGCCGCACCTGGGACGACAGCTTC





AACAACAAGGTGCTGGTGCTGGGCAGCGAGAACCAGAACAAGGGCAACCA





GACCCCCTACGAGTACTTCAACGGCAAGGACAACAGCCGCGAGTGGCAGG





AGTTCAAGGCCCGCGTGGAGACCAGCCGCTTCCCCCGCAGCAAGAAGCAG





CGCATCCTGCTGCAGAAGTTCGACGAGGACGGCTTCAAGGAGCGCAACCT





GAACGACACCCGCTACGTGAACCGCTTCCTGTGCCAGTTCGTGGCCGACC





GCATGCGCCTGACCGGCAAGGGCAAGAAGCGCGTGTTCGCCAGCAACGGC





CAGATCACCAACCTGCTGCGCGGCTTCTGGGGCCTGCGCAAGGTGCGCGC





CGAGAACGACCGCCACCACGCCCTGGACGCCGTGGTGGTGGCCTGCAGCA





CCGTGGCCATGCAGCAGAAGATCACCCGCTTCGTGCGCTACAAGGAGATG





AACGCCTTCGACGGTAAAACCATCGACAAGGAGACCGGCGAGGTGCTGCA





CCAGAAGACCCACTTCCCCCAGCCCTGGGAGTTCTTCGCCCAGGAGGTGA





TGATCCGCGTGTTCGGCAAGCCCGACGGCAAGCCCGAGTTCGAGGAGGCC





GACACCCCCGAGAAGCTGCGCACCCTGCTGGCCGAGAAGCTGAGCAGCCG





CCCTGAGGCCGTGCACGAGTACGTGACTCCTCTGTTCGTGAGCCGCGCCC





CCAACCGCAAGATGAGCGGTCAGGGTCACATGGAGACCGTGAAGAGCGCC





AAGCGCCTGGACGAGGGCGTGAGCGTGCTGCGCGTGCCCCTGACCCAGCT





GAAGCTGAAGGACCTGGAGAAGATGGTGAACCGCGAGCGCGAGCCCAAGC





TGTACGAGGCCCTGAAGGCCCGCCTGGAGGCCCACAAGGACGACCCCGCC





AAGGCCTTCGCCGAGCCCTTCTACAAGTACGACAAGGCCGGCAACCGCAC





CCAGCAGGTGAAGGCCGTGCGCGTGGAGCAGGTGCAGAAGACCGGCGTGT





GGGTGCGCAACCACAACGGCATCGCCGACAACGCCACCATGGTGCGCGTG





GACGTGTTCGAGAAGGGCGACAAGTACTACCTGGTGCCCATCTACAGCTG





GCAGGTGGCCAAGGGCATCCTGCCCGACCGCGCCGTGGTGCAGGGCAAGG





ACGAGGAGGACTGGCAGCTGATCGACGACAGCTTCAACTTCAAGTTCAGC





CTGCACCCCAACGACCTGGTGGAGGTGATCACCAAGAAGGCCCGCATGTT





CGGCTACTTCGCCAGCTGCCACCGCGGCACCGGCAACATCAACATCCGCA





TCCACGACCTGGACCACAAGATCGGCAAGAACGGCATCCTGGAGGGCATC





GGCGTGAAGACCGCCCTGAGCTTCCAGAAGTACCAGATCGACGAGCTGGG





CAAGGAGATCCGCCCCTGCCGCCTGAAGAAGCGCCCTCCTGTGCGCTAA






Provided below is the corresponding amino acid sequence of a N. meningitidis Cas9 molecule.









(SEQ ID NO: 25)


MAAFKPNPINYILGLDIGIASVGWAMVEIDEDENPICLIDLGVRVFERAE





VPKTGDSLAMARRLARSVRRLTRRRAHRLLRARRLLKREGVLQAADFDEN





GLIKSLPNTPWQLRAAALDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGET





ADKELGALLKGVADNAHALQTGDFRTPAELALNKFEKESGHIRNQRGDYS





HTFSRKDLQAELILLFEKQKEFGNPHVSGGLKEGIETLLMTQRPALSGDA





VQKMLGHCTFEPAEPKAAKNTYTAERFIWLTKLNNLRILEQGSERPLTDT





ERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEASTLMEM





KAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRLK





DRIQPEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYG





DHYGKKNTEEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPAR





IHIETAREVGKSFKDRKEIEKRQEENRKDREKAAAKFREYFPNFVGEPKS





KDILKLRLYEQQHGKCLYSGKEINLGRLNEKGYVEIDHALPFSRTWDDSF





NNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFKARVETSRFPRSKKQ





RILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKRVFASNG





QITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEM





NAFDGKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEA





DTPEKLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSA





KRLDEGVSVLRVPLTQLKLKDLEKMVNREREPKLYEALKARLEAHKDDPA





KAFAEPFYKYDKAGNRTQQVKAVRVEQVQKTGVWVRNHNGIADNATMVRV





DVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGKDEEDWQLIDDSFNFKFS





LHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHKIGKNGILEGI





GVKTALSFQKYQIDELGKEIRPCRLKKRPPVR*






Provided below is an amino acid sequence of a S. aureus Cas9 molecule.









(SEQ ID NO: 26)


MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSK





RGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKL





SEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYV





AELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDT





YIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYA





YNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIA





KEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQ





IAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI





NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVV





KRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQ





TNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNP





FNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKIS





YETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTR





YATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKH





HAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEY





KEIPITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTL





IVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDE





KNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNS





RNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEA





KKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDIT





YREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQII





KKG*






If any of the above Cas9 sequences are fused with a peptide or polypeptide at the C-terminus (e.g., an eiCas9 fused with a transcription repressor at the C-terminus), it is understood that the stop codon will be removed.


Other Cas Molecules


Various types of Cas molecules can be used to practice the inventions disclosed herein. In an embodiment, Cas molecules of Type II Cas systems are used. In an embodiment, Cas molecules of other Cas systems are used. For example, Type I or Type III Cas molecules may be used. Exemplary Cas molecules (and Cas systems) are described, e.g., in Haft et al., PLoS COMPUTATIONAL BIOLOGY 2005, 1(6): e60 and Makarova et al., NATURE REVIEW MICROBIOLOGY 2011, 9:467-477, the contents of both references are incorporated herein by reference in their entirety. Exemplary Cas molecules (and Cas systems) are also shown in Table









TABLE 11-1







Cas Systems















Structure of
Families (and






encoded protein
superfamily) of



Gene
System type
Name from Haft
(PDB
encoded



name*
or subtype
et al.§
accessions)′∥
protein#**
Representatives





cas1
Type I
cas1
3GOD, 3LFX
COGI518
SERP2463, SPyI047



Type II

and 2YZS

andygbT



Type III






cas2
Type I
cas2
2IVY, 2I8E and
COGI343 and
SERP2462, SPyI048,



Type II

3EXC
COG3512
SPyI723 (N-terminal



Type III



domain) and ygbF


cas3′
Type I**
cas3
NA
COGI203
APEI232 and ygcB


cas3″
Subtype I-A
NA
NA
COG2254
APEI231 and BH0336



Subtype I-B






cas4
Subtype I-A
cas 4 and csa1
NA
COGI468
APE1239 and BH0340



Subtype I-B







Subtype I-C







Subtype I-D







Subtype II-B






cas5
Subtype I-A
cas5a, cas5d,
3KG4
COGI688
APEI234, BH0337,



Subtype I-B
cas5e, cas5h,

(RAMP)
devS and ygcl



Subtype I-C
cas5p, cas5t and






Subtype I-E
cmx5





cas6
Subtype I-A
cas6 and cmx6
314H
COGI583 and
PFI131 and slr7014



Subtype I-B


COG555I




Subtype I-D


(RAMP)




Subtype III-A







Subtype III-B






cas6e
Subtype I-E
cse3
1WJ9
(RAMP)
ygcH


cas6f
Subtype I-F
csy4
2XLJ
(RAMP)
y1727


cas7
Subtype I-A
csa2, csd2, cse4,
NA
COGI857 and
devR and ygcl



Subtype I-B
csh2, cspl and

COG3649




Subtype I-C
cst2

(RAMP)




Subtype I-E






cas8al
Subtype I-A**
cm:xl, est], csx8,
NA
BH0338-like
LA319I§§ and




csxl3 and


PG2018§§




CXXC-CXXC





cas8a2
Subtype I-A**
csa4 and csx9
NA
PH0918
AF0070, AFI873,







MJ0385, PF0637,







PH0918 and SS0140I


cas8b
Subtype I-B**
cshl and
NA
BH0338-like
MTH1090 and




TMI802


TMI802


cas8c
Subtype I-c**
csdl and csp2
NA
BH0338-like
BH0338


cas9
Type II**
csnl and csxl2
NA
COG3513
FTN_0757 and







SPyI046


caslO
Type III**
cmr2, csml and
NA
COGI353
MTH326, Rv2823c§§




csxll


and TMI 794§§


caslOd
Subtype I-D**
csc3
NA
COGI353
slr70I 1


csyl
Subtype I-pH
csyl
NA
y1724-like
y1724


csy2
Subtype I-F
csy2
NA
(RAMP)
y1725


csy3
Subtype I-F
csy3
NA
(RAMP)
y1726


csel
Subtype I-E**
csel
NA
YgcL-like
ygcL


cse2
Subtype I-E
cse2
2ZCA
YgcK-like
ygcK


cscl
Subtype I-D
cscl
NA
alr1563-like
alr1563






(RAMP)



csc2
Subtype I-D
cscl and csc2
NA
COGI337
slr7012






(RAMP)



csa5
Subtype I-A
csa5
NA
AFI870
AFI870, MJ0380,







PF0643 and SS01398


csn2
Subtype II-A
csn2
NA
SPyI049-like
SPyI049


csm2
Subtype III-A**
csm2
NA
COGI421
MTH108I and







SERP2460


csm3
Subtype III-A
csc2 and csm3
NA
COGI337
MTH1080 and






(RAMP)
SERP2459


csm4
Subtype III-A
csm4
NA
COGI567
MTH1079 and






(RAMP)
SERP2458


csm5
Subtype III-A
csm5
NA
COGI332
MTH1078 and






(RAMP)
SERP2457


csm6
Subtype III-A
APE2256 and
2WTE
COGI517
APE2256 and




csm6


SS01445


cmrl
Subtype III-B
cmrl
NA
COGI367
PFI130






(RAMP)



cmr3
Subtype III-B
cmr3
NA
COGI769
PFI128






(RAMP)



cmr4
Subtype III-B
cmr4
NA
COGI336
PFI126






(RAMP)



cmr5
Subtype III-B**
cmr5
2ZOP and 20EB
COG3337
MTH324 and PFI125


cmr6
Subtype III-B
cmr6
NA
COGI604
PFI124






(RAMP)



csbl
Subtype I-u
GSU0053
NA
(RAMP)
Balac_I306 and







GSU0053


csb2
Subtype I-u§§
NA
NA
(RAMP)
Balac_ I305 and







GSU0054


csb3
Subtype I-u
NA
NA
(RAMP)
Balac_I303§§


csxl7
Subtype I-u
NA
NA
NA
Btus_2683


csxl4
Subtype I-u
NA
NA
NA
GSU0052


csxlO
Subtype I-u
csxlO
NA
(RAMP)
Caur_2274


csxl6
Subtype III-U
VVAI548
NA
NA
VVAI548


csaX
Subtype III-U
csaX
NA
NA
SS01438


csx3
Subtype III-U
csx33
NA
NA
AFI864


csxl
Subtype III-U
csa3, csxl, csx2,
IXMX and 2171
COGI517 and
MJ1666, NEOI 13,




DXTHG,

COG4006
PFI127 and TMI812




NEO113 and







TIGR02710





csxl5
Unknown
NA
NA
TTE2665
TTE2665


csfl
Type U
csfl
NA
NA
TTE_I038


csj2
Type U
csj2
NA
(RAMP)
AFE_I039


csf3
Type U
csf3
NA
(RAMP)
AFE_I040


csf4
Type U
csf4
NA
NA
AFE_I037










III. Functional Analysis of Candidate Molecules


Candidate Cas9 molecules, candidate gRNA molecules, candidate Cas9 molecule/gRNA molecule complexes, can be evaluated by art-known methods or as described herein. For example, exemplary methods for evaluating the endonuclease activity of Cas9 molecule are described, e.g., in Jinek et al., SCIENCE 2012; 337(6096):816-821.


Binding and Cleavage Assay: Testing the Endonuclease Activity of Cas9 Molecule


The ability of a Cas9 molecule/gRNA molecule complex to bind to and cleave a target nucleic acid can be evaluated in a plasmid cleavage assay. In this assay, synthetic or in vitro-transcribed gRNA molecule is pre-annealed prior to the reaction by heating to 95° C. and slowly cooling down to room temperature. Native or restriction digest-linearized plasmid DNA (300 ng (−8 nM)) is incubated for 60 min at 37° C. with purified Cas9 protein molecule (50-500 nM) and gRNA (50-500 nM, 1:1) in a Cas9 plasmid cleavage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 0.5 mM DTT, 0.1 mM EDTA) with or without 10 mM MgCh. The reactions are stopped with 5×DNA loading buffer (30% glycerol, 1.2% SDS, 250 mM EDTA), resolved by a 0.8 or 1% agarose gel electrophoresis and visualized by ethidium bromide staining. The resulting cleavage products indicate whether the Cas9 molecule cleaves both DNA strands, or only one of the two strands. For example, linear DNA products indicate the cleavage of both DNA strands. Nicked open circular products indicate that only one of the two strands is cleaved.


Alternatively, the ability of a Cas9 molecule/gRNA molecule complex to bind to and cleave a target nucleic acid can be evaluated in an oligonucleotide DNA cleavage assay. In this assay, DNA oligonucleotides (10 pmol) are radiolabeled by incubating with 5 units T4 polynucleotide kinase and −3-6 pmol (−20-40 mCi) [y-32P]-ATP in 1× T4 polynucleotide kinase reaction buffer at 37° C. for 30 min, in a 50 μL reaction. After heat inactivation (65° C. for 20 min), reactions are purified through a column to remove unincorporated label. Duplex substrates (100 nM) are generated by annealing labeled oligonucleotides with equimolar amounts of unlabeled complementary oligonucleotide at 95° C. for 3 min, followed by slow cooling to room temperature. For cleavage assays, gRNA molecules are annealed by heating to 95° C. for 30 s, followed by slow cooling to room temperature. Cas9 (500 nM final concentration) is pre-incubated with the annealed gRNA molecules (500 nM) in cleavage assay buffer (20 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgC12, 1 mM DTT, 5% glycerol) in a total volume of 9 μl. Reactions are initiated by the addition of 1 μl target DNA (10 nM) and incubated for 1 hat 37° C. Reactions are quenched by the addition of 20 μl of loading dye (5 mM EDTA, 0.025% SDS, 5% glycerol in formamide) and heated to 95° C. for 5 min. Cleavage products are resolved on 12% denaturing polyacrylamide gels containing 7 M urea and visualized by phosphor imaging. The resulting cleavage products indicate that whether the complementary strand, the non-complementary strand, or both, are cleaved.


One or both of these assays can be used to evaluate the suitability of a candidate gRNA molecule or candidate Cas9 molecule.


Binding Assay: Testing the binding of Cas9 molecule to target DNA


Exemplary methods for evaluating the binding of Cas9 molecule to target DNA are described, e.g., in Jinek et al., SCIENCE 2012; 337(6096):816-821.


For example, in an electrophoretic mobility shift assay, target DNA duplexes are formed by mixing of each strand (10 nmol) in deionized water, heating to 95° C. for 3 min and slow cooling to room temperature. All DNAs are purified on 8% native gels containing 1×TBE. DNA bands are visualized by UV shadowing, excised, and eluted by soaking gel pieces in DEPC-treated H2O. Eluted DNA is ethanol precipitated and dissolved in DEPC-treated H2O. DNA samples are 5′ end labeled with [y-32P]-ATP using T4 polynucleotide kinase for 30 min at 37° C. Polynucleotide kinase is heat denatured at 65° C. for 20 min, and unincorporated radiolabel is removed using a column. Binding assays are performed in buffer containing 20 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCh, 1 mM DTT and 10% glycerol in a total volume of 10 μl. Cas9 protein molecule is programmed with equimolar amounts of pre-annealed gRNA molecule and titrated from 100 pM to 1 μM. Radiolabeled DNA is added to a final concentration of 20 pM. Samples are incubated for 1 hat 37° C. and resolved at 4° C. on an 8% native polyacrylamide gel containing 1×TBE and 5 mM MgCh. Gels are dried and DNA visualized by phosphorimaging.


IV. Template Nucleic Acids (Genome Editing Approaches)


The terms “template nucleic acid” and “swap nucleic acid” are used interchangeably and have identical meaning in this document and its priority documents.


Mutations in a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII, may be corrected using one of the approaches discussed herein. In an embodiment, a mutation in a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII, is corrected by homology directed repair (HDR) using a template nucleic acid (see Section IV, 1). In an embodiment, a mutation in a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII, is corrected by Non-Homologous End Joining (NHEJ) repair using a template nucleic acid (see Section IV.2).


IV.1 HDR Repair and Template Nucleic Acids


As described herein, nuclease-induced homology directed repair (HDR) can be used to alter a target sequence and correct (e.g., repair or edit) a mutation in the genome. While not wishing to be bound by theory, it is believed that alteration of the target sequence occurs by homology-directed repair (HDR) with a donor template or template nucleic acid. For example, the donor template or the template nucleic acid provides for alteration of the target sequence. It is contemplated that a plasmid donor can be used as a template for homologous recombination. It is further contemplated that a single stranded donor template can be used as a template for alteration of the target sequence by alternate methods of homology directed repair (e.g., single strand annealing) between the target sequence and the donor template. Donor template-effected alteration of a target sequence depends on cleavage by a Cas9 molecule. Cleavage by Cas9 can comprise a double strand break or two single strand breaks.


In an embodiment, a mutation can be corrected by either a single double-strand break or two single strand breaks. In an embodiment, a mutation can be corrected by (1) a single double-strand break, (2) two single strand breaks, (3) two double stranded breaks with a break occurring on each side of the target sequence, (4) one double stranded breaks and two single strand breaks with the double strand break and two single strand breaks occurring on each side of the target sequence or (5) four single stranded breaks with a pair of single stranded breaks occurring on each side of the target sequence.


Double Strand Break Mediated Correction


In an embodiment, double strand cleavage is effected by a Cas9 molecule having cleavage activity associated with an HNH-like domain and cleavage activity associated with a RuvC-like domain, e.g., an N-terminal RuvC-like domain, e.g., a wild type Cas9. Such an embodiment requires only a single gRNA.


Single Strand Break Mediated Correction


In an embodiment, two single strand breaks, or nicks, are effected by a Cas9 molecule having nickase activity, e.g., cleavage activity associated with an HNH-like domain or cleavage activity associated with an N-terminal RuvC-like domain. Such an embodiment requires two gRNAs, one for placement of each single strand break. In an embodiment, the Cas9 molecule having nickase activity cleaves the strand to which the gRNA hybridizes, but not the strand that is complementary to the strand to which the gRNA hybridizes. In an embodiment, the Cas9 molecule having nickase activity does not cleave the strand to which the gRNA hybridizes, but rather cleaves the strand that is complementary to the strand to which the gRNA hybridizes.


In an embodiment, the nickase has HNH activity, e.g., a Cas9 molecule having the RuvC activity inactivated, e.g., a Cas9 molecule having a mutation at D1O, e.g., the D1OA mutation. D1OA inactivates RuvC; therefore, the Cas9 nickase has (only) HNH activity and will cut on the strand to which the gRNA hybridizes (e.g., the complementary strand, which does not have the NGG PAM on it). In an embodiment, a Cas9 molecule having an H840, e.g., an H840A, mutation can be used as a nickase. H840A inactivates HNH; therefore, the Cas9 nickase has (only) RuvC activity and cuts on the non-complementary strand (e.g., the strand that has the NGG PAM and whose sequence is identical to the gRNA).


In an embodiment, in which a nickase and two gRNAs are used to position two single strand nicks, one nick is on the + strand and one nick is on the − strand of the target nucleic acid. The PAMs are outwardly facing. The gRNAs can be selected such that the gRNAs are separated by, from about 0-50, 0-100, or 0-200 nucleotides. In an embodiment, there is no overlap between the target sequence that is complementary to the targeting domains of the two gRNAs. In an embodiment, the gRNAs do not overlap and are separated by as much as 50, 100, or 200 nucleotides. In an embodiment, the use of two gRNAs can increase specificity, e.g., by decreasing off-target binding (Ran et al., CELL 2013).


In an embodiment, a single nick can be used to induce HDR. It is contemplated herein that a single nick can be used to increase the ratio of HR to NHEJ at a given cleavage site.


Placement of the Double Strand Break or a Single Strand Break Relative to Target Position


The double strand break or single strand break in one of the strands should be sufficiently close to target position such that correction occurs. In an embodiment, the distance is not more than 50, 100, 200, 300, 350 or 400 nucleotides. While not wishing to be bound by theory, it is believed that the break should be sufficiently close to target position such that the break is within the region that is subject to exonuclease-mediated removal during end resection. If the distance between the target position and a break is too great, the mutation may not be included in the end resection and, therefore, may not be corrected, as donor sequence may only be used to correct sequence within the end resection region.


In an embodiment, in which a gRNA (unimolecular (or chimeric) or modular gRNA) and Cas9 nuclease induce a double strand break for the purpose of inducing HOR-mediated correction, the cleavage site is between 0-200 bp (e.g., 0 to 175, 0 to 150, 0 to 125, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 25 to 200, 25 to 175, 25 to 150, 25 to 125, 25 to 100, 25 to 75, 25 to 50, 50 to 200, 50 to 175, 50 to 150, 50 to 125, 50 to 100, 50 to 75, 75 to 200, 75 to 175, 75 to 150, 75 to 125, 75 to 100 bp) away from the target position. In an embodiment, the cleavage site is between 0-100 bp (e.g., 0 to 75, 0 to 50, 0 to 25, 25 to 100, 25 to 75, 25 to 50, 50 to 100, 50 to 75 or 75 to 100 bp) away from the target position.


In an embodiment, in which two gRNAs (independently, unimolecular (or chimeric) or modular gRNA) complexing with Cas9 nickases induce two single strand breaks for the purpose of inducing HOR-mediated correction, the closer nick is between 0-200 bp (e.g., 0 to 175, 0 to 150, 0 to 125, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 25 to 200, 25 to 175, 25 to 150, 25 to 125, 25 to 100, 25 to 75, 25 to 50, 50 to 200, 50 to 175, 50 to 150, 50 to 125, 50 to 100, 50 to 75, 75 to 200, 75 to 175, 75 to 150, 75 to 125, 75 to 100 bp) away from the target position and the two nicks will ideally be within 25-55 bp of each other (e.g., 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 30 to 55, 30 to 50, 30 to 45, 30 to 40, 30 to 35, 35 to 55, 35 to 50, 35 to 45, 35 to 40, 40 to 55, 40 to 50, 40 to 45 bp) and no more than 100 bp away from each other (e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20, 10 or 5 bp away from each other). In an embodiment, the cleavage site is between 0-100 bp (e.g., 0 to 75, 0 to 50, 0 to 25, 25 to 100, 25 to 75, 25 to 50, 50 to 100, 50 to 75 or 75 to 100 bp) away from the target position.


In one embodiment, two gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to position a double-strand break on both sides of a target position. In an alternate embodiment, three gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to position a double strand break (i.e., one gRNA complexes with a cas9 nuclease) and two single strand breaks or paired single stranded breaks (i.e., two gRNAs complex with Cas9 nickases) on either side of the target position (e.g., the first gRNA is used to target upstream (i.e., 5′) of the target position and the second gRNA is used to target downstream (i.e., 3′) of the target position). In another embodiment, four gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to generate two pairs of single stranded breaks (i.e., two pairs of two gRNAs complex with Cas9 nickases) on either side of the target position (e.g., the first gRNA is used to target upstream (i.e., 5′) of the target position and the second gRNA is used to target downstream (i.e., 3′) of the target position). The double strand break(s) or the closer of the two single strand nicks in a pair will ideally be within 0-500 bp of the target position (e.g., no more than 450, 400, 350, 300, 250, 200, 150, 100, 50 or 25 bp from the target position). When nickases are used, the two nicks in a pair are within 25-55 bp of each other (e.g., between 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 50 to 55, 45 to 55, 40 to 55, 35 to 55, 30 to 55, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 35 to 45, or 40 to 45 bp) and no more than 100 bp away from each other (e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20 or 10 bp).


In one embodiment, two gRNAs. e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to position a double-strand break on both sides of a target position. In an alternate embodiment, three gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to position a double strand break (i.e., one gRNA complexes with a cas9 nuclease) and two single strand breaks or paired single stranded breaks (i.e., two gRNAs complex with Cas9 nickases) on either side of the target position (e.g., the first gRNA is used to target upstream (i.e., 5′) of the mutation in a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII and the second gRNA is used to target downstream (i.e., 3′) of the mutation in a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VI-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII). In another embodiment, four gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to generate two pairs of single stranded breaks (i.e., two pairs of two gRNAs complex with Cas9 nickases) on either side of the target position (e.g., the first gRNA is used to target upstream (i.e., 5′) of the mutation in a gene or pathway described herein, and the second gRNA is used to target downstream (i.e., 3′) of the mutation in a gene or pathway described herein). The double strand break(s) or the closer of the two single strand nicks in a pair will ideally be within 0-500 bp of the target position (e.g., no more than 450, 400, 350, 300, 250, 200, 150, 100, 50 or 25 bp from the target position). When nickases are used, the two nicks in a pair are within 25-55 bp of each other (e.g., between 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 50 to 55, 45 to 55, 40 to 55, 35 to 55, 30 to 55, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 35 to 45, or 40 to 45 bp) and no more than 100 bp away from each other (e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20 or 10 bp).


Length of the Homology Arms


The homology arm should extend at least as far as the region in which end resection may occur, e.g., in order to allow the resected single stranded overhang to find a complementary region within the donor template. The overall length could be limited by parameters such as plasmid size or viral packaging limits. In an embodiment, a homology arm does not extend into repeated elements, e.g., ALU repeats, LINE repeats.


Exemplary homology arm lengths include a least 50, 100, 250, 500, 750 or 1000 nucleotides.


Target position, as used herein, refers to a site on a target nucleic acid (e.g., the chromosome) that is modified by a Cas9 molecule-dependent process. For example, the target position can be a modified Cas9 molecule cleavage of the target nucleic acid and template nucleic acid directed modification, e.g., correction, of the target position. In an embodiment, a target position can be a site between two nucleotides, e.g., adjacent nucleotides, on the target nucleic acid into which one or more nucleotides is added. The target position may comprise one or more nucleotides that are altered, e.g., corrected, by a template nucleic acid. In an embodiment, the target position is within a target sequence (e.g., the sequence to which the gRNA binds). In an embodiment, a target position is upstream or downstream of a target sequence (e.g., the sequence to which the gRNA binds).


A template nucleic acid, as that term is used herein, refers to a nucleic acid sequence which can be used in conjunction with a Cas9 molecule and a gRNA molecule to alter the structure of a target position. The term “template nucleic acid” is synonymous with the term “swap nucleic acid” used in the priority document and herein. The terms “template nucleic acid” and “swap nucleic acid” have exactly the same meaning and can be used interchangeably. In an embodiment, the target nucleic acid is modified to have some or all of the sequence of the template nucleic acid, typically at or near cleavage site(s). In an embodiment, the template nucleic acid is single stranded. In an alternate embodiment, the template nucleic acid is double stranded. In an embodiment, the template nucleic acid is DNA, e.g., double stranded DNA. In an alternate embodiment, the template nucleic acid is single stranded DNA.


In an embodiment, the template nucleic acid alters the structure of the target position by participating in a homology directed repair event. In an embodiment, the template nucleic acid alters the sequence of the target position. In an embodiment, the template nucleic acid results in the incorporation of a modified, or non-naturally occurring, nucleotide into the target nucleic acid.


Typically, the template sequence undergoes a breakage mediated or catalyzed recombination with the target sequence. In an embodiment, the template nucleic acid includes sequence that corresponds to a site on the target sequence that is cleaved by an eaCas9 mediated cleavage event. In an embodiment, the template nucleic acid includes sequence that corresponds to both, a first site on the target sequence that is cleaved in a first Cas9 mediated event, and a second site on the target sequence that is cleaved in a second Cas9 mediated event.


In an embodiment, the template nucleic acid can include sequence which results in an alteration in the coding sequence of a translated sequence, e.g., one which results in the substitution of one amino acid for another in a protein product, e.g., transforming a mutant allele into a wild type allele, transforming a wild type allele into a mutant allele, and/or introducing a stop codon, insertion of an amino acid residue, deletion of an amino acid residue, or a nonsense mutation.


In an embodiment, the template nucleic acid can include sequence which results in an alteration in a non-coding sequence, e.g., an alteration in an exon or in a 5′ or 3′ non-translated or non-transcribed region. Such alterations include an alteration in a control element, e.g., a promoter, enhancer, and an alteration in a cis-acting or trans-acting control element.


A template nucleic acid having homology with a target position in a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VI-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII, can be used to alter the structure of a target sequence. The template sequence can be used to alter an unwanted structure, e.g., an unwanted or mutant nucleotide.


The template nucleic acid can include sequence which, when integrated, results in:

    • decreasing the activity of a positive control element;
    • increasing the activity of a positive control element;
    • decreasing the activity of a negative control element;
    • increasing the activity of a negative control element;
    • decreasing the expression of a gene;
    • increasing the expression of a gene;
    • increasing resistance to a disorder or disease;
    • increasing resistance to viral entry;
    • correcting a mutation or altering an unwanted amino acid residue
    • conferring, increasing, abolishing or decreasing a biological property of a gene product, e.g., increasing the enzymatic activity of an enzyme, or increasing the ability of a gene product to interact with another molecule.


The template nucleic acid can include sequence which results in:

    • a change in sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more nucleotides of the target sequence.


In an embodiment, the template nucleic acid is 20+/−10, 30+/−10, 40+/−10, 50+/−10, 60+/−10, 70+/−10, 80+/−10, 90+/−10, 100+/−10, 110+/−10, 120+/−10, 130+/−10, 140+/−10, 150+/−10, 160+/−10, 170+/−10, 180+/−10, 190+/−10, 200+/−10, 210+/−10, of 220+/−10 nucleotides in length.


In an embodiment, the template nucleic acid is 30+/−20, 40+/−20, 50+/−20, 60+/−20, 70+/−20, 80+/−20, 90+/−20, 100+/−20, 110+/−20, 120+/−20, 130+/−20, 140+/−20, 150+/−20, 160+/−20, 170+/−20, 180+/−20, 190+/−20, 200+/−20, 210+/−20, of 220+/−20 nucleotides in length.


In an embodiment, the template nucleic acid is 10 to 1,000, 20 to 900, 30 to 800, 40 to 700, 50 to 600, 50 to 500, 50 to 400, 50 to 300, 50 to 200, or 50 to 100 nucleotides in length.


A template nucleic acid comprises the following components:


[5′ homology arm]-[replacement sequence]-[3′ homology arm].


The homology arms provide for recombination into the chromosome, thus replacing the undesired element, e.g., a mutation or signature, with the replacement sequence. In an embodiment, the homology arms flank the most distal cleavage sites.


In an embodiment, the 3′ end of the 5′ homology arm is the position next to the 5′ end of the replacement sequence. In an embodiment, the 5′ homology arm can extend at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 5′ from the 5′ end of the replacement sequence.


In an embodiment, the 5′ end of the 3′ homology arm is the position next to the 3′ end of the replacement sequence. In an embodiment, the 3′ homology arm can extend at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 3′ from the 3′ end of the replacement sequence.


It is contemplated herein that one or both homology arms may be shortened to avoid including certain sequence repeat elements, e.g., Alu repeats, LINE elements. For example, a 5′ homology arm may be shortened to avoid a sequence repeat element. In an embodiment, a 3′ homology arm may be shortened to avoid a sequence repeat element. In an embodiment, both the 5′ and the 3′ homology arms may be shortened to avoid including certain sequence repeat elements.


It is contemplated herein that template nucleic acids for correcting a mutation may designed for use as a single-stranded oligonucleotide (ssODN). When using a ssODN, 5′ and 3′ homology arms may range up to about 200 base pairs (bp) in length, e.g., at least 25, 50, 75, 100, 125, 150, 175, or 200 bp in length. Longer homology arms are also contemplated for ssODNs as improvements in oligonucleotide synthesis continue to be made.


In an embodiment, an ssODN may be used to correct a mutation in a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII.


IV.2 NHEJ Approaches for Gene Targeting


As described herein, nuclease-induced non-homologous end-joining (NHEJ) can be used to target gene-specific knockouts. Nuclease-induced NHEJ can also be used to remove (e.g., delete) sequence in a gene of interest.


While not wishing to be bound by theory, it is believed that, in an embodiment, the genomic alterations associated with the methods described herein rely on nuclease-induced NHEJ and the error-prone nature of the NHEJ repair pathway. NHEJ repairs a double-strand break in the DNA by joining together the two ends; however, generally, the original sequence is restored only if two compatible ends, exactly as they were formed by the double-strand break, are perfectly ligated. The DNA ends of the double-strand break are frequently the subject of enzymatic processing, resulting in the addition or removal of nucleotides, at one or both strands, prior to rejoining of the ends. This results in the presence of insertion and/or deletion (indel) mutations in the DNA sequence at the site of the NHEJ repair. Two-thirds of these mutations typically alter the reading frame and, therefore, produce a non-functional protein. Additionally, mutations that maintain the reading frame, but which insert or delete a significant amount of sequence, can destroy functionality of the protein. This is locus dependent as mutations in critical functional domains are likely less tolerable than mutations in non-critical regions of the protein.


The indel mutations generated by NHEJ are unpredictable in nature; however, at a given break site certain indel sequences are favored and are over represented in the population, likely due to small regions of microhomology. The lengths of deletions can vary widely; most commonly in the 1-50 bp range, but they can easily reach greater than 100-200 bp. Insertions tend to be shorter and often include short duplications of the sequence immediately surrounding the break site. However, it is possible to obtain large insertions, and in these cases, the inserted sequence has often been traced to other regions of the genome or to plasmid DNA present in the cells.


Because NHEJ is a mutagenic process, it can also be used to delete small sequence motifs as long as the generation of a specific final sequence is not required. If a double-strand break is targeted near to a short target sequence, the deletion mutations caused by the NHEJ repair often span, and therefore remove, the unwanted nucleotides. For the deletion of larger DNA segments, introducing two double-strand breaks, one on each side of the sequence, can result in NHEJ between the ends with removal of the entire intervening sequence. Both of these approaches can be used to delete specific DNA sequences; however, the error-prone nature of NHEJ may still produce indel mutations at the site of repair.


Both double strand cleaving eaCas9 molecules and single strand, or nickase, eaCas9 molecules can be used in the methods and compositions described herein to generate NHEJ-mediated indels. NHEJ-mediated indels targeted to the gene, e.g., a coding region, e.g., an early coding region of a gene of interest can be used to knockout (i.e., eliminate expression of) a gene of interest. For example, early coding region of a gene of interest includes sequence immediately following a transcription start site, within a first exon of the coding sequence, or within 500 bp of the transcription start site (e.g., less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp).


Placement of Double Strand or Single Strand Breaks Relative to the Target Position


In an embodiment, in which a gRNA and Cas9 nuclease generate a double strand break for the purpose of inducing NHEJ-mediated indels, a gRNA, e.g., a unimolecular (or chimeric) or modular gRNA molecule, is configured to position one double-strand break in close proximity to a nucleotide of the target position. In an embodiment, the cleavage site is between 0-500 bp away from the target position (e.g., less than 500, 400, 300, 200, 100, 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 bp from the target position).


In an embodiment, in which two gRNAs complexing with Cas9 nickases induce two single strand breaks for the purpose of inducing NHEJ-mediated indels, two gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to position two single-strand breaks to provide for NHEJ repair a nucleotide of the target position. In an embodiment, the gRNAs are configured to position cuts at the same position, or within a few nucleotides of one another, on different strands, essentially mimicking a double strand break. In an embodiment, the closer nick is between 0-30 bp away from the target position (e.g., less than 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 bp from the target position), and the two nicks are within 25-55 bp of each other (e.g., between 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 50 to 55, 45 to 55, 40 to 55, 35 to 55, 30 to 55, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 35 to 45, or 40 to 45 bp) and no more than 100 bp away from each other (e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20 or 10 bp). In an embodiment, the gRNAs are configured to place a single strand break on either side of a nucleotide of the target position.


Both double strand cleaving eaCas9 molecules and single strand, or nickase, eaCas9 molecules can be used in the methods and compositions described herein to generate breaks both sides of a target position. Double strand or paired single strand breaks may be generated on both sides of a target position (e.g., of a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, IX-1, IX-1A, IX-3, or XII-1, or in Section VIII, to remove the nucleic acid sequence between the two cuts (e.g., the region between the two breaks is deleted). In one embodiment, two gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to position a double-strand break on both sides of a target position (e.g., the first gRNA is used to target upstream (i.e., 5′) of the mutation in a gene or pathway described herein, and the second gRNA is used to target downstream (i.e., 3′) of the mutation in a gene or pathway described herein). In an alternate embodiment, three gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to position a double strand break (i.e., one gRNA complexes with a cas9 nuclease) and two single strand breaks or paired single stranded breaks (i.e., two gRNAs complex with Cas9 nickases) on either side of a target position (e.g., the first gRNA is used to target upstream (i.e., 5′) of the mutation in a gene or pathway described herein, and the second gRNA is used to target downstream (i.e., 3′) of the mutation in a gene or pathway described herein). In another embodiment, four gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to generate two pairs of single stranded breaks (i.e., two pairs of two gRNAs complex with Cas9 nickases) on either side of the target position (e.g., the first gRNA is used to target upstream (i.e., 5′) of the mutation in a gene or pathway described herein, and the second gRNA is used to target downstream (i.e., 3′) of the mutation in a gene or pathway described herein). The double strand break(s) or the closer of the two single strand nicks in a pair will ideally be within 0-500 bp of the target position (e.g., no more than 450, 400, 350, 300, 250, 200, 150, 100, 50 or 25 bp from the target position). When nickases are used, the two nicks in a pair are within 25-55 bp of each other (e.g., between 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 50 to 55, 45 to 55, 40 to 55, 35 to 55, 30 to 55, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 35 to 45, or 40 to 45 bp) and no more than 100 bp away from each other (e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20 or 10 bp).


IV.3 Targeted Knockdown


Unlike CRISPR/Cas-mediated gene knockout, which permanently eliminates expression by mutating the gene at the DNA level, CRISPR/Cas knockdown allows for temporary reduction of gene expression through the use of artificial transcription factors. Mutating key residues in both DNA cleavage domains of the Cas9 protein (e.g. the D1OA and H840A mutations) results in the generation of a catalytically inactive Cas9 (eiCas9 which is also known as dead Cas9 or dCas9). A catalytically inactive Cas9 complexes with a gRNA and localizes to the DNA sequence specified by that gRNA's targeting domain, however, it does not cleave the target DNA. Fusion of the dCas9 to an effector domain, e.g., a transcription repression domain, enables recruitment of the effector to any DNA site specified by the gRNA. While it has been show that the eiCas9 itself can block transcription when recruited to early regions in the coding sequence, more robust repression can be achieved by fusing a transcriptional repression domain (for example KRAB, SID or ERD) to the Cas9 and recruiting it to the promoter region of a gene. It is likely that targeting DNAseI hypersensitive regions of the promoter may yield more efficient gene repression or activation because these regions are more likely to be accessible to the Cas9 protein and are also more likely to harbor sites for endogenous transcription factors. Especially for gene repression, it is contemplated herein that blocking the binding site of an endogenous transcription factor would aid in downregulating gene expression. In another embodiment, an eiCas9 can be fused to a chromatin modifying protein. Altering chromatin status can result in decreased expression of the target gene.


In an embodiment, a gRNA molecule can be targeted to a known transcription response elements (e.g., promoters, enhancers, etc.), a known upstream activating sequences (UAS), and/or sequences of unknown or known function that are suspected of being able to control expression of the target DNA.


CRISPR/Cas-mediated gene knockdown can be used to reduce expression of an unwanted allele or transcript. Contemplated herein are scenarios wherein permanent destruction of the gene is not ideal. In these scenarios, site-specific repression may be used to temporarily reduce or eliminate expression. It is also contemplated herein that the off-target effects of a Cas-repressor may be less severe than those of a Cas-nuclease as a nuclease can cleave any DNA sequence and cause mutations whereas a Cas-repressor may only have an effect if it targets the promoter region of an actively transcribed gene. However, while nuclease-mediated knockout is permanent, repression may only persist as long as the Cas-repressor is present in the cells. Once the repressor is no longer present, it is likely that endogenous transcription factors and gene regulatory elements would restore expression to its natural state.


IV.4 Examples of gRNAs in Genome Editing Methods


gRNA molecules as described herein can be used with Cas9 molecules that generate a double strand break or a single strand break to alter the sequence of a target nucleic acid, e.g., a target position or target genetic signature, gRNA molecules useful in these methods are described below.


In an embodiment, the gRNA, e.g., a chimeric gRNA, is configured such that it comprises one or more of the following properties;


a) it can position, e.g., when targeting a Cas9 molecule that makes double strand breaks, a double strand break (i) within 50, 100, 150 or 200 nucleotides of a target position, or (ii) sufficiently close that the target position is within the region of end resection;


b) it has a targeting domain of at least 15, 16, 17, 18, 19 or 20, nucleotides, e.g., a targeting domain of (i) 17, (ii) 18, or (iii) 20 nucleotides; and


c)

    • (i) the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from a naturally occurring S. pyogenes. S. thermophilus, S. aureus, or N. meningitidis tail and proximal domain, or a sequence that differs by no more than 1, 2, 3, 4, 5; 6, 7, 8, 9 or 10 nucleotides therefrom:
      • (ii) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from the corresponding sequence of a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis gRNA, or a sequence that differs by no more than 1, 2, 3, 4, 5; 6, 7, 8, 9 or 10 nucleotides therefrom;
      • (iii) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain, e.g., at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides from the corresponding sequence of a naturally occurring S. pyogenes. S. thermophilus, S. aureus, or N. meningitidis gRNA, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom;
      • iv) the tail domain is at least 10, 15, 20, 25, 30, 35 or 40 nucleotides in length, e.g., it comprises at least 10, 15, 20, 25, 30, 35 or 40 nucleotides from a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail domain; or, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom; or
      • (v) the tail domain comprises 15, 20, 25, 30, 35, 40 nucleotides or all of the corresponding portions of a naturally occurring tail domain, e.g., a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail domain.


In an embodiment, the gRNA is configured such that it comprises properties: a and b(i).


In an embodiment, the gRNA is configured such that it comprises properties: a and b(ii).


In an embodiment, the gRNA is configured such that it comprises properties: a and b(iii).


In an embodiment, the gRNA is configured such that it comprises properties: a and c.


In an embodiment, the gRNA is configured such that in comprises properties: a, b, and c.


In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(i), and c(i).


In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(i), and c(ii).


In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(iii), and c(i).


In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(iii), and c(ii).


In an embodiment, the gRNA, e.g., a chimeric gRNA, is configured such that it comprises one or more of the following properties;


a) it can position, e.g., when targeting a Cas9 molecule that makes single strand breaks, a single strand break (i) within 50, 100, 150 or 200 nucleotides of a target position, or (ii) sufficiently close that the target position is within the region of end resection:


b) it has a targeting domain of at least 15, 16, 17, 18, 19, or 20, nucleotides, e.g., a targeting domain of (i) 17, (ii) 18, or (iii) 20 nucleotides; and


c)

    • (i) the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail and proximal domain, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom;
    • (ii) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from the corresponding sequence of a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis gRNA, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom;
    • (iii) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain, e.g., at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides from the corresponding sequence of a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis gRNA, or a sequence that differs by no more than 1, 2, 3, 4, 5; 6, 7, 8, 9 or 10 nucleotides therefrom;
    • iv) the tail domain is at least 10, 15, 20, 25, 30, 35 or 40 nucleotides in length, e.g., it comprises at least 10, 15, 20, 25, 30, 35 or 40 nucleotides from a naturally occurring S. pyogenes, S. thermophilus. S. aureus, or N. meningitidis tail domain; or, a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom; or
    • (v) the tail domain comprises 15, 20, 25, 30, 35, 40 nucleotides or all of the corresponding portions of a naturally occurring tail domain, e.g., a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail domain.


In an embodiment, the gRNA is configured such that it comprises properties: a and b(i).


In an embodiment, the gRNA is configured such that it comprises properties: a and b(ii).


In an embodiment, the gRNA is configured such that it comprises properties: a and b(iii).


In an embodiment, the gRNA is configured such that it comprises properties: a and c.


In an embodiment, the gRNA is configured such that in comprises properties: a, b, and c.


In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(i), and c(i).


In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(i), and c(ii).


In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(iii), and c(i).


In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(iii), and c(ii).


In an embodiment, the gRNA is used with a Cas9 nickase molecule having HNH activity, e.g., a Cas9 molecule having the RuvC activity inactivated, e.g., a Cas9 molecule having a mutation at D10, e.g., the D1OA mutation.


In an embodiment, the gRNA is used with a Cas9 nickase molecule having RuvC activity, e.g., a Cas9 molecule having the HNH activity inactivated, e.g., a Cas9 molecule having a mutation at H840, e.g., a H840A.


In an embodiment, a pair of gRNAs, e.g., a pair of chimeric gRNAs, comprising a first and a second gRNA, is configured such that they comprises one or more of the following properties;


a) one or both of the gRNAs can position, e.g., when targeting a Cas9 molecule that makes single strand breaks, a single strand break within (i) 50, 100, 150 or 200 nucleotides of a target position, or (ii) sufficiently close that the target position is within the region of end resection;


b) one or both have a targeting domain of at least 17 nucleotides, e.g., a targeting domain of (i) 17 or (ii) 18 nucleotides;


c) one or both:

    • (i) the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from a naturally occurring S. pyogenes, S. thermophilus. S. aureus, or N. meningitidis tail and proximal domain, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom;
    • (ii) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3′ to the last nucleotide of the second complementarity domain, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides from the corresponding sequence of a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis gRNA, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom;
    • (iii) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3′ to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain, e.g., at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides from the corresponding sequence of a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis gRNA, or a sequence that differs by no more than 1, 2, 3, 4, 5; 6, 7, 8, 9 or 10 nucleotides therefrom;
    • iv) the tail domain is at least 10, 15, 20, 25, 30, 35 or 40 nucleotides in length, e.g., it comprises at least 10, 15, 20, 25, 30, 35 or 40 nucleotides from a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail domain; or, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom, or
    • (v) the tail domain comprises 15, 20, 25, 30, 35, 40 nucleotides or all of the corresponding portions of a naturally occurring tail domain, e.g., a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail domain;


d) the gRNAs are configured such that, when hybridized to target nucleic acid, they are separated by 0-50, 0-100, 0-200, at least 10, at least 20, at least 30 or at least 50 nucleotides;


e) the breaks made by the first gRNA and second gRNA are on different strands; and


f) the PAMs are facing outwards.


In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a and b(i).


In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a and b(ii).


In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a and b(iii).


In an embodiment, one or both of the gRNAs configured such that it comprises properties: a and c.


In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a, b, and c.


In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a(i), b(i), and c(i).


In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a(i), b(i), and c(ii).


In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a(i), b(i), c, and d.


In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a(i), b(i), c, and e.


In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a(i), b(i), c, d, and e.


In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a(i), b(iii), and c(i).


In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a(i), b(iii), and c(ii).


In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a(i), b(iii), c, and d.


In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a(i), b(iii), c, and e.


In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a(i), b(iii), c, d, and e.


In an embodiment, the gRNAs are used with a Cas9 nickase molecule having HNH activity, e.g., a Cas9 molecule having the RuvC activity inactivated, e.g., a Cas9 molecule having a mutation at D10, e.g., the D1OA mutation.


In an embodiment, the gRNAs are used with a Cas9 nickase molecule having RuvC activity, e.g., a Cas9 molecule having the HNH activity inactivated, e.g., a Cas9 molecule having a mutation at H840, e.g., a H840A.


V. Constructs/Component


The components, e.g., a Cas9 molecule or gRNA molecule, or both, can be delivered, formulated, or administered in a variety of forms, see, e.g., Table V-1a and Table V-1b. When a component is delivered encoded in DNA the DNA will typically include a control region, e.g., comprising a promoter, to effect expression. Useful promoters for Cas9 molecule sequences include CMV, EF-1a, MSCV, PGK, CAG control promoters. Useful promoters for gRNAs include H1, EF-1a and U6 promoters. Promoters with similar or dissimilar strengths can be selected to tune the expression of components. Sequences encoding a Cas9 molecule can comprise a nuclear localization signal (NLS), e.g., an SV40 NLS. In an embodiment, a promoter for a Cas9 molecule or a gRNA molecule can be, independently, inducible, tissue specific, or cell specific.


Table V-1a and Table V-1b provide examples of how the components can be formulated, delivered, or administered.









TABLE V-1a







Element










Cas9

Template



Mole-
gRNA
Nucleic



cule(s)
molecule(s)
Acid
Comments





DNA
DNA
DNA
In an embodiment, a Cas9 molecule, typically an





eaCas9 molecule, and a gRNA are transcribed





from DNA. In this embodiment, they are





encoded on separate molecules. In an





embodiment, the donor template is provided as a





separate DNA molecule. A governing gRNA





molecule can also be present. It can be encoded





on the molecule that encodes the Cas9 molecule





or the gRNA molecule or can be on a third





nucleic acid molecule. The governing gRNA





molecule can be a Cas9-targeting gRNA





molecule or a gRNA-targeting gRNA molecule.





In an embodiment, both are present. In an





embodiment, the governing gRNA molecule is a





Cas9-targeting gRNA molecule which targets, by





binding and/or cleavage, the sequence that





encodes the Cas9 molecule and results in





substantial reduction of the production of Cas9





molecule. In an embodiment, the governing





gRNA molecule is a gRNA-targeting gRNA





molecule which targets, by binding and/or





cleavage, the sequence that encodes the gRNA





molecule and results in substantial reduction of





the production of gRNA molecule.









DNA
DNA
In an embodiment, a Cas9 molecule, typically an













eaCas9 molecule, and a gRNA are transcribed





from DNA. In this embodiment, they are





encoded on separate molecules. In this





embodiment, the donor template is provided on





the same DNA molecule that encodes thegRNA.





A governing gRNA molecule can also be





present. It can be encoded on the molecule that





encodes the Cas9 molecule and the gRNA





molecule or can be on a second nucleic acid





molecule. The governing gRNA molecule can





be a Cas9-targeting gRNA molecule or a gRNA-





targeting gRNA molecule. In an embodiment,





both are present. In an embodiment, the





governing gRNA molecule is a Cas9-targeting





gRNA molecule which targets, by binding and/or





cleavage, the sequence that encodes the Cas9





molecule and results in substantial reduction of





the production of Cas9 molecule. In an





embodiment, the governing gRNA molecule is a





gRNA-targeting gRNA molecule which targets,





by binding and/or cleavage, the sequence that





encodes the gRNA molecule and results in





substantial reduction of the production of gRNA





molecule.









DNA
DNA
In an embodiment, a Cas9 molecule,typically an













eaCas9 molecule, and a gRNA are transcribed





from DNA, here from a single molecule. In this





embodiment, the donor template is provided as a





separate DNA molecule. A governing gRNA





molecule can also be present. It can be encoded





on the molecule that encodes the Cas9 molecule





and the gRNA molecule or can be on a second





nucleic acid molecule. The governing gRNA





molecule can be a Cas9-targeting gRNA





molecule or a gRNA-targeting gRNA molecule.





In an embodiment, both are present. In an





embodiment, the governing gRNA molecule is a





Cas9-targeting gRNA molecule which targets, by





binding and/or cleavage, the sequence that





encodes the Cas9 molecule and results in





substantial reduction of the production of Cas9





molecule. In an embodiment, the governing





gRNA molecule is a gRNA-targeting gRNA





molecule which targets, by binding and/or





cleavage, the sequence that encodes the gRNA





molecule and results in substantial reduction of





the production of gRNA molecule.


DNA
DNA
DNA
In an embodiment, a Cas9 molecule, typically an





eaCas9 molecule, and a gRNA are transcribed





from DNA. In this embodiment, they are





encoded on separate molecules. In this





embodiment, the donor template is provided on





the same DNA molecule that encodes the Cas9.





A governing gRNA molecule can also be





present. It can be encoded on the molecule that





encodes the Cas9 molecule or the gRNA





molecule or can be on a third nucleic acid





molecule. The governing gRNA molecule can





be a Cas9-targeting gRNA molecule or a gRNA-





targeting gRNA molecule. In an embodiment,





both are present. In an embodiment, the





governing gRNA molecule is a Cas9-targeting





gRNA molecule which targets, by binding and/or





cleavage, the sequence that encodes the Cas9





molecule and results in substantial reduction of





the production of Cas9 molecule. In an





embodiment, the governing gRNA molecule is a





gRNA-targeting gRNA molecule which targets,





by binding and/or cleavage, the sequence that





encodes the gRNA molecule and results in





substantial reduction of the production of gRNA





molecule.


DNA
RNA
DNA
In an embodiment, a Cas9 molecule, typically an





eaCas9 molecule, is transcribed from DNA, and





a gRNA is provided as in vitro transcribed or





synthesized RNA. In this embodiment, the





donor template is provided as a separate DNA





molecule. In an embodiment, the gRNA





comprises one or more modifications, e.g., as





described in Section X. A governing gRNA





molecule can also be present. It can be encoded





on the molecule that encodes the Cas9 molecule





or can be on a second nucleic acid molecule. In





an embodiment, the governing gRNA molecule





is a Cas9-targeting gRNA molecule which





targets, by binding and/or cleavage, the sequence





that encodes the Cas9 molecule and results in





substantial reduction of the production of Cas9





molecule.


DNA
RNA
DNA
In an embodiment, a Cas9 molecule, typically an





eaCas9 molecule, is transcribed from DNA, and





a gRNA is provided as in vitro transcribed or





synthesized RNA. In this embodiment, the





donor template is provided on the same DNA





molecule that encodes the Cas9. In an





embodiment, the gRNA comprises one or more





modifications, e.g., as described in Section X. A





governing gRNA molecule can also be present.





It can be encoded on the molecule that encodes





the Cas9 molecule or can be on a second nucleic





acid molecule. In an embodiment the governing





gRNA molecule is a Cas9-targeting gRNA





molecule which targets, by binding and/or





cleavage, the sequence that encodes the Cas9





molecule and results in substantial reduction of





the production of Cas9 molecule.


mRNA
RNA
DNA
In an embodiment, a Cas9 molecule, typically an





eaCas9 molecule, is translated from in vitro





transcribed mRNA, and a gRNA is provided as





n vitro transcribed or synthesized RNA. In this





embodiment, the donor template is provided as a





DNA molecule. In an embodiment, the gRNA





comprises one or more modifications, e.g., as





described in Section X. In an embodiment, the





mRNA comprises one or more modifications,





e.g., as described in Section X.


mRNA
DNA
DNA
In an embodiment, a Cas9 molecule, typically a





eaCas molecule, is translated from in vitro





transcribed mRNA, and a gRNA is transcribed





from DNA. In this embodiment, the donor





template is provided as a separate DNA





molecule. In an embodiment, the mRNA





comprises one or more modifications, e.g., as





described in Section X. A governing gRNA





molecule can also be present. It can he encoded





on the molecule that encodes the gRNA





molecule or can be on a second nucleic acid





molecule. In an embodiment, the governing





gRNA molecule is a gRNA-targeting gRNA





molecule which targets, by binding and/or





cleavage, the sequence that encodes the gRNA





molecule and results in substantial reduction of





the production of gRNA molecule.









mRNA
DNA
In an embodiment, a Cas9 molecule, typically an













eaCas9 molecule, is translated from in vitro





transcribed mRNA, and a gRNA is transcribed





from DNA. In this embodiment, the donor





template is provided on the same DNA molecule





that encodes the gRNA In an embodiment, the





mRNA comprise one or more modifications,





e.g., as described in Section X. A governing





gRNA molecule can also be present. It can be





encoded on the molecule that encodes the gRNA





molecule or can be on a second nucleic acid





molecule. In an embodiment, the governing





gRNA molecule is a gRNA-targeting gRNA





molecule which targets, by binding and/or





cleavage, the sequence that encodes the gRNA





molecule and results in substantial reduction of





the production of gRNA molecule.


Protein
DNA
DNA
In an embodiment, a Cas9 molecule, typically an





eaCas9 molecule, is provided as a protein, and a





gRNA is transcribed from DNA. In this





embodiment, the donor template is provided as a





separate DNA molecule. A governing gRNA





molecule can also be present. It can be encoded





on the molecule that encodes the gRNA





molecule or can be on a second nucleic acid





molecule. In an embodiment the governing





gRNA molecule is a gRNA-targeting gRNA





molecule which targets, by binding and/or





cleavage, the sequence that encodes the gRNA





molecule and results in substantial reduction of





the production of gRNA molecule.









Protein
DNA
In an embodiment, a Cas9 molecule, typically an













eaCas9 molecule, is provided as a protein, and a





gRNA is transcribed from DNA. In this





embodiment, the donor template is provided on





the same DNA molecule that encodes the gRNA.





A governing gRNA molecule can also be





present. It can be encoded on the molecule that





encodes the gRNA molecule or can be on a





second nucleic acid molecule. In an embodiment





the governing gRNA molecule is a gRNA-





targeting gRNA molecule which targets, by





binding and/or cleavage, the sequence that





encodes the gRNA molecule and results in





substantial reduction of the production of gRNA





molecule.


Protein
RNA
DNA
In an embodiment, an eaCas9 molecule is





provided as a protein, and a gRNA is provided as





transcribed or synthesized RNA. In this





embodiment, the donor template is provided as a





DNA molecule. In an embodiment, the gRNA





comprises one or more modifications, e.g., as





described in Section X.
















TABLE V-1b







Element










Cas9
gRNA




Molecule(s)
molecule(s)
Payload
Comments





DNA
DNA
Yes
In this embodiment, a Cas9 molecule, typically





an eiCas9 molecule, and a gRNA are transcribed





from DNA. Here they are provided on separate





molecules. A governing gRNA molecule can





also be present. II can be encoded on the





molecule that encodes the Cas9 molecule or the





gRNA molecule or can be on athird nucleic acid





molecule. The governing gRNA molecule can





be a Cas9-targeting gRNA or molecule or a





gRNA-targeting gRNA molecule. In an





embodiment, both are present. In an





embodiment, the governing gRNA molecule is a





Cas9-targeting gRNA molecule which targets,





by binding and/or cleavage, the sequence that





encodes the Cas9 molecule and results in





substantial reduction of the production of Cas9





molecule. In an embodiment, the governing





gRNA molecule is a gRNA-targeting gRNA





molecule which targets, by binding and/or





cleavage, the sequence that encodes the gRNA





molecule and results in substantial reduction of





the production of gRNA molecule.









DNA
Yes
Similar to above, but in this embodiment, a Cas9













molecule, typically an eiCas9 molecule, and a





gRNA are transcribed from a single molecule. A





governing gRNA molecule can also be present.





It can be encoded on the molecule that encodes





the Cas9 molecule or the gRNA molecule or can





be on a second nucleic acid molecule. The





governing gRNA molecule can be a Cas9-





targeting RNA molecule or a gRNA-targeting





gRNA molecule. In an embodiment, both are





present. In an embodiment, the governing





gRNA molecule is a Cas9-targeting gRNA





molecule which targets, by binding and/or





cleavage, the sequence that encodes the Cas9





molecule and results in substantial reduction of





the production of Cas9 molecule. In an





embodiment, the governing gRNA molecule is a





gRNA-targeting gRNA molecule which targets,





by binding and/or cleavage, the sequence that





encodes the gRNA molecule and results in





substantial reduction of the production of gRNA





molecule.


DNA
RNA
Yes
In this embodiment, a Cas9 molecule, typically





an eiCas9 molecule, is transcribed from DNA.





A gRNA is provided as RNA. In an





embodiment, the gRNA comprises one or more





modifications, e.g., as described in Section X.





A governing gRNA molecule can also be





present. It can be encoded on the molecule that





encodes the Cas9 molecule or can be on a





second nucleic acid molecule. In an





embodiment the governing gRNA molecule is a





Cas9-targeting gRNA molecule which targets,





by binding and/or cleavage, the sequence that





encodes the Cas9 molecule and results in





substantial reduction of the production of Cas9





molecule.


mRNA
RNA
Yes
In this embodiment, a Cas9 molecule, typically





an eiCas9 molecule, is provided as encoded in





mRNA. A gRNA is provided as RNA. In an





embodiment, the gRNA comprises one or more





modifications, e.g., as described in Section X.





In an embodiment, the mRNA comprises one or





more modifications, e.g., as described in section





X.


Protein
DNA
Yes
In this embodiment a Cas9 molecule, typically





an eiCas9 molecule, is provided as a protein. A





gRNA is provided encoded in DNA. A





governing gRNA molecule can also be present.





It can be encoded on the molecule that encodes





the gRNA molecule or can be on a second





nucleic acid molecule. In an embodiment the





governing gRNA molecule is a gRNA-targeting





gRNA molecule which targets, by binding





and/or cleavage, the sequence that encodes the





gRNA molecule and results in substantial





reduction of the production of the gRNA





molecule.


Protein
RNA
Yes
In this embodiment, a Cas9 molecule, typically





an eiCas9 molecule, is provided as a protein. A





gRNA is provided as RNA. In an embodiment,





the gRNA comprises one or more modifications,





e.g., as described in Section X.









In an embodiment, the components of a Cas system are delivered in vivo, e.g., using a method describe herein. In another embodiment, the components a Cas system are delivered ex vivo, e.g., using a method described herein.


Table V-2 summarizes various delivery methods the components of a Cas system, e.g., the Cas9 molecule component and the gRNA molecule component are described herein, e.g., in Table V-2.













TABLE V-2






Delivery






into Non-
Duration
Genome
Type of



Dividing
of Ex-
Integra-
Molecule


Delivery Vector/Mode
Cells
pression
tion
Delivered







Physical (eg,
YES
Transient
NO
Nucleic


electroporation,



Acids


particle gun, Calcium



and


Phosphate



Proteins


transfection)
















Viral
Retrovirus
NO
Stable
YES
RNA



Lentivirus
YES
Stable
YES/NO
RNA






with mod-







ifications




Adenovirus
YES
Transient
NO
DNA



Adeno-
YES
Stable
NO
DNA



Associated







Virus (AAV)







Vaccinia
YES
Very
NO
DNA



Virus

Transient





Herpes
YES
Stable
NO
DNA



Simplex







Virus






Non-
Cationic
YES
Transient
Depends
Nucleic


Viral
Liposomes


on what is
Acids






delivered
and







Proteins



Polymeric
YES
Transient
Depends
Nucleic



Nanoparticles


on what is
Acids






delivered
and







Proteins


Bio-
Attenuated
YES
Transient
NO
Nucleic


logical
Bacteria



Acids


Non-
Engineered
YES
Transient
NO
Nucleic


Viral
Bacterio-



Acids


Delivery
phages






Vehicles
Mammalian
YES
Transient
NO
Nucleic



Virus-like



Acids



Particles







Biological
YES
Transient
NO
Nucleic



liposomes:



Acids



Erythrocyte







Ghosts and







Exosomes









DNA-Based Delivery of a Cas9 Molecule and or a gRNA Molecule


DNA encoding Cas9 molecules (e.g., eaCas9 molecules or eiCas9 molecules), gRNA molecules, and/or template nucleic acids, can be administered to subjects or delivered into cells by art-known methods or as described herein. For example, Cas9-encoding and/or gRNA-encoding DNA can be delivered, e.g., by vectors (e.g., viral or non-viral vectors), non-vector based methods (e.g., using naked DNA or DNA complexes), or a combination thereof.


In an embodiment, the DNA includes a nucleic acid that encodes a governing gRNA molecule. The governing gRNA molecule can complex with the Cas9 molecule to inactivate or silence a component of the system, e.g., the nucleic acid that encodes the Cas9 molecule or the nucleic acid that encodes the gRNA molecule. In either case, the governing gRNA, e.g., a Cas9-targeting gRNA molecule, or a gRNA targeting gRNA molecule, limits the effect of the Cas9/gRNA complex mediated gene targeting, and can place temporal limits on activity or reduce off-target activity.


In an embodiment, the Cas9- and/or gRNA-encoding DNA is delivered by a vector (e.g., viral vector/virus or plasmid).


A vector can comprise a sequence that encodes a Cas9 molecule and/or a gRNA molecule. A vector can also comprise a sequence encoding a signal peptide (e.g., for nuclear localization, nucleolar localization, mitochondrial localization), fused, e.g., to a Cas9 molecule sequence. For example, a vector can comprise a nuclear localization sequence (e.g., from SV40) fused to the sequence encoding the Cas9 molecule.


One or more regulatory/control elements, e.g., a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, internal ribosome entry sites (IRES), a 2A sequence, and a splice acceptor or donor can be included in the vectors. In an embodiment, the promoter is recognized by RNA polymerase II (e.g., a CMV promoter). In an embodiment, the promoter is recognized by RNA polymerase III (e.g., a U6 promoter). In an embodiment, the promoter is a regulated promoter (e.g., inducible promoter). In an embodiment, the promoter is a constitutive promoter. In an embodiment, the promoter is a tissue specific promoter. In an embodiment, the promoter is a viral promoter. In an embodiment, the promoter is a non-viral promoter.


In an embodiment, the vector or delivery vehicle is a viral vector (e.g., for generation of recombinant viruses). In an embodiment, the virus is a DNA virus (e.g., dsDNA or ssDNA virus). In an embodiment, the virus is an RNA virus (e.g., an ssRNA virus). Exemplary viral vectors/viruses include, e.g., retroviruses, lentiviruses, adenovirus, adeno-associated virus (AAV), vaccinia viruses, poxviruses, and herpes simplex viruses. In an embodiment, the viral vector, e.g., an AAV, comprises a sequence that encodes a governing gRNA molecule, e.g., a Cas9-targeting gRNA molecule or a gRNA-targeting gRNA molecule.


In an embodiment, the viral vector has the ability of cell type and/or tissue type recognition. For example, the viral vectors can be pseudotyped with different/alternative viral envelope glycoproteins; engineered with cell type-specific receptors (e.g., genetically modification of viral envelope glycoproteins to incorporate targeting ligands such as peptide ligands, single chain antibodies, growth factors); and/or engineered to have a molecular bridge with dual specificities with one end recognizing viral glycoproteins and the other end recognizing a moiety of the target cell surface (e.g., ligand-receptor, monoclonal antibodies, avidin-biotin and chemical conjugation).


In an embodiment, the viral vector achieves cell type specific expression. For example, tissue-specific promoter can be constructed to restrict expression of the transgene (Cas 9 and gRNA) in only the target cells. The specificity of the vectors can also be mediated by microRNA-dependent control of transgene expression. In an embodiment, the viral vector has increased efficiency of fusion of viral vector and target cell membrane. For example, fusion proteins such as fusion-competent hemagglutin (HA) can be incorporated to increase viral uptake into cells. In an embodiment, the viral vector has the ability of nuclear localization. For example, certain viruses that require the breakdown of the cell wall (during cell division) will not infect non-diving cell. Incorporated nuclear localization peptides into the matrix proteins of the virus allow transduction into non-proliferating cells.


In an embodiment, the virus infects dividing cells. In an embodiment, the virus infects non-dividing cells. In an embodiment, the virus infects both dividing and non-dividing cells. In an embodiment, the virus can integrate into the host genome. In an embodiment, the virus is engineered to have reduced immunity, e.g., in human. In an embodiment, the virus is replication-competent. In an embodiment, the virus is replication-defective, e.g., having one or more coding regions for the genes necessary for additional rounds of virion replication and/or packaging replaced with other genes or deleted. In an embodiment, the virus causes transient expression of the Cas9 molecule and/or the gRNA molecule. In an embodiment, the virus causes long-lasting, e.g., at least 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months, 9 months, 1 year, 2 years, or permanent expression, of the Cas9 molecule and/or the gRNA molecule. The packaging capacity of the viruses may vary, e.g., from at least about 4 kb to at least about 30 kb, e.g., at least about 5 kb, 10 kb, 15 kb, 20 kb, 25 kb, 30 kb, 35 kb, 40 kb, 45 kb, or 50 kb.


In an embodiment, the Cas9- and/or gRNA-encoding DNA is delivered by a recombinant retrovirus. In an embodiment, the retrovirus (e.g., Moloney murine leukemia virus) comprises a reverse transcriptase, e.g., that allows integration into the host genome. In an embodiment, the retrovirus is replication-competent. In an embodiment, the retrovirus is replication-defective, e.g., having one of more coding regions for the genes necessary for additional rounds of virion replication and packaging replaced with other genes, or deleted.


In an embodiment, the Cas9- and/or gRNA-encoding DNA is delivered by a recombinant lentivirus. For example, the lentivirus is replication-defective, e.g., does not comprise one or more genes required for viral replication.


In an embodiment, the Cas9- and/or gRNA-encoding DNA is delivered by a recombinant adenovirus. In an embodiment, the adenovirus is engineered to have reduced immunity in human.


In an embodiment, the Cas9- and/or gRNA-encoding DNA is delivered by a recombinant AAV. In an embodiment, the AAV can incorporate its genome into that of a host cell, e.g., a target cell as described herein. In an embodiment, the AAV is a self-complementary adeno-associated virus (scAAV), e.g., a scAAV that packages both strands which anneal together to form double stranded DNA. AAV serotypes that may be used in the disclosed methods include, e.g., AAV1, AAV2, modified AAV2 (e.g., modifications at Y444F, Y500F, Y730F and/or S662V), AAV3, modified AAV3 (e.g., modifications at Y705F, Y73F and/or T492V), AAV4, AAV5, AAV6, modified AAV6 (e.g., modifications at S663V and/or T492V), AAV8, AAV 8.2, AAV9, AAV rh 10, and pseudo typed AAV, such as AAV2/8, AAV2/5 and AAV2/6 can also be used in the disclosed methods.


In an embodiment, the Cas9- and/or gRNA-encoding DNA is delivered by a hybrid virus, e.g., a hybrid of one or more of the viruses described herein.


A packaging cell is used to form a virus particle that is capable of infecting a host or target cell. Such a cell includes a 293 cell, which can package adenovirus, and a j/2 cell or a PA317 cell, which can package retrovirus. A viral vector used in gene therapy is usually generated by a producer cell line that packages a nucleic acid vector into a viral particle. The vector typically contains the minimal viral sequences required for packaging and subsequent integration into a host or target cell (if applicable), with other viral sequences being replaced by an expression cassette encoding the protein to be expressed. For example, an AAV vector used in gene therapy typically only possesses inverted terminal repeat (ITR) sequences from the AAV genome which are required for packaging and gene expression in the host or target cell. The missing viral functions are supplied in trans by the packaging cell line. Henceforth, the viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line is also infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV.


In an embodiment, the viral vector has the ability of cell type and/or tissue type recognition. For example, the viral vector can be pseudotyped with a different/alternative viral envelope glycoprotein: engineered with a cell type-specific receptor (e.g., genetic modification of the viral envelope glycoproteins to incorporate targeting ligands such as a peptide ligand, a single chain antibody, a growth factor); and/or engineered to have a molecular bridge with dual specificities with one end recognizing a viral glycoprotein and the other end recognizing a moiety of the target cell surface (e.g., ligand-receptor, monoclonal antibody, avidin-biotin and chemical conjugation).


In an embodiment, the viral vector achieves cell type specific expression. For example, a tissue-specific promoter can be constructed to restrict expression of the transgene (Cas 9 and gRNA) in only the target cell. The specificity of the vector can also be mediated by microRNA-dependent control of transgene expression. In an embodiment, the viral vector has increased efficiency of fusion of the viral vector and a target cell membrane. For example, a fusion protein such as fusion-competent hemagglutin (HA) can be incorporated to increase viral uptake into cells. In an embodiment, the viral vector has the ability of nuclear localization. For example, aviruse that requires the breakdown of the cell wall (during cell division) and therefore will not infect a non-diving cell can be altered to incorporate a nuclear localization peptide in the matrix protein of the virus thereby enabling the transduction of non-proliferating cells. In an embodiment, the Cas9- and/or gRNA-encoding DNA is delivered by a non-viral vector or non-vector based method (e.g., using naked DNA or DNA complexes). For example, the DNA can be delivered, e.g., by organically modified silica or silicate (Ormosil), electroporation, gene gun, sonoporation, magnetofection, lipid-mediated transfection, dendrimers, inorganic nanoparticles, calcium phosphates, or a combination thereof. In an embodiment, the DNA is delivered by an inorganic nanoparticle (e.g., attached to the payload to the surface of the nanoparticle). Exemplary inorganic nanoparticles include, e.g., magnetic nanoparticles (e.g., Fe3Mn02), silica (e.g., can integrate multi-functionality, e.g., conjugate the outer surface of the nanoparticle with a positively charged polymer (e.g., polyethylenimine, polylysine, polyserine) which allows for attachment (e.g., conjugation or entrapment) of payload and internal magnetic component, mesaporous silica nanoparticles with a positive charged polymer loaded with chloroquine to enhance transfection of the non-viral vector in vitro, high density lipoproteins and gold nanoparticles, gold nanoparticles coated with payload which gets released when nanoparticles are exposed to increased temperature by exposure to near infrared light, gold, iron or silver nanoparticles with surface modified with polylysine or another charge polymer to capture the nucleic acid cargo. In an embodiment, the DNA is delivered by an organic nanoparticle (e.g., entrapment of the payload inside the nanoparticle). Exemplary organic nanoparticles include, e.g., SNALP liposomes that contain cationic lipids together with neutral helper lipids which are coated with polyethylene glycol (PEG) and protamine and nucleic acid complex coated with lipid coating.


In an embodiment, the delivery vehicle is a physical vehicle. In an embodiment, the vehicle is low density ultrasound. For example, microbubbles containing payload (e.g., made of biocompatible material such protein, surfactant, or biocompatible polymer or lipid shell) can be used and the microbubbles can be destructed by a focused ultrasound bean during microvascular transit. In an embodiment, the vehicle is electroporation. For example, naked nucleic acids or proteins can be delivered by electroporation, e.g., into cell suspensions or tissue environment, such as retina and embryonic tissue. In an embodiment, the vehicle is needle or jet injection. For example, naked nucleic acids or protein can be injected into, e.g., muscular, liver, skin, brain or heart tissue.


In an embodiment, the Cas9- and/or gRNA-encoding DNA is delivered by a combination of a vector and a non-vector based method. For example, a virosome comprises a liposome combined with an inactivated virus (e.g., HIV or influenza virus), which can result in more efficient gene transfer, e.g., in a respiratory epithelial cell than either a viral or a liposomal method alone.


In an embodiment, the delivery vehicle is a non-viral vector. In an embodiment, the non-viral vector is an inorganic nanoparticle (e.g., attached to the payload to the surface of the nanoparticle). Exemplary inorganic nanoparticles include, e.g., magnetic nanoparticles (e.g., Fe3Mn02, or silica. The outer surface of the nanoparticle can be conjugated with a positively charged polymer (e.g., polyethylenimine, polylysine, polyserine) which allows for attachment (e.g., conjugation or entrapment) of payload. In an embodiment, the non-viral vector is an organic nanoparticle (e.g., entrapment of the payload inside the nanoparticle). Exemplary organic nanoparticles include, e.g., SNALP liposomes that contain cationic lipids together with neutral helper lipids which are coated with polyethylene glycol (PEG) and protamine and nucleic acid complex coated with lipid coating.


Exemplary lipids for gene transfer are shown in Table V-3.









TABLE V-3







Lipids Used for Gene Transfer









Lipid
Abbreviation
Feature





1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine
DOPC
Helper


1,2-Dioleoyl-sn-glycero-3-phosphatidylethanolamine
DOPE
Helper


Cholesterol

Helper


N-[1-(2,3-Dioleyloxy)prophyl]N,N,N-trimethylammonium
DOTMA
Cationic


chloride




1,2-Dioleoyloxy-3-trimethylammonium-propane
DOTAP
Cationic


Dioctadecylamidoglycylspermine
DOGS
Cationic


N-(3-Aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-
GAP-DLRIE
Cationic


propanaminium bromide




Cetyltrimethylammonium bromide
CTAB
Cationic


6-Lauroxyhexyl omithinate
LHON
Cationic


1-(2,3-Dioleoyoxypropyl)-2,4,6-trimethylpyridinium
20c
Cationic


2,3-Dioleyloxy-N-[2(sperminecarboxamido-ethyl]-N,N-dimethyl-
DOSPA
Cationic


1-propanaminium trifluoroacetate




1,2-Dioleyl-3-trimethylammonium-propane
DOPA
Cationic


N-(2-Hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy-l-
MDRIE
Cationic


propanaminium bromide




Dimyristooxypropyl dimethyl hydroxyethyl ammonium bromide
DMRI
Cationic


3-[N-(N′,N′-Dimethylaminoethane)-carbamoyl]cholesterol
DC-Chol
Cationic


Bis-guanidium-tren-cholesterol
BGTC
Cationic


1,3-Diodeoxy-2-(6-carboxy-spermyl)-propylamide
DOSPER
Cationic


Dimethyloctadecylammonium bromide
DDAB
Cationic


Dioctadecylamidoglicylspermidin
DSL
Cationic


rac-[(2,3-Dioctadecyloxypropyl)(2-hydroxyethyl)]-
CLIP-1
Cationic


dimethylammonium chloride




rac-[2(2,3-Dihexadecyloxypropyl-
CLIP-6
Cationic


oxymethyloxy)ethyl]trimethylammonium bromide




Ethyldimyristoylphosphatidycholine
EDMPC
Cationic


1,2-Distearyloxy-N,N-dimethyl-3-aminopropane
DSDMA
Cationic


1,2-Dimyristoyl-trimethylammonium propane
DMTAP
Cationic


0,0′-Dimyristyl-N-lysyl aspartate
DMKE
Cationic


1,2-Distearoyl-sn-glycero-3-ethylphosphocholine
DSEPC
Cationic


N-Palmitoyl D-erythro-sphingosyl carbamoyl-spermine
CCS
Cationic


N-t-Butyl-NO-tetradecyl-3-tetradecylaminopropionamidine
diC14-amidine
Cationic


Octadecenolyoxy[ethyl-2-heptadecenyl-3 hydroxyethyl]
DOTIM
Cationic


imidazolinium chloride




N1-Cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine
CDAN
Cationic


2-(3-[Bis(3-amino-propyl)-amino]propylamino)-N-
RPR209120
Cationic


ditetradecylcarbamoylme-ethyl-acetamide




1,2-dilinoleyloxy-3-dimethylaminopropane
DLinDMA
Cationic


2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane
DLin-KC2-
Cationic



DMA



dilinoleyl-methyl-4-dimethylaminobutyrate
DLin-MC3-
Cationic



DMA









Exemplary polymers for gene transfer are shown below in Table V-4.









TABLE V-4







Polymers Used for Gene Transfer










Polymer
Abbreviation







Poly(ethylene)glycol
PEG



Polyethylenimine
PEI



Dithiobis(succinimidylpropionate)
DSP



Dimethyl-3,3′-dithiobispropionimidate
DTBP



Poly(ethylene imine)biscarbamate
PEIC



Poly(L-lysine)
PLL



Histidine modified PLL




Poly(N-vinylpyrrolidone)
PVP



Poly(propylenimine)
PPI



Poly(amidoamine)
PAMAM



Poly(amidoethylenimine)
SS-PAEI



Triethylenetetramine
TETA



Poly(-aminoester)




Poly(4-hydroxy-L-proline ester)
PHP



Poly(allylamine)




Poly(a-[4-aminobutyl]-L-glycolic acid)
PAGA



Poly(D,L-lactic-co-glycolie acid)
PLGA



Poly(N-ethyl-4-vinylpyridinium bromide)




Poly(phosphazene)s
PPZ



Poly(phosphoester)s
PPE



Poly(phosphoramidate)s
PPA



Poly(N-2-hydroxypropylmethacrylamide)
pHPMA



Poly (2-dimethylamino)ethyl methacrylate)
pDMAEMA



Poly(2-aminoethyl propylene phosphate)
PPE-EA



Chitosan




Galactosylated chitosan




N-Dodacylated chitosan




Histone




Collagen




Dextran-spermine
D-SPM










In an embodiment, the vehicle has targeting modifications to increase target cell uptake of nanoparticles and liposomes, e.g., cell specific antigens, monoclonal antibodies, single chain antibodies, aptamers, polymers, sugars, and cell penetrating peptides. In an embodiment, the vehicle uses fusogenic and endosome-destabilizing peptides/polymers. In an embodiment, the vehicle undergoes acid-triggered conformational changes (e.g., to accelerate endosomal escape of the cargo). In an embodiment, a stimuli-cleavable polymer is used, e.g., for release in a cellular compartment. For example, disulfide-based cationic polymers that are cleaved in the reducing cellular environment can be used.


In an embodiment, liposomes are used for delivery, e.g., to blood or bone marrow, e.g., as a way of targeting hematopoietic stem cells (HSCs) and progenitors. For example, long-term treatment can be enabled by direct delivery using liposomes for conditions where obtaining HSCs is difficult (e.g., HSCs are not stable or HSCs are rare). These conditions can include, e.g., sickle cell anemia, Fanconi anemia, and aplastic anemia. In an embodiment, liposomes are used for delivery to localized specific tissues, e.g., to liver or lung, via intravenous delivery or via localized injection to target organ or its blood flow. For example, long-term treatment can be enable to concentrate effect in that specific organ or tissue type. These conditions can include urea cycle disorders, alpha-I-anti-trypsin or cystic fibrosis.


In an embodiment, the delivery vehicle is a biological non-viral delivery vehicle. In an embodiment, the vehicle is an attenuated bacterium (e.g., naturally or artificially engineered to be invasive but attenuated to prevent pathogenesis and expressing the transgene (e.g., Listeria monocytogenes, certain Salmonella strains, Bifidobacterium longum, and modified Escherichia coli), bacteria having nutritional and tissue-specific tropism to target specific tissues, bacteria having modified surface proteins to alter target tissue specificity). In an embodiment, the vehicle is a genetically modified bacteriophage (e.g., engineered phages having large packaging capacity, less immunogenic, containing mammalian plasmid maintenance sequences and having incorporated targeting ligands). In an embodiment, the vehicle is a mammalian virus-like particle. For example, modified viral particles can be generated (e.g., by purification of the “empty” particles followed by ex vivo assembly of the virus with the desired cargo). The vehicle can also be engineered to incorporate targeting ligands to alter target tissue specificity. In an embodiment, the vehicle is a biological liposome. For example, the biological liposome is a phospholipid-based particle derived from human cells (e.g., erythrocyte ghosts, which are red blood cells broken down into spherical structures derived from the subject (e.g., tissue targeting can be achieved by attachment of various tissue or cell-specific ligands), or secretory exosomes-subject (i.e., patient) derived membrane-bound nanovesicle (30-100 nm) of endocytic origin (e.g., can be produced from various cell types and can therefore be taken up by cells without the need of for targeting ligands).


In an embodiment, delivery of Cas components by nanoparticles in the bone marrow is an in vivo approach to curing blood and immune diseases.


In an embodiment, the components of a Cas system, e.g., the Cas9 molecule component and the gRNA molecule component described herein is delivered by nucleofection. For example, Nucleofector™ (Lonza Cologne AG) is a transfection technology that can be used for delivery to primary cells and difficult-to-transfect cell lines. It is a non-viral method based on a combination of electrical parameters and cell-type specific solutions. It allows transfected nucleic acids to directly enter the nucleus (e.g., without relying on cell division for the transfer of nucleic acids into the nucleus), providing the ability to transfect non-dividing cells, such as neurons and resting blood cells. In an embodiment, nucleofection is used as an ex vivo delivery method.


In an embodiment, the components of a Cas system, e.g., the Cas9 molecule component and the gRNA molecule component described herein is delivered by methods utilizing endogenous receptor-mediate transporters, e.g., antibody-based molecular Trojan Horses (ArmaGen). Such methods can allow for non-invasive delivery of therapeutics to locations that are otherwise difficult to reach, e.g., brain (e.g., to cross blood brain barrier (BBB), e.g., via endogenous receptor-mediated transport processes).


In an embodiment, one or more nucleic acid molecules (e.g., DNA molecules) other than the components of a Cas system, e.g., the Cas9 molecule component and/or the gRNA molecule component described herein, are delivered. In an embodiment, the nucleic acid molecule is delivered at the same time as one or more of the components of the Cas system are delivered. In an embodiment, the nucleic acid molecule is delivered before or after (e.g., less than about 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 4 weeks) one or more of the components of the Cas system are delivered. In an embodiment, the nucleic acid molecule is delivered by a different means than one or more of the components of the Cas system, e.g., the Cas9 molecule component and/or the gRNA molecule component, are delivered. The nucleic acid molecule can be delivered by any of the delivery methods described herein. For example, the nucleic acid molecule can be delivered by a viral vector, e.g., an integration-deficient lentivirus, and the Cas9 molecule component and/or the gRNA molecule component can be delivered by electroporation, e.g., such that the toxicity caused by nucleic acids (e.g., DNAs) can be reduced. In an embodiment, the nucleic acid molecule encodes a therapeutic protein, e.g., a protein described herein. In an embodiment, the nucleic acid molecule encodes an RNA molecule, e.g., an RNA molecule described herein.


Delivery of RNA Encoding a Cas9 Molecule


RNA encoding Cas9 molecules (e.g., eaCas9 molecules, eiCas9 molecules or eiCas9 fusion proteins) and/or gRNA molecules, can be delivered into cells, e.g., target cells described herein, by art-known methods or as described herein. For example, Cas9-encoding and/or gRNA-encoding RNA can be delivered, e.g., by microinjection, electroporation, lipid-mediated transfection, peptide-mediated delivery, or a combination thereof.


Delivery Cas9 Molecule Protein


Cas9 molecules (e.g., eaCas9 molecules, eiCas9 molecules or eiCas9 fusion proteins) can be delivered into cells by art-known methods or as described herein. For example, Cas9 protein molecules can be delivered, e.g., by microinjection, electroporation, lipid-mediated transfection, peptide-mediated delivery, or a combination thereof. Delivery can be accompanied by DNA encoding a gRNA or by a gRNA.


Route of Administration


Systemic modes of administration include oral and parenteral routes. Parenteral routes include, by way of example, intravenous, intrarterial, intraosseous, intramuscular, intradermal, subcutaneous, intranasal and intraperitoneal routes. Components administered systemically may be modified or formulated to target the components to a specific organ or cell type.


Local modes of administration include, by way of example, intrathecal, intracerebroventricular, intraparenchymal (e.g., localized intraparenchymal delivery to the striatum (e.g., into the caudate or into the putamen)), cerebral cortex, precentral gyrus, hippocampus (e.g., into the dentate gyrus or CA3 region), temporal cortex, amygdala, frontal cortex, thalamus, cerebellum, medulla, hypothalamus, tectum, tegmentum or substantia nigra intraocular, intraorbital, subconjuctival, intravitreal, subretinal or transscleral routes. In an embodiment, significantly smaller amounts of the components (compared with systemic approaches) may exert an effect when administered locally (for example, intraparenchymal or intravitreal) compared to when administered systemically (for example, intravenously). Local modes of administration can reduce or eliminate the incidence of potentially toxic side effects that may occur when therapeutically effective amounts of a component are administered systemically.


In an embodiment, components described herein are delivered by intraparenchymal injection into discrete regions of the brain, including, e.g., regions comprising medium spiny neurons, or regions comprising cortical neurons. Injections may be made directly into more than one region of the brain.


In an embodiment, components described herein are delivered by subretinally, e.g., by subretinal injection. Subretinal injections may be made directly into the macular, e.g., submacular injection.


In an embodiment, components described herein are delivered by intravitreal injection. Intravitreal injection has a relatively low risk of retinal detachment risk. In an embodiment, a nanoparticle or viral vector, e.g., AAV vector, e.g., an AAV2 vector, e.g., a modified AAV2 vector, is delivered intravitreally.


In an embodiment, a nanoparticle or viral vector, e.g., AAV vector, delivery is via intraparenchymal injection.


Methods for administration of agents to the eye are known in the medical arts and can be used to administer components described herein. Exemplary methods include intraocular injection (e.g., retrobulbar, subretinal, submacular, intravitreal and intrachoridal), iontophoresis, eye drops, and intraocular implantation (e.g., intravitreal, sub-Tenons and sub-conjunctival).


Administration may be provided as a periodic bolus (for example, subretinally, intravenously or intravitreally) or as continuous infusion from an internal reservoir (for example, from an implant disposed at an intra- or extra-ocular location (see, U.S. Pat. Nos. 5,443,505 and 5,766,242)) or from an external reservoir (for example, from an intravenous bag). Components may be administered locally, for example, by continuous release from a sustained release drug delivery device immobilized to an inner wall of the eye or via targeted transscleral controlled release into the choroid (see, for example, PCT/US00/00207. PCT/US02/14279. Ambati et al., (2000) INVEST. OPHTHALMOL. VIS. SCI, 41:1181-1185, and Ambati et al., (2000) INVEST. OPHTHALMOL. VIS. SCI, 41:1186-1191). A variety of devices suitable for administering components locally to the inside of the eye are known in the art. See, for example, U.S. Pat. Nos. 6,251,090, 6,299,895, 6,416,777, 6,413,540, and PCT/US00/28187.


In addition, components may be formulated to permit release over a prolonged period of time. A release system can include a matrix of a biodegradable material or a material which releases the incorporated components by diffusion. The components can be homogeneously or heterogeneously distributed within the release system. A variety of release systems may be useful, however, the choice of the appropriate system will depend upon rate of release required by a particular application. Both non-degradable and degradable release systems can be used. Suitable release systems include polymers and polymeric matrices, non-polymeric matrices, or inorganic and organic excipients and diluents such as, but not limited to, calcium carbonate and sugar (for example, trehalose). Release systems may be natural or synthetic. However, synthetic release systems are preferred because generally they are more reliable, more reproducible and produce more defined release profiles. The release system material can be selected so that components having different molecular weights are released by diffusion through or degradation of the material.


Representative synthetic, biodegradable polymers include, for example: polyamides such as poly(amino acids) and poly(peptides); polyesters such as poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), and poly(caprolactone); poly(anhydrides); polyorthoesters; polycarbonates; and chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), copolymers and mixtures thereof. Representative synthetic, non-degradable polymers include, for example: polyethers such as poly(ethylene oxide), poly(ethylene glycol), and poly(tetramethylene oxide); vinyl polymers-polyacrylates and polymethacrylates such as methyl, ethyl, other alkyl, hydroxyethyl methacrylate, acrylic and methacrylic acids, and others such as poly(vinyl alcohol), poly(vinyl pyrolidone), and poly(vinyl acetate); poly(urethanes); cellulose and its derivatives such as alkyl, hydroxyalkyl, ethers, esters, nitrocellulose, and various cellulose acetates; polysiloxanes; and any chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), copolymers and mixtures thereof.


Poly(lactide-co-glycolide) microsphere can also be used for intraocular injection. Typically the microspheres are composed of a polymer of lactic acid and glycolic acid, which are structured to form hollow spheres. The spheres can be approximately 15-30 microns in diameter and can be loaded with components described herein.


Bi-Modal or Differential Delivery of Components


Separate delivery of the components of a Cas system, e.g., the Cas9 molecule component and the gRNA molecule component, and more particularly, delivery of the components by differing modes, can enhance performance, e.g., by improving tissue specificity and safety.


In an embodiment, the Cas9 molecule and the gRNA molecule are delivered by different modes, or as sometimes referred to herein as differential modes. Different or differential modes, as used herein, refer modes of delivery that confer different pharmacodynamic or pharmacokinetic properties on the subject component molecule, e.g., a Cas9 molecule, gRNA molecule, or template nucleic acid. For example, the modes of delivery can result in different tissue distribution, different half-life, or different temporal distribution, e.g., in a selected compartment, tissue, or organ.


Some modes of delivery, e.g., delivery by a nucleic acid vector that persists in a cell, or in progeny of a cell, e.g., by autonomous replication or insertion into cellular nucleic acid, result in more persistent expression of and presence of a component. Examples include viral, e.g., adeno associated virus or lentivirus, delivery.


By way of example, the components, e.g., a Cas9 molecule and a gRNA molecule, can be delivered by modes that differ in terms of resulting half life or persistent of the delivered component the body, or in a particular compartment, tissue or organ. In an embodiment, a gRNA molecule can be delivered by such modes. The Cas9 molecule component can be delivered by a mode which results in less persistence or less exposure of its to the body or a particular compartment or tissue or organ.


More generally, in an embodiment, a first mode of delivery is used to deliver a first component and a second mode of delivery is used to deliver a second component. The first mode of delivery confers a first pharmacodynamic or pharmacokinetic property. The first pharmacodynamic property can be, e.g., distribution, persistence, or exposure, of the component, or of a nucleic acid that encodes the component, in the body, a compartment, tissue or organ. The second mode of delivery confers a second pharmacodynamic or pharmacokinetic property. The second pharmacodynamic property can be, e.g., distribution, persistence, or exposure, of the component, or of a nucleic acid that encodes the component, in the body, a compartment, tissue or organ.


In an embodiment, the first pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure, is more limited than the second pharmacodynamic or pharmacokinetic property.


In an embodiment, the first mode of delivery is selected to optimize, e.g., minimize, a pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure.


In an embodiment, the second mode of delivery is selected to optimize, e.g., maximize, a pharmacodynamic or pharmcokinetic property, e.g., distribution, persistence or exposure.


In an embodiment, the first mode of delivery comprises the use of a relatively persistent element, e.g., a nucleic acid, e.g., a plasmid or viral vector, e.g., an AAV or lentivirus. As such vectors are relatively persistent product transcribed from them would be relatively persistent.


In an embodiment, the second mode of delivery comprises a relatively transient element, e.g., an RNA or protein.


In an embodiment, the first component comprises gRNA, and the delivery mode is relatively persistent, e.g., the gRNA is transcribed from a plasmid or viral vector, e.g., an AAV or lentivirus. Transcription of these genes would be of little physiological consequence because the genes do not encode for a protein product, and the gRNAs are incapable of acting in isolation. The second component, a Cas9 molecule, is delivered in a transient manner, for example as mRNA or as protein, ensuring that the full Cas9 molecule/gRNA molecule complex is only present and active for a short period of time.


Furthermore, the components can be delivered in different molecular form or with different delivery vectors that complement one another to enhance safety and tissue specificity.


Use of differential delivery modes can enhance performance, safety and efficacy. For example, the likelihood of an eventual off-target modification can be reduced. Delivery of immunogenic components, e.g., Cas9 molecules, by less persistent modes can reduce immunogenicity, as peptides from the bacterially-derived Cas enzyme are displayed on the surface of the cell by MHC molecules. A two-part delivery system can alleviate these drawbacks.


Differential delivery modes can be used to deliver components to different, but overlapping target regions. The formation active complex is minimized outside the overlap of the target regions. Thus, in an embodiment, a first component, e.g., a gRNA molecule is delivered by a first delivery mode that results in a first spatial, e.g., tissue, distribution. A second component, e.g., a Cas9 molecule is delivered by a second delivery mode that results in a second spatial, e.g., tissue, distribution. In an embodiment, the first mode comprises a first element selected from a liposome, nanoparticle, e.g., polymeric nanoparticle, and a nucleic acid, e.g., viral vector. The second mode comprises a second element selected from the group. In an embodiment, the first mode of delivery comprises a first targeting element, e.g., a cell specific receptor or an antibody, and the second mode of delivery does not include that element. In an embodiment, the second mode of delivery comprises a second targeting element, e.g., a second cell specific receptor or second antibody.


When the Cas9 molecule is delivered in a virus delivery vector, a liposome, or polymeric nanoparticle, there is the potential for delivery to and therapeutic activity in multiple tissues, when it may be desirable to only target a single tissue. A two-part delivery system can resolve this challenge and enhance tissue specificity. If the gRNA molecule and the Cas9 molecule are packaged in separated delivery vehicles with distinct but overlapping tissue tropism, the fully functional complex is only be formed in the tissue that is targeted by both vectors.


VI. Payloads


Cas9 molecules, typically eiCas9 molecules and gRNA molecules, e.g., an eiCas9 molecule/gRNA molecule complex, can be used to deliver a wide variety of payloads. In an embodiment, the payload is delivered to target nucleic acids or to chromatin, or other components, near or associated with a target nucleic acid.


While not wishing to be bound by theory, it is believed that the sequence specificity of the gRNA molecule of an eiCas9 molecule/gRNA molecule complex contributes to a specific interaction with the target sequence, thereby effecting the delivery of a payload associated with, e.g., covalently or noncovalently coupled to, the Cas9 molecule/gRNA molecule complex.


In an embodiment, the payload is covalently or non-covalently coupled to a Cas9, e.g., an eiCas9 molecule. In an embodiment, the payload is covalently or non-covalently coupled to a gRNA molecule. In an embodiment, the payload is linked to a Cas9 molecule, or gRNA molecule, by a linker, e.g., a linker which comprises a bond cleavable under physiological conditions. In an embodiment the bond is not cleavable or is only poorly cleavable, under physiological conditions. In an embodiment, “covalently coupled” means as part of a fusion protein containing a Cas9 molecule.


Delivery of Multiple Payloads


In an embodiment, a first payload molecule is delivered by a first Cas9 molecule and a second payload molecule is delivered by a second Cas9 molecule. In an embodiment, the first and second payloads are the same. In an embodiment, first and second Cas9 molecules are the same, e.g. are from the same species, have the same PAM, and/or have the same sequence. In an embodiment, first and second Cas9 molecules are different, e.g. are from different species, have the different PAMs, and/or have different sequences. Examples of configurations are provided in Table VI-1. Typically the Cas9 molecules of Table VI-1 are eiCas9 molecules. In an embodiment, a Cas9 molecule is selected such that payload delivery and cleavage are both effected. In an embodiment, multiple payloads, e.g., two payloads, is delivered with a single Cas9 molecule.









TABLE VI-1







Configurations for delivery of payloads by more than one Cas9


molecule/gRNA molecule complex











First Cas9
Second Cas9
First
Second



molecule
molecule
Payload
Payload
Comments





C1
C1
P1
P1
In this embodiment, both Cas9 molecules are






the same, as are both payloads. In an






embodiment, the first and second Cas9






molecule are guided by different gRNA






molecules.


C1
C1
P1
P2
In this embodiment, both Cas9 molecules are






the same but each delivers a different






Payloads. In an embodiment, the first and






second Cas9 molecule are guided by different






gRNA molecules.


C1
C2
P1
P1
In this embodiment, the Cas9 molecules are






different but each delivers the same payload.






In an embodiment, the first and second Cas9






molecule are guided by different gRNA






molecules.


C1
C2
P1
P2
In this embodiment, the Cas9 molecules are






different as are the payloads. In an






embodiment, the first and second Cas9






molecule are guided by different gRNA






molecules.









In an embodiment, two different drugs are delivered. In an embodiment, a first payload, e.g., a drug, coupled by a first linker to a first Cas9 molecule and a second payload, e.g., a drug, coupled by a second linker to a second Cas9 molecule are delivered. In an embodiment, the first and second payloads are the same, and, in an embodiment, are coupled to the respective Cas9 molecule by different linkers, e.g., having different release kinetics. In an embodiment, the first and second payloads are different, and, in an embodiment, are coupled to the respective Cas9 molecule by the same linker. In an embodiment, the first and second payload interact. E.g., the first and second payloads form a complex, e.g., a dimeric or multimeric complex, e.g., a dimeric protein. In an embodiment, the first payload can activate the second payload, e.g., the first payload can modify, e.g., cleave or phosphorylate, the second payload. In an embodiment the first payload interacts with the second payload to modify, e.g., increase or decrease, an activity of the second payload.


A payload can be delivered in vitro, ex vivo, or in vivo.


Classes of Payloads


A payload can comprise a large molecule or biologics (e.g., antibody molecules), a fusion protein, an amino acid sequence fused, as a fusion partner, to a Cas9 molecule, e.g., an eiCas9 molecule, an enzyme, a small molecules (e.g., HDAC and other chromatin modifiers/inhibitors, exon skipping molecules, transcription inhibitors), a microsatellite extension inhibitor, a carbohydrate, and DNA degraders (e.g., in an infectious disease or “foreign” DNA setting), a nucleic acid, e.g., a DNA, RNA, mRNA, siRNA, RNAi, or an antisense oligonucleotide.


Table VI-2 provides exemplary classes of payloads.









TABLE VI-2





Exemplary Classes of Payloads



















Large Molecules




Small Molecules




Polymers




Biologics




Proteins and polypeptides, e.g., antibodies,




enzymes, structural peptides, ligands, receptors,




fusion proteins, fusion partners (as a fusion protein




with a Cas9, e.g., and eiCas9)




Carbohydrates




HDAC and other chromatin modifiers/inhibitors




Exon skipping molecules,




Transcription inhibitors




Microsatellite extension inhibitors




Entities that degrade DNA










Large Molecules


In an embodiment a payload comprises a polymer, e.g., a biological polymer, e.g., a protein, nucleic acid, or carbohydrate.


In an embodiment the payload comprises a protein, biologic, or other large molecule (i.e., a molecule having a molecular weight of at least, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kD). In an embodiment a payload comprises a polymer, e.g., a biological polymer, e.g., a protein, nucleic acid, or carbohydrate. The polymer can be a naturally occurring or non-naturally occurring polymer. In an embodiment, the payload is a natural product. For example, the natural product can be a large molecule or a small molecule.


Polypeptides, Proteins


In an embodiment the payload comprises a protein or polypeptide, e.g., a protein or polypeptide covalently or non-covalently coupled to a Cas9 molecule.


In an embodiment, the protein or polypeptide is dimeric or multimeric, and each subunit is delivered by a Cas9 molecule. In an embodiment, a first protein and second protein are delivered by one or more Cas9 molecules, e.g., each by a separate Cas9 molecule or both by the same Cas9 molecule.


In an embodiment, the protein or polypeptide is linked to a Cas9 molecule by a linker, e.g., a linker which comprises a bond cleavable under physiological conditions. In an embodiment, a linker is a linker from Section XI herein. In an embodiment, the bond is not cleavable under physiological conditions.


Specific Binding Ligands, Antibodies


In an embodiment the payload comprises a ligand, e.g., a protein, having specific affinity for a counter ligand. In an embodiment, the ligand can be a receptor (or the ligand for a receptor), or an antibody.


In an embodiment a payload comprises an antibody molecule. Exemplary antibody molecules include, e.g., proteins or polypeptides that include at least one immunoglobulin variable domain. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term “antibody” encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab′)2, Fd fragments, Fv fragments, scFv, and domain antibodies (dAb) fragments (de Wildt et al., EUR J IMMUNOL, 1996; 26(3):629-639)). For example, antigen-binding fragments of antibodies can include, e.g., (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab′h fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) NATURE 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) that retains functionality. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules known as single chain Fv (scFv). See, e.g., U.S. Pat. Nos. 5,260,203, 4,946,778, and 4,881,175; Bird et al., (1988) SCIENCE 242:423-426; and Huston et al., (1988) PROC. NATL. ACAD. Ser. USA 85:5879-5883. An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof). Antibodies may be from any source, but primate (human and non-human primate) and primatized are preferred. In an embodiment, the antibody is a human antibody or humanized antibody.


In an embodiment, the antibody molecule is a single-domain antibody (e.g., an sdAb, e.g., a nanobody), e.g., an antibody fragment consisting of a single monomeric variable antibody domain. In an embodiment, the molecular weight of the single-domain antibody is about 12-15 kDa. For example, the single-domain antibody can be engineered from heavy-chain antibodies found in camelids (e.g., VHH fragments). Cartilaginous fishes also have heavy-chain antibodies (IgNAR, ‘immunoglobulin new antigen receptor’), from which single-domain antibodies called VNAR fragments can be obtained. An alternative approach is to split the dimeric variable domains from common immunoglobulin G (IgG), e.g., from humans or mice, into monomers. Single-domain antibodies derived from either heavy or light chain can be obtained to bind specifically to target epitopes. For example, a single-domain antibody can be a peptide chain of about 110 amino acids long, comprising one variable domain (VH) of a heavy-chain antibody, or of a common IgG.


Single-domain antibodies can have similar affinity to antigens as whole antibodies. They can also be more heat-resistant and/or stable towards detergents and high concentrations of urea. Those, e.g., derived from camelid and fish antibodies can be less lipophilic and more soluble in water, owing to their complementarity determining region 3 (CDR3), which forms an extended loop covering the lipophilic site that normally binds to a light chain. In an embodiment, the single-domain antibody does not show complement system triggered cytotoxicity, e.g., because they lack an Fe region. Single-domain antibodies, e.g., camelid and fish derived sdAbs, can bind to hidden antigens that may not be accessible to whole antibodies, for example to the active sites of enzymes. This property can result from their extended CDR3 loop, which is able to penetrate such sites.


A single-domain antibody can be obtained by immunization of, e.g., dromedaries, camels, llamas, alpacas or sharks with the desired antigen and subsequent isolation of the mRNA coding for heavy-chain antibodies. By reverse transcription and polymerase chain reaction, a gene library of single-domain antibodies containing several million clones is produced. Screening techniques like phage display and ribosome display help to identify the clones binding the antigen.


A different method uses gene libraries from animals that have not been immunized beforehand. Such naive libraries usually contain only antibodies with low affinity to the desired antigen, making it necessary to apply affinity maturation by random mutagenesis as an additional step.


When the most potent clones have been identified, their DNA sequence can be optimized, for example to improve their stability towards enzymes. Another goal is humanization to prevent immunological reactions of the human organism against the antibody. The final step is the translation of the optimized single-domain antibody in E. coli, Saccharomyces cerevisiae or other suitable organisms.


Alternatively, single-domain antibodies can be made from common murine or human IgG with four chains. The process is similar, comprising gene libraries from immunized or naive donors and display techniques for identification of the most specific antigens. Monomerization is usually accomplished by replacing lipophilic by hydrophilic amino acids. If affinity can be retained, the single-domain antibodies can likewise be produced in E. coli. S. cerevisiae or other organisms.


In an embodiment, a payload comprises a transcription activator protein or domain, e.g., a VP16 protein or domain, or a transcription repressor protein or domain.


Fusion Proteins and Fusion Partners


In an embodiment the payload comprises a fusion protein. Exemplary fusion proteins include a first and second fusion partner, which can possess different functional properties or which can be derived from different proteins. In an embodiment, the fusion protein can comprise a first fusion partner that binds a nucleic acid and a second fusion partner that that comprises an enzymatic activity or that promotes or inhibits gene expression. In an embodiment, the payload itself is a fusion protein. In an embodiment, the payload is fused to a Cas9 molecule.


For example, the fusion protein can contain a segment that adds stability and/or deliverability to the fused protein. In an embodiment, the fusion protein can be a protein described herein (e.g., a receptor) fused to an immunoglobulin fragment (e.g., Fe fragment), transferring, or a plasma protein, e.g., albumin. The fusion protein can also contain a segment that adds toxicity to the fused protein (e.g. conveyed by toxins, enzymes or cytokines). Fusion proteins can also be used to enable delivery and/or targeting routes (e.g., by HIV-1 TAT protein). Other examples include, e.g., fusions that allow for mutivalency, such as streptavidin fusions, or fusions of two active components (e.g., with or without a cleavable linker in between).


In an embodiment, the protein or polypeptide is a fusion partner with a Cas9 molecule, e.g., an eiCas9 molecule.


In an embodiment, a payload comprises fusion partner with a Cas9 molecule comprising a transcription activator protein or domain, e.g., a VP16 protein or domain, or a transcription repressor protein or domain.


Enzymes


In an embodiment a payload comprises an enzyme. Exemplary enzymes include, e.g., oxidoreductases (e.g., catalyze oxidation/reduction reactions), transferases (e.g., transfer a functional group (e.g. a methyl or phosphate group)), hydrolases (e.g., catalyze the hydrolysis of various bonds), lyases (e.g., cleave various bonds by means other than hydrolysis and oxidation), isomerases (catalyze isomerization changes within a single molecule), and ligases (e.g., join two molecules with covalent bonds). In an embodiment an enzymes mediates or is associated with one or more functions in the cell nucleus, e.g., DNA synthesis, transcription, epigenetic modification of DNA and histones, RNA post-transcriptional modification, cell cycle control, DNA damage repair, or genomic instability.


Small Molecules


In an embodiment a payload comprises a small molecule compounds.


In an embodiment a small molecule is a regulator of a biological process. For example, a small molecule can bind to a second molecule, e.g., biopolymer, e.g., a carbohydrate, protein, polypeptide, or a nucleic acid, and in an embodiment, alter one or more of the structure, distribution, activity, or function of the second molecule. In an embodiment, the size of the small molecule is on the order of 10−9 m. In an embodiment, the molecular weight of the small molecule is, e.g., between 200 amu and 500 amu, between 300 amu and 700 amu, between 500 amu and 700 amu, between 700 amu and 900 amu, or between 500 amu and 900 amu.


Exemplary small molecules include histone deacetylase (HDAC) inhibitors (e.g., suberoylanilide hydroxamic acid (SAHA), or romidepsin), histone methyltransferase inhibitors (DNA methyltransferase inhibitors (e.g., azacitidine (or 5-azacitidine), decitabine (or 5-aza-2′-deoxycytidine), or DNA replication inhibitors. Small molecules can also include, e.g., small nucleic acid molecules (1-4 bases depending upon the base. e.g., that would be under 2 kD) and peptides.


Microsatellite Extension Inhibitors


In an embodiment a payload comprises a microsatellite extension inhibitor. In an embodiment, the microsatellite extension inhibitor is a DNA mismatch repair protein. Exemplary DNA mismatch repair proteins that can be delivered by the molecules and methods described herein include, e.g., MSH2, MSH3, MSH6, MLH1, MLH3, PMS1, PMS2.


Signal generators, radionuclides, reporter molecules, diagnostic probes


In an embodiment a payload comprises a molecule that generates a signal. Such payloads are useful, e.g., in research, therapeutic (e.g., cancer therapy) and diagnostic applications. In an embodiment, the signal comprises: an electromagnetic emission, e.g., in the infrared, visible, or ultraviolet range; a particle, e.g., a product of radioactive decay, e.g., an alpha, beta, or gamma particle; a detectable substrate, e.g., a colored substrate; a reaction product, e.g., the product of an enzymatic reaction; or a ligand detectable by a specific binding agent, e.g., an antibody; or a dye. In an embodiment the signal comprises a fluorescent emission, e.g., by a fluorescent protein. Exemplary fluorescent proteins include, Blue/UV Proteins (e.g., TagBFP, mTagBFP, Azurite, EBFP2, mKalamal, Sirius, Sapphire, T-Sapphire), Cyan Proteins (e.g., ECFP, Cerulean, SCFP3A, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP, mTFP1), Green Proteins (e.g., EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi. Clover, mNeonGreen), Yellow Proteins (e.g., EYFP, Citrine, Venus, SYFP2, TagYFP), Orange Proteins (e.g., Monomeric Kusabira-Orange, mKOK, mK02, mOrange, m0range2), Red Proteins (mRaspberry, mCherry, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mRuby2), Far-Red Proteins (e.g., mPlum, HcRed-Tandem, mKate2, mNeptune, NirFP, TagRFP657, IFP1.4, iRFP), Long Stokes Shift Proteins (e.g., mKeima Red, LSS-mKate1, LSS-mKate2, mBeRFP), Photoactivatible Proteins (e.g., PA-GFP, PAmCherry1, PATagRFP), Photoconvertible Proteins (e.g., Kaede (green), Kaede (red), KikGR1 (green), KikGR1 (red), PS-CFP2, mEos2 (green), mEos2 (red), mEos3.2 (green), mEos3.2 (red), PSmOrange), Photoswitchable Proteins (e.g., Dronpa).


In an embodiment, a signal producing moiety is provided as the fusion partner of a Cas9 molecule, e.g., an eiCas9 molecule.


Signal generators or reporters, useful, e.g., for labeling polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., indium (111In), iodine (1311 or 1251), yttrium (9°Y), lutetium (177Lu), actinium (225Ac), bismuth (212Bi or 213Bi), sulfur (35S), carbon (14C), tritium (3H), rhodium (188Rh), technetium (99 mTc), praseodymium, or phosphorous (32P) or a positron-emitting radionuclide, e.g., carbon-11 (11C), potassium-40 (40K), nitrogen-13 (13N), oxygen-15 (15O), fluorine-18 (18F), and iodine-121 (1211)), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups (which can be detected by a marked avidin, e.g., a molecule containing a streptavidin moiety and a fluorescent marker or an enzymatic activity that can be detected by optical or calorimetric methods), and predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In an embodiment, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.


In an embodiment, a payload comprises a radionuclide. The radionuclide can be incorporated into the gRNA molecule, the Cas9 molecule, or into a payload molecule. Exemplary radionuclides include, e.g., beta emitters, alpha emitters or gamma emitters. In an embodiment the radionuclide is iodine, e,g., 131 I or 125I, yttnum, e.g., 90Y, lutetmm, e.g., 177Lu, Actinium, e.g., 225Ac, bismuth, e.g., 212Bi or 213Bi), sulfur, e.g., 35S), carbon, e.g., 14C, tritium, 3H), rhodium, e.g., 188Rh, technetium, e.g., 99Tc, praseodymium, or phosphorous, e.g., 32P.


Modulators of DNA and Chromatin Structure


In an embodiment, a payload comprises an endogenous or exogenous modulator of DNA structure. A modulator, as is typical of payloads, can be delivered in vitro, ex vivo, or in vivo.


In an embodiment, the payload comprises a modulator of an epigenetic state or characteristic of DNA. In an embodiment an epigenetic state or characteristic can be altered to treat a disorder, or to influence the developmental or other state of a cell.


In an embodiment, the epigenetic state or characteristic comprises DNA methylation. For example, the payloads described herein can modulate the addition of methyl groups to DNA, e.g., to convert cytosine to 5-methylcytosine, e.g., at CpG sites.


Aberrant DNA methylation patterns (e.g., hypermethylation and hypomethylation compared to normal tissue) are associated with various diseases and conditions, e.g., cancer. The modulators described herein can be used to reactivate transcriptionally silenced genes or to inhibit transcriptionally hyperactive genes, e.g., to treat diseases, e.g., cancer.


DNA methylation can affect gene transcription. Genes with high levels of 5-methylcytosine, e.g., in their promoter region, can be transcriptionally less active or silent. Thus, methods described herein can be used to target and suppress transcriptional activity, e.g., of genes described herein.


In an embodiment, the modulator promotes maintenance of DNA methylation. For example, the modulators can have DNA methyltransferase (DNMT) activity or modulate DNMT activity, e.g., to maintain DNA methylation or reduce passive DNA demethylation, e.g., after DNA replication.


In an embodiment, the modulator promotes de nova DNA methylation. For example, the modulators described herein can have de nova DNA methyltransferase (DNMT) (e.g., DNMT3a, DNMT3b, DNMT3L) activity or modulate de nova DNMT (e.g., DNMT3a, DNMT3b, DNMT3L) activity, e.g., to produce DNA methylation patterns, e.g., early in development.


Epigenetic changes in DNA (e.g., methylation), can be evaluated by art-known methods or as described herein. Exemplary methods for detecting DNA methylation include, e.g., Methylation-Specific PCR (MSP), whole genome bisulfite sequencing (BS-Seq), HELP (Hpal1 tiny fragment Enrichment by Ligation-mediated PCR) assay, ChIP-on-chip assays, restriction landmark genomic scanning, Methylated DNA immunoprecipitation (MeDIP), pyrosequencing of bisulfite treated DNA, molecular break light assay for DNA adenine methyltransferase activity, methyl sensitive Southern Blotting, separation of native DNA into methylated and unmethylated fractions using MethylCpG Binding Proteins (MBPs) and fusion proteins containing just the Methyl Binding Domain (MBD).


In an embodiment, the modulator cleaves DNA. For example, a modulator can catalyze the hydrolytic cleavage of phosphodiester linkages in the DNA backbone. In an embodiment, the modulator (e.g., DNase I) cleaves DNA preferentially at phosphodiester linkages adjacent to a pyrimidine nucleotide, yielding 5′-phosphate-terminated polynucleotides with a free hydroxyl group on position 3′. In an embodiment, the modulator (e.g., DNase II) hydrolyzes deoxyribonucleotide linkages in DNA, yielding products with 3′-phosphates. In an embodiment, the modulator comprises endodeoxyribonuclease activity. In an embodiment, the modulator comprises exodeoxyribonuclease activity (e.g., having 3′ to 5′ or 5′ to 3′ exodeoxyribonuclease activity). In an embodiment, the modulator recognizes a specific DNA sequence (e.g., a restriction enzyme). In an embodiment, the modulator does not cleave DNA in a sequence-specific manner. A modulator can cleave single-stranded DNA (e.g., having nickase activity), double-stranded DNA, or both.


In an embodiment, modulator affects, e.g., alters or preserves, tertiary or quaternary DNA structure. For example, the modulators described herein can modulate tertiary structure, e.g., handedness (right or left), length of the helix tum, number of base pairs per tum, and/or difference in size between the major and minor grooves. In an embodiment, the modulator mediates the formation of B-DNA, A-DNA, and/or Z-DNA. The modulators described herein can also modulate quaternary structure, e.g., the interaction of DNA with other molecules (DNA or non-DNA molecules, e.g., histones), e.g., in the form of chromatin. In an embodiment, the modulator that mediate or modify tertiary or quaternary DNA structure comprises DNA helicases activity or modulates DNA helicase activity.


In an embodiment, the modulator promotes or inhibits DNA damage response and/or repa1r. For example, a modulator can promote one or more DNA damage response and repair mechanisms, e.g., direct reversal, base excision repair (BER), nucleotide excision repair (NER) (e.g., global genomic repair (GG-NER), transcription-coupled repair (TC-NER)), mismatch repair (MMR), non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), homologous recombination, and/or translesion synthesis (TLS). In an embodiment, a modulator promotes the step of damage recognition. In an embodiment, a modulator promotes the step of DNA repair.


Aberrant DNA damage repair is associated with various diseases and conditions, e.g., aging, hereditary DNA repair disorders, and cancer. For example, DNA repair gene mutations that can increase cancer risk include, e.g., BRCA1 and BRCA2 (e.g., involved in homologous recombination repair (HRR) of double-strand breaks and daughter strand gaps, e.g., in breast and ovarian cancer); ATM (e.g., different mutations reduce HRR, single strand annealing (SSA), NHEJ or homology-directed DSBR (HDR), e.g., in leukemia, lymphoma, and breast cancer), NBS (e.g., involved in NHEJ, e.g., in lymphoid malignancies); MREl 1 (e.g., involved in HRR, e.g., in breast cancer); BLM (e.g., involved in HRR, e.g., in leukemia, lymphoma, colon, breast, skin, auditory canal, tongue, esophagus, stomach, tonsil, larynx, lung, and uterus cancer); WRN (e.g., involved in HRR, NHEJ, long-patch BER, e.g., in soft tissue sarcomas, colorectal, skin, thyroid, and pancreatic cancer); RECQ4 (RECQL4) (e.g., involved in HRR, e.g., causing Rothmund-Thomson syndrome (RTS), RAPADILINO syndrome or Baller Gerold syndrome, cutaneous carcinomas, including basal cell carcinoma, squamous cell carcinoma, and Bowen's disease); FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, and FANCN (e.g., involved in HRR and TLS, e.g., in leukemia, liver tumors, solid tumors in many locations), XPC and XPE(DDB2) (e.g., involved in NER (GGR type), e.g., in skin cancer (melanoma and non-melanoma)); XPA, XPB, XPD, XPF, and XPG (e.g., involved in NER (both GGR type and TCR type), e.g., in skin cancer (melanoma and non-melanoma) and central nervous system); XPV (POLH) (e.g., involved in TLS, e.g., in skin cancer (melanoma and non-melanoma)); hMSH2, hMSH6, hMLH1, and hPMS2 (involved in MMR, e.g., in colorectal, endometrial and ovarian cancer); MUTYH (e.g., involved in BER of A mispaired with 80H-dG, as well as mispairs with G. FapydG and C, e.g., in colon cancer)


Modulators can be used to treat a disease or condition associated with aberrant DNA damage repair, e.g., by modulating one or more DNA damage repair mechanisms described herein.


In an embodiment, the modulator is selected from, or modulates, one or more proteins involved in direct reversal, e.g., methyl guanine methyl transferase (MGMT).


In an embodiment, the modulator is selected from, or modulates, one or more proteins involved in BER, e.g., DNA glycosylase, AP endonuclease, DNA polymerase, DNA ligase.


In an embodiment, the modulator is selected from, or modulates, one or more proteins involved in GG-NER, e.g., XPC, HR23b, CAK, TFIIH, XPA, RPA, XPG, XPF, ERCC1, TFIIH, PCNA, RFC, ADN Pol, and LigaseI.


In an embodiment, the modulator is selected from, or modulates, one or more proteins involved in TC-NER, e.g., CSB, XPA, RPA, XPG, XPF, ERCC1, CSA-CNS, TFIIH, CAK, PCNA, RFC, Ligase I, and RNA PolymeraseII.


In an embodiment, the modulator is selected from, or modulates, one or more DNA mismatch repair proteins.


In an embodiment, the modulator is selected from, or modulates, one or more proteins involved in NHEJ, e.g., Ku70/80, DNA-PKcs, DNA Ligase IV, XRCC4, XLF, Artemis, DNA polymerase mu, DNA polymerase lambda, PNKP, Aprataxin, and APLF.


In an embodiment, the modulator is selected from, or modulates, one or more proteins involved in homologous recombination, e.g., as described herein.


In an embodiment, the modulator is selected from, or modulates, one or more proteins involved in TLS, e.g., DNA polymerase eta, iota, kappa, zeta, and PCNA.


In an embodiment, a modulator can modulate global response to DNA damage. e.g., DNA damage checkpoints and/or transcriptional responses to DNA damage. For example, DNA damage checkpoints can occur at the G1/S and 02/M boundaries. An intra-S checkpoint can also exist. Checkpoint activation can be modulated by two master kinases, ATM and ATR. ATM can respond to DNA double-strand breaks and disruptions in chromatin structure and ATR can respond to stalled replication forks. These kinases can phosphorylate downstream targets in a signal transduction cascade, e.g., leading to cell cycle arrest. A class of checkpoint mediator proteins (e.g., BRCA1, MDC1, and 53BP1), which transmit the checkpoint activation signal to downstream proteins, can be modulated. Exemplary downstream proteins that can be modulated include, e.g., p53, p21, and cyclin/cyclin-dependent kinase complexes.


In an embodiment, the modulator modulates nuclear DNA damage response and repair. In an embodiment, the modulator modulates mitochondrial DNA damage response and repair.


In an embodiment, the modulator promotes or inhibits DNA replication. For example, a modulator can promote or inhibit one or more stages of DNA replication, e.g., initiation (e.g., assembly of pre-replicative complex and/or initiation complex), elongation (e.g., formation of replication fork), and termination (e.g., formation of replication fork barrier). In an embodiment, the modulator is selected from, or modulates, one or more proteins involved in initiation, e.g., the origin recognition complex (ORC), CDC6, CDT1, minichromosome maintenance proteins (e.g., MCM2, MCM3, MCM4, MCM5, MCM6, MCM7, and MCM1O), CDC45, CDK, DDK, CDC101, CDC102, CDC103, and CDC105. In an embodiment, the modulator is selected from, or modulates, one or more proteins involved in elongation, e.g., DNA helicases, DNA polymerase, PCNA, CDC45-MCM-GINS helicase complex, and Replication Factor C complex.


In an embodiment, the modulator is selected, from or modulates, one or more proteins involved in termination, e.g., type II topoisomerase and telomerase. In an embodiment, the modulator is selected from, or modulates, one or more replication checkpoint proteins, e.g., ATM, ATR, ATRIP, TOPBP1, RAD9, HUS1, Rad1, and CHK1.


In an embodiment, the payload comprises a modulator of nuclear DNA replication. In an embodiment, the modulator promotes or inhibits mitochondrial DNA replication.


Defects in DNA replication can be associated with various diseases and conditions, e.g., cancer and neurological diseases (e.g., Alzheimer's disease). Defects in mitochondrial DNA replication can also be associated with diseases and conditions. e.g., mtDNA depletion syndromes (e.g., Alpers or early infantile hepatocerebral syndromes) and mtDNA deletion disorders (e.g., progressive external ophthalmoplegia (PEO), ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)). A modulator can be used to treat a disease or condition associated with aberrant DNA replication, e.g., by modulating DNA replication as described herein.


Exemplary endogenous or exogenous modulators of DNA structure are described herein, e.g., in Table VI-3.










TABLE VI-3







DNA2
DNA replication helicase/nuclease 2


DNAAF1
dynein, axonemal, assembly factor 1


DNAAF2
dynein, axonemal, assembly factor 2


DNAAF3
dynein, axonemal, assembly factor 3


DNAHl
dynein, axonemal, heavy chain 1


DNAH2
dynein, axonemal, heavy chain 2


DNAH3
dynein, axonemal, heavy chain 3


DNAH5
dynein, axonemal, heavy chain 5


DNAH6
dynein, axonemal, heavy chain 6


DNAH7
dynein, axonemal, heavy chain 7


DNAH8
dynein, axonemal, heavy chain 8


DNAH9
dynein, axonemal, heavy chain 9


DNAH1O
dynein, axonemal, heavy chain 10


DNAH1OOS
dynein, axonemal, heavy chain 10 opposite strand


DNAHll
dynein, axonemal, heavy chain 11


DNAH12
dynein, axonemal, heavy chain 12


DNAH14
dynein, axonemal, heavy chain 14


DNAH17
dynein, axonemal, heavy chain 17


DNAH17-AS1
DNAHl 7 antisense RNA 1


DNAi1
dynein, axonemal, intermediate chain 1


DNAI2
dynein, axonemal, intermediate chain 2


DNAJB8-AS1
DNAJB8 antisense RNA 1


DNAJC3-AS1
DNAJC3 antisense RNA 1 (head to head)


DNAJC9-AS1
DNAJC9 antisense RNA 1


DNAJC25-
DNAJC25-GNG 10 readthrough


GNGlO



DNAJC27-AS1
DNAJC27 antisense RNA 1


DNAL1
dynein, axonemal, light chain 1


DNAL4
dynein, axonemal, light chain 4


DNALi1
dynein, axonemal, light intermediate chain 1


DNASE1
deoxyribonuclease I


DNASElL1
deoxyribonuclease I-like 1


DNASE1L2
deoxyribonuclease I-like 2


DNASE1L3
deoxyribonuclease I-like 3


DNASE2
deoxyribonuclease II, lysosomal


DNASE2B
deoxyribonuclease II beta


CD226
CD226 molecule


FAM120A
family with sequence similarity 120A


GAK
cyclin G associated kinase


GCFC2
GC-rich sequence DNA-binding factor 2


MCMlO
minichromosome maintenance complex component 10


PRKDC
protein kinase, DNA-activated, catalytic polypeptide


SACS
spastic ataxia of Charlevoix-Saguenay (sacsin)


SCNNlD
sodium channel, non-voltage-gated 1, delta subunit


SPATS2L
spermatogenesis associated, serine-rich 2-like


MT7SDNA
mitochondrially encoded 7S DNA


DCLRElA
DNA cross-link repair lA


DCLRElB
DNA cross-link repair lB


DCLRElC
DNA cross-link repair lC


DDIT3
DNA-damage-inducible transcript 3


DDIT4
DNA-damage-inducible transcript 4


DDIT4L
DNA-damage-inducible transcript 4-like


DFFA
DNA fragmentation factor, 45 kDa, alpha polypeptide


DFFB
DNA fragmentation factor, 40 kDa, beta poly-peptide (caspase-activated



DNase)


DMAPl
DNA methyltransferase 1 associated protein 1


DMCl
DNA meiotic recombinase 1


DNMTl
DNA (cytosine-5-)-methyltransferase 1


DNMT3A
DNA (cytosine-5-)-methyltransferase 3 alpha


DNMT3B
DNA (cytosine-5-)-methyltransferase 3 beta


DNMT3L
DNA (cytosine-5-)-methyltransferase 3-like


DNTT
DNA nucleotidylexotransferase


DRAMl
DNA-damage regulated autophagy modulator 1


DRAM2
DNA-damage regulated autophagy modulator 2


DSCCl
DNA replication and sister chromatid cohesion 1


ZBPl
Z-DNA binding protein 1


SON
SON DNA binding protein


TARDBP
TAR DNA binding protein


BMF
Bcl2 modifying factor


CENPBDl
CENPB DNA-binding domains containing 1


UNG
uracil-DNA glycosylase


PDRGl
p53 and DNA-damage regulated 1


TDG
thymine-DNA glycosylase


TDPl
tyrosyl-DNA phosphodiesterase 1


TDP2
tyrosyl-DNA phosphodiesterase 2


AHDCl
AT hook, DNA binding motif, containing 1


GMNN
geminin, DNA replication inhibitor


PRIM1
primase, DNA, polypeptide 1 (49 kDa)


PRIM2
primase, DNA, polypeptide 2 (58 kDa)


HELB
helicase (DNA) B


LIGl
ligase I, DNA, ATP-dependent


SUMFl
sulfatase modifying factor 1


SUMF2
sulfatase modifying factor 2


LIG4
ligase IV, DNA, ATP-dependent


LIG3
ligase III, DNA, ATP-dependent


MDCl
mediator of DNA-damage checkpoint 1


MMS22L
MMS22-like, DNA repair protein


POLA1
polymerase (DNA directed), alpha 1, catalytic subunit


POLA2
polymerase (DNA directed), alpha 2, accessory subunit


POLB
polymerase (DNA directed), beta


POLDl
polymerase (DNA directed), delta 1, catalytic subunit


POLD2
polymerase (DNA directed), delta 2, accessory subunit


POLD3
polymerase (DNA-directed), delta 3, accessory subunit


POLD4
polymerase (DNA-directed), delta 4, accessory subunit


POLDIP2
polymerase (DNA-directed), delta interacting protein 2


POLDIP3
polymerase (DNA-directed), delta interacting protein 3


POLE
polymerase (DNA directed), epsilon, catalytic subunit


POLE2
polymerase (DNA directed), epsilon 2, accessory subunit


POLE3
polymerase (DNA directed), epsilon 3, accessory subunit


POLE4
polymerase (DNA-directed), epsilon 4, accessory subunit


POLO
polymerase (DNA directed), gamma


POLG2
polymerase (DNA directed), gamma 2, accessory subunit


POLH
polymerase (DNA directed), eta


POLI
polymerase (DNA directed) iota


POLK
polymerase (DNA directed) kappa


POLL
polymerase (DNA directed), lambda


POLM
polymerase (DNA directed), mu


POLN
polymerase (DNA directed) nu


POLQ
polymerase (DNA directed), theta


IDl
inhibitor of DNA binding 1, dominant negative helix-loop-helix protein


ID2
inhibitor of DNA binding 2, dominant negative helix-loop-helix protein


ID3
inhibitor of DNA binding 3, dominant negative helix-loop-helix protein


ID4
inhibitor of DNA binding 4, dominant negative helix-loop-helix protein


OGGl
8-oxoguanine DNA glycosylase


MSANTDl
Myb/SANT-like DNA-binding domain containing 1


MSANTD2
Myb/SANT-like DNA-binding domain containing 2


MSANTD3
Myb/SANT-like DNA-binding domain containing 3


MSANTD4
Myb/SANT-like DNA-binding domain containing 4 with coiled-coils


PIFl
PIFl 5′-to-3′ DNA helicase


TONSL
tonsoku-like, DNA repair protein


MPG
N-methylpurine-DNA glycosylase


TOPl
topoisomerase (DNA) I


TOPlMT
topoisomerase (DNA) I, mitochondrial


TOP2A
topoisomerase (DNA) II alpha 170 kDa


TOP2B
topoisomerase (DNA) II beta 180 kDa


TOP3A
topoisomerase (DNA) III alpha


TOP3B
topoisomerase (DNA) III beta


TOPBPl
topoisomerase (DNA) II binding protein 1


DDBl
damage-specific DNA binding protein 1, 127 kDa


DDB2
damage-specific DNA binding protein 2, 48 kDa


SSBPl
single-stranded DNA binding protein 1, mitochondrial


SSBP2
single-stranded DNA binding protein 2


SSBP3
single stranded DNA binding protein 3


SSBP4
single stranded DNA binding protein 4


GADD45A
growth arrest and DNA-damage-inducible, alpha


GADD45B
growth arrest and DNA-damage-inducible, beta


GADD45G
growth arrest and DNA-damage-inducible, gamma


GADD45GIP1
growth arrest and DNA-damage-inducible, gamma interacting protein 1


MGMT
0-6-methylguanine-DNA methyltransferase


REVl
REVl, polymerase (DNA directed)


RECQL
RecQ protein-like (DNA helicase Q1-like)


CCDC6
coiled-coil domain containing 6


KLRKl
killer cell lectin-like receptor subfamily K, member 1


N6AMT1
N-6 adenine-specific DNA methyltransferase 1 (putative)


N6AMT2
N-6 adenine-specific DNA methyltransferase 2 (putative)


POLR2A
polymerase (RNA) II (DNA directed) polypeptide A, 220 kDa


POLR2B
polymerase (RNA) II (DNA directed) polypeptide B, 140 kDa


POLR2C
polymerase (RNA) II (DNA directed) polypeptide C, 33 kDa


POLR2D
polymerase (RNA) II (DNA directed) polypeptide D


POLR2E
polymerase (RNA) II (DNA directed) polypeptide E, 25 kDa


POLR2F
polymerase (RNA) II (DNA directed) polypeptide F


POLR2G
polymerase (RNA) II (DNA directed) polypeptide G


POLR2H
polymerase (RNA) II (DNA directed) polypeptide H


POLR2I
polymerase (RNA) II (DNA directed) polypeptide I, 14.5 kDa


POLR2J
polymerase (RNA) H (DNA directed) polypeptide J, 13.3 kDa


POLR2J2
polymerase (RNA) II (DNA directed) polypeptide J2


POLR2J3
polymerase (RNA) II (DNA directed) polypeptide J3


POLR2K
polymerase (RNA) II (DNA directed) polypeptide K, 7.0 kDa


POLR2L
polymerase (RNA) II (DNA directed) polypeptide L, 7.6 kDa


POLR2M
polymerase (RNA) II (DNA directed) polypeptide M


TRDMTl
tRNA aspartic acid methyltransferase 1


CHDl
chromodomain helicase DNA binding protein 1


CHDlL
chromodomain helicase DNA binding protein 1-like


CHD2
chromodomain helicase DNA binding protein 2


CHD3
chromodomain helicase DNA binding protein 3


CHD4
chromodomain helicase DNA binding protein 4


CHD5
chromodomain helicase DNA binding protein 5


CHD6
chromodomain helicase DNA binding protein 6


CHD7
chromodomain helicase DNA binding protein 7


CHD8
chromodomain helicase DNA binding protein 8


CHD9
chromodomain helicase DNA binding protein 9


KLLN
killin, p53-regulated DNA replication inhibitor


POLR3A
polymerase (RNA) III (DNA directed) polypeptide A, 155 kDa


POLR3B
polymerase (RNA) III (DNA directed) polypeptide B


POLR3C
polymerase (RNA) III (DNA directed) polypeptide C (62 kD)


POLR3D
polymerase (RNA) III (DNA directed) polypeptide D, 44 kDa


POLR3E
polymerase (RNA) III (DNA directed) polypeptide E (80 kD)


POLR3F
polymerase (RNA) III (DNA directed) polypeptide F, 39 kDa


POLR3G
polymerase (RNA) III (DNA directed) polypeptide G (32 kD)


POLR3GL
polymerase (RNA) III (DNA directed) polypeptide G (32 kD)-like


POLR3H
polymerase (RNA) III (DNA directed) polypeptide H (22.9 kD)


POLR3K
polymerase (RNA) III (DNA directed) polypeptide K, 12.3 kDa


WDHDl
WD repeat and HMG-box DNA binding protein 1


PGAPl
post-GPI attachment to proteins 1


PGAP2
post-GPI attachment to proteins 2


PGAP3
post-GPI attachment to proteins 3


REV3L
REV3-like, polymerase (DNA directed), zeta, catalytic subunit


CDTl
chromatin licensing and DNA replication factor 1


PANDAR
promoter of CDKNlA antisense DNA damage activated RNA


APEXl
APEX nuclease (multifunctional DNA repair enzyme) 1


CHMPlA
charged multivesicular body protein lA


CHMPlB
charged multivesicular body protein lB


CHMP2A
charged multivesicular body protein 2A


CHMP2B
charged multivesicular body protein 2B


CHMP4A
charged multivesicular body protein 4A


CHMP4B
charged multivesicular body protein 4B


CHMP4C
charged multivesicular body protein 4C


CHMP5
charged multivesicular body protein 5


CHMP6
charged multivesicular body protein 6


POLRMT
polymerase (RNA) mitochondrial (DNA directed)


SPIDR
scaffolding protein involved in DNA repair


MCIDAS
multiciliate differentiation and DNA synthesis associated cell cycle protein


PAPD7
PAP associated domain containing 7


RFX8
RFX family member 8, lacking RFX DNA binding domain


DEK
DEK oncogene


NUBl
negative regulator of ubiquitin-like proteins 1


PAXBPl
PAX3 and PAX7 binding protein 1


RAMPl
receptor (G protein-coupled) activity modifying protein 1


RAMP2
receptor (G protein-coupled) activity modifying protein 2


RAMP3
receptor (G protein-coupled) activity modifying protein 3


RC3H2
ring finger and CCCH-type domains 2


ARHGAP35
Rho GTPase activating protein 35


SMUGl
single-strand-selective monofunctional uracil-DNA glycosylase 1


CXXCl
CXXC finger protein 1


FAM50A
family with sequence similarity 50, member A


FANCG
Fanconi anemia, complementation group G


GLI3
GLI family zinc finger 3


GTF2H5
general transcription factor IIH, polypeptide 5


LAGE3
L antigen family, member 3


MYCNOS
MYCN opposite strand/antisense RNA


NFRKB
nuclear factor related to kappaB binding protein


RAD51D
RAD51 paralog D


RFX2
regulatory factor X, 2 (influences HLA class II expression)


RFXANK
regulatory factor X-associated ankyrin-containing protein


RRPl
ribosomal RNA processing 1


SPRTN
SprT-like N-terminal domain


XRCC4
X-ray repair complementing defective repair in Chinese hamster cells 4


CDKllA
cyclin-dependent kinase 1 lA


CDKllB
cyclin-dependent kinase 1 lB


LURAPIL
leucine rich adaptor protein 1-like


MAD2L2
MAD2 mitotic arrest deficient-like 2 (yeast)


PRDM2
PR domain containing 2, with ZNF domain


NABP2
nucleic acid binding protein 2


NABPl
nucleic acid binding protein 1


PPP1R15A
protein phosphatase 1, regulatory subunit 15A


TATDNl
TatD DNase domain containing 1


TATDN2
TatD DNase domain containing 2


TATDN3
TatD DNase domain containing 3


CEBPB
CCAAT/enhancer binding protein (C/EBP), beta


INIP
INTS3 and NABP interacting protein


INTS3
integrator complex subunit 3


SDIM1
stress responsive DNAJB4 interacting membrane protein 1


DHX9
DEAH (Asp-Glu-Ala-His) (SEQ ID NO: 39) box helicase 9


SATBl
SATB homeobox 1


FENl
flap structure-specific endonuclease 1


HCST
hematopoietic cell signal transducer


TYROBP
TYRO protein tyrosine kinase binding protein


APA
ankyloblepharon filiforme adnatum


C9orf169
chromosome 9 open reading frame 169


TSP02
translocator protein 2


TCIRGl
T-cell, immune regulator 1, ATPase, H+ transporting, lysosomal VO



subunit A3


Clorf61
chromosome 1 open reading frame 61


HLA-DOA
major histocompatibility complex, class II, DO alpha


SPINK13
serine peptidase inhibitor, Kazal type 13 (putative)









In an embodiment, the payload comprises a modulator of an epigenetic state or characteristic of a component of chromatin, e.g., a chromatin associated protein, e.g., a histone. For example, the epigenetic state or characteristic can comprise histone acetylation, deacetylation, methylation (e.g., mono, di, or tri-methylation), demethylation, phosphorylation, dephosphorylation, ubiquitination (e.g., mono or polyubiquitination), deubiquitination, sumoylation. ADP-ribosylation, deimination, or a combination thereof.


In an embodiment, the modulator is selected from, or modulates, one or more histone modifying enzymes. In an embodiment, the histone modifying enzyme is a histone methyltransferase (HMT). In an embodiment, the histone modifying enzyme is a histone demethyltransferase (HDMT). In an embodiment, the histone modification enzyme is a histone acetyltransferase (HAT). In an embodiment, the histone modifying enzyme is a histone deacetylase (HDAC). In an embodiment, the histone modification enzyme is a kinase. In an embodiment, the histone modifying enzyme is a phosphatase. In an embodiment, the histone modifying enzyme is ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), or ubiquitin ligases (E3s). In an embodiment, the histone modifying enzyme is a deubiquitinating (DUB) enzyme.


In an embodiment, histone modifications involved in regulation of gene transcription are modulated. For example, mono-methylation of H3K4, H3K9, H3K27, H3K79, H4K20, H2BK5, di-methylation of H3K79, tri-methylation of H3K4, H3K79, H3K36, and acetylation of H3K9, H3K14, H3K27, can be associated with transcription activation. As another example, di-methylation of H3K9, H3K27, and tri-methylation of H3K9, H3K27, H3K79, H2BK5 can be associated with transcription repression. In an embodiment, the modulator modulates trimethylation of H3 lysine 4 (H3K4Me3) and/or trimethylation of H3 lysine 36 (H3K36Me3), e.g., in active genes. In an embodiment, the modulator modulates trimethylation of H3 lysine 27 (H3K27Me3), di- and tri-methylation of H3 lysine 9 (H3K9Me2/3), and/or trimethylation of H4 lysine 20 (H4K20Me3), e.g., in repressed genes. In an embodiment, the modulator modulates both activating (e.g., H3K4Me3) and repressing (e.g., H3K27Me3) marks, e.g., in stem cells.


In an embodiment, histone modifications involved in DNA damage response and repair are modulated. For example, the modulators described herein can modulate phosphorylation of H2AX at Serine 139 and/or acetylation of H3 lysine 56 (H3K56Ac).


Aberrant histone modifications are associated with various diseases and conditions, e.g., cancer, cardiovascular disease, and neurodegenerative disorder. The modulators described herein can be used to treat a disease or condition described herein, e.g., by modulating one or more histone modifications, as described herein.


Epigenetic changes in histones can be evaluated by art-known methods or as described herein. Exemplary methods for detecting histone modifications include, e.g., chromatin immunoprecipitation (Ch1P) using antibodies against modified histones, e.g., followed by quantitative PCR.


Exemplary endogenous or exogenous modulators of chromatin structure are described herein, e.g., in Table VI-4.












TABLE VI-4





Approved





Symbol
Approved Name
Synonyms
Ref Seq IDs







SUV39Hl
suppressor of variegation 3-9
KMTlA
NM_003173



homolog 1 (Drosophila)




SUV39H2
suppressor of variegation 3-9
FLJ23414, KMTlB
NM_024670



homolog 2 (Drosophila)




EHMT2
euchromatic histone-1ysine N-
G9A, Em: AF134726.3,
NM_006709



methyltransferase 2
NG36/G9a, KMTlC



EHMTl
euchromatic histone-1ysine N-
Eu-HMTasel,
NM_024757



methyltransferase 1
FLJ12879, KIAA1876,





bA188C12.1, KMTlD



SETDBl
SET domain, bifurcated 1
KGlT, KIAA0067,





ESET, KMTlE,





TDRD21



SETDB2
SET domain, bifurcated 2
CLLD8, CLLL8,
NM_031915




KMTlF



KMT2A
lysine (K)-specific methyltransferase
TRXl, HRX, ALL-1,
NM_005933



2A
HTRXl, CXXC7,





MLLlA



KMT2B
lysine (K)-specific methyltransferase
KIAA0304, MLL2,
NM_014727



2B
TRX2, HRX2, WBP7,





MLL1B, MLL4



KMT2C
lysine (K)-specific methyltransferase
KIAA1506, HALR




2C




KMT2D
lysine (K)-specific methyltransferase
ALR, MLL4,




2D
CAGL114



KMT2E
lysine (K)-specific methyltransferase
HDCMC04P




2E




SETDlA
SET domain containing lA
KIAA0339, Setl,
NM_014712




KMT2F



SETDlB
SET domain containing lB
KIAA1076, SetlB,
XM_037523




KMT2G



ASHIL
ashl (absent, small, or homeotic)-like
huASHl, ASHl,
NM_018489



(Drosophila)
ASHlLl, KMT2H



SETD2
SET domain containing 2
HYPB, HIF-1,
NM_014159




KIAA1732, FLJ23184,





KMT3A



NSDl
nuclear receptor binding SET domain
ARA267, FLJ22263,
NM_172349



protein 1
KMT3B



SMYD2
SET and MYND domain containing
HSKM-B, ZMYND14,
NM_020197



2
KMT3C



SMYDl
SET and MYND domain containing
BOP, ZMYND22,
XM_097915



1
KMT3D



SMYD3
SET and MYND domain containing
KMT3E
NM_022743



3




DOTIL
DOTl-like histone H3K79
KIAA1814, DOTl,
NM_032482



methyltransferase
KMT4



SETD8
SET domain containing (lysine
SETS, SET07, PR-
NM_020382



methyltransferase) 8
Set7, KMT5A



SUV420H
suppressor of variegation 4-20
CGI-85, KMT5B
NM_017635


1
homolog 1 (Drosophila)




SUV420H
suppressor of variegation 4-20
MGC2705, KMT5C
NM_032701


2
homolog 2 (Drosophila)




EZH2
enhancer of zeste homolog 2
EZHl, ENX-1, KMT6,




(Drosophila)
KMT6A



EZHl
enhancer of zeste homolog 1
KIAA0388, KMT6B
NM_001991



(Drosophila)




SETD7
SET domain containing (lysine
KIAA1717, SET7,
NM_030648



methyltransferase) 7
SET7/9, Set9, KMT7



PRDM2
PR domain containing 2, with ZNF
RIZ, RIZ1, RIZ2,
NM_012231



domain
KMT8, MTB-ZF,





HUMHOXY1



HATl
histone acetyltransferase 1
KATl
NM_003642


KAT2A
K(lysine) acetyltransferase 2A
GCN5, PCAF-b
NM_021078


KAT2B
K(lysine) acetyltransferase 2B
P/CAF, GCN5,
NM_003884




GCN5L



CREBBP
CREB binding protein
RTS, CBP, KAT3A
NM_004380


EP300
ElA binding protein p300
p300, KAT3B
NM_001429


TAFl
TAFl RNA polymerase II, TATA
NSCL2, TAFII250,
NM_004606



box binding protein (TBP)-associated
KAT4, DYT3/TAF1




factor, 250 kDa




KATS
K(lysine) acetyltransferase 5
TIP60, PLIP, cPLA2,
NM_006388




HTATIPl, ESAl,





ZC2HC5



KAT6A
K(lysine) acetyltransferase 6A
MOZ, ZC2HC6A
NM_006766


KAT6B
K(lysine) acetyltransferase 6B
querkopf, qkf, Morf,
NM_012330




MOZ2, ZC2HC6B



KAT7
K(lysine) acetyltransferase 7
HBOA, HBOl,
NM_007067




ZCZHC7



KATS
K(lysine) acetyltransferase 8
MOP, FLJ14040,
NM_032188




hMOF, ZC2HC8



ELP3
elongator acetyltransferase complex
FLJ10422, KAT9
NM_018091



subunit 3




GTF3C4
general transcription factor IIIC,
TFIIIC90, KAT12




polypeptide 4, 90 kDa




NCOAl
nuclear receptor coactivator 1
SRCl, F-SRC-1,
NM_147223




NCoA-1, KAT13A,





RIP160, bHLHe74



NCOA3
nuclear receptor coactivator 3
RAC3, AIB1, ACTR,
NM_006534




p/CIP, TRAM-1,





CAGH16, TNRC16,





KAT13B, bHLHe42,





SRC-3, SRC3



NCOA2
nuclear receptor coactivator 2
TIF2, GRIPl, NCoA-2,





KAT13C, bHLHe75



CLOCK
clock circadian regulator
KIAA0334, KAT13D,
NM_004898




bHLHe8



KDMlA
lysine (K)-specific demethylase lA
KIAA0601, BHCl 10,
NM_015013




LSD1



KDMlB
lysine (K)-specific demethylase lB
FLJ34109, FLJ33898,
NM_153042




dJ298J15.2,





bA204B7.3, FLJ43328,





LSD2



KDM2A
lysine (K)-specific demethylase 2A
KIAA1004, FBLll,
NM_012308




LILINA,





DKFZP434Ml 735,





FBL7, FLJ00115,





CXXC8, JHDMlA



KDM2B
lysine (K)-specific demethylase 2B
PCCX2, CXXC2,
NM_032590




FbllO, JHDMlB



KDM3A
lysine (K)-specific demethylase 3A
TSGA, KIAA0742,
NM_018433




JHMD2A



KDM3B
lysine (K)-specific demethylase 3B
KIAA1082, NET22
NM_016604


KDM4A
lysine (K)-specific demethylase 4A
KIAA0677, JHDM3A,
NM_014663




TDRD14A



KDM4B
lysine (K)-specific demethylase 4B
KIAA0876, TDRD14B
NM_015015


KDM4C
lysine (K)-specific demethylase 4C
GASCl, KIAA0780,
NM_015061




TDRD14C



KDM4D
lysine (K)-specific demethylase 4D
FLJ10251
NM_018039


KDM4E
lysine (K)-specific demethylase 4E
JMJD2E
NM_OOl 161630


KDM5A
lysine (K)-specific demethylase 5A

NM_005056


KDM5B
lysine (K)-specific demethylase 5B
RBBP2H1A, PLU-1,
NM_006618




CT31



KDM5C
lysine (K)-specific demethylase 5C
DXS1272E, XE169
NM_004187


KDM5D
lysine (K)-specific demethylase 5D
KIAA0234
NM_004653


KDM6A
lysine (K)-specific demethylase 6A

NM_021140


KDM6B
lysine (K)-specific demethylase 6B
KIAA0346
XM_043272


JHDMlD
jumonji C domain containing histone
KIAA1718
NM_030647



demethylase 1 homolog D (S.





cerevisiae)




PHF8
PHD finger protein 8
ZNF422, KIAAl 111,
NM_015107




JHDMlF



PHF2
PHD finger protein 2
KIAA0662, JHDMlE,
NM_005392




CENP-35



KDM8
lysine (K)-specific demethylase 8
FLJ13798
NM_024773









Modulators of Gene Expression


In an embodiment a payload comprises a modulator of gene expression. A modulator of gene expression can be delivered in vitro, ex vivo, or in vivo.


In an embodiment, the payload comprises a transcription factor. Transcription factors can bind to specific DNA sequences (e.g., an enhancer or promoter region) adjacent to the genes that they regulate. For example, transcription factors can stabilize or inhibit the binding of RNA polymerase to DNA, catalyze the acetylation or deacetylation of histone proteins (e.g., directly or by recruiting other proteins with such catalytic activity), or recruit coactivator or corepressor proteins to the transcription factor/DNA complex. Modulators of gene expression also include, e.g., any proteins that interact with transcription factors directly or indirectly.


In an embodiment, the transcription factor is a general transcription factor, e.g., is ubiquitous and interacts with the core promoter region surrounding the transcription start site(s) of many, most or all class II genes. Exemplary general transcription factors include, e.g., TFIIA. TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. In an embodiment, the transcription factor is an upstream transcription factor, e.g., binds upstream of the initiation site to stimulate or repress transcription. In an embodiment, the transcription factor is a specific transcription factor, e.g., a transcription factor dependent on a recognition sequence present in the proximity of the gene. Exemplary specific transcription factors include, e.g., SP1, AP-1, C/EBP, heat shock factor, ATF/CREB, -Myc, OCT-1, and NF-1.


In an embodiment, the transcription factor is constitutively active, e.g., a general transcription factor, SP1, NF-1, or CCAAT. In an embodiment, the transcription factor is conditionally active, e.g. it requires activation, e.g., developmental (e.g., GATA, HNF, PIT-1, MyoD, MyfS, Hox, Winged Helix), signal-dependent (e.g., extracellular ligand (endocrine or paracrine)-dependent, intracellular ligand (autocrine)-dependent (e.g., SREBP, p53, orphan nuclear receptors), cell membrane receptor-dependent (e.g., resident nuclear factors (e.g., CREB, AP-1, Mef2) or latent cytoplasmic factors (e.g., STAT, R-SMAD, NF-KB, Notch, TUBBY, NFAT).


Other exemplary transcription factors are described herein, e.g., in Table VI-5.









TABLE VI-5







Selected Transcription Factors with Anotations








Transcription



factor family



(#genes/



family)
Comments





AF-4(4)
Exemplary diseases include acute lymphoblastic



leukemia (AF4 and AFF3) and mental retardation



(FMR2).


CBF(1)
Exemplary functions include regulator of hematopoiesis.



For example, CBF, is also involved in the chondrocyte



differentiation and ossification.


CSL(2)
Exemplary functions include universal transcriptional



effector of Notch signaling. For example, Notch



signaling is dysregulated in many cancers and faulty



notch signaling is implicated in many diseases.



Exemplary disease include T-ALL (T-cell acute



lymphoblastic leukemia), CADASIL (Cerebral



Autosomal-Dominant Arteriopathy with Sub-cortical



Infarcts and Leukoencephalopathy), MS (Multiple



Sclerosis), Tetralogy of Fallot, Alagille syndrome.


ETS(29)
Exemplary functions include regulation of cellular



differentiation, cell cycle control, cell migration,



cell proliferation, apoptosis (programmed cell death)



and angiogenesis. Exemplary diseases include dieases



associated with cancer, such as through gene fusion,



e.g., prostate cancer.


HMGI/
Overexpression in certain cancers


HMGY(2)



MH1(8)
Exemplary diseases include cancer, fibrosis and



autoimmune diseases.


Nuclear
Exemplary functions include superfamily of


orphan
transcription regulators that are involved in widely


receptor(3)
diverse physiological functions, including control of



embryonic development, cell differentiation and



homeostasis. Exemplary diseases include



inflammation, cancer, and metabolic disorders.


PC4(1)
Exemplary functions include replication, DNA



repair and transcription.


RFX(8)
Exemplary functions include regulation of



development and function of cilia. Exemplary



diseases include Bardet-Biedl syndrome.


STAT(7)
Exemplary functions include regulation of many



aspects of growth, survival and differentiation in



cells. Exemplary diseases include angiogenesis,



enhanced survival of tumors and immunosuppression.


Thyroid
Involved in widely diverse physiological functions,


hormone
including control of embryonic development, cell


receptor(25)
differentiation and homeostasis


zf-C2HC(6)
Highly transcribed in the developing nervous system.



Exemplary diseases include Duane Radial Ray



Syndrome.


Androgen
Exemplary functions include diverse physiological


receptor(1)
functions, including control of embryonic development,



cell differentiation and homeostasis. Exemplary



diseases include X-linked spinal, bulbar muscular



atrophy and prostate cancer.


CG-1(2)
Exemplary functions include calcium signaling by



direct binding of calmodulin.


CTF/NF1(4)
Exemplary functions include both viral DNA



replication and regulation of gene expression.



Exemplary diseases include leukemia, juvenile



myelomonocytic.


Fork
Involvement in early developmental decisions of


head(49)
cell fates during embryogenesis. Exemplary



diseases include lymphedema-distichiasis,



developmental verbal dyspraxia, autoimmune



diseases.


Homeobox
Exemplary functions include involvement in a


(205)
wide range of critical activities during development.



Exemplary diseases include limb malformations,



eye disorders, and abnormal head, face, and tooth



development. Additionally, increased or decreased



activity of certain homeobox genes has been



associated with several forms of cancer.


MYB(25)
Exemplary functions include regulator of



proliferation, differentiation and cell fate.



Exemplary diseases include cancer (e.g.,



oncogenic disease).


Oestrogen
Control of embryonic development, cell


receptor(1)
differentiation and homeostasis. Exemplary



diseases include estrogen resistance, familial



breast cancer, migrane, myocardial infaction.


POU(21)
Wide variety of functions, related to the function



of the neuroendocrine system and the



development of an organism. Exemplary



diseases include non-syndromic deafness.


RHD(lO)
Exemplary diseases include autoimmune



arthritis, asthma, septic shock, lung fibrosis,



glomerulonephritis, atherosclerosis, and AIDS.


T-box(17)



TSC22(4)



zf-



GATA(14)



AP-2(5)



COE(4)



CUT(7)



GCM(2)



HSF(8)



NDT80/



PhoG(1)



Other nuclear



receptor(2)



PPAR



receptor(3)



ROR



receptor(4)



TEA(4)



Tub(5)



zf-LITAF-



like(2)



ARID(15)



COUP(3)



DM(7)



GCR(1)



HTH(2)



NF-YA(1)



Others(3)



Progesterone



receptor(1)



Runt(3)



TF_bZIP(46)



ZBTB(48)



zf-MIZ(7)



bHLH(106)



CP2(7)



E2F(ll)



GTF2I(5)



IRF(9)



NF-YB/C(2)



P53(3)



Prox1(2)



SAND(8)



TF_Otx(3)



zf-BED(5)



zf-NF-Xl(2)



C/EBP(lO)



CSD(8)



Ecdystd



receptor(2)



HMG(50)



MBD(9)



Nrfl(l)



PAX(9)



Retinoic acid



receptor(7)



SRF(6)



THAP(l2)



zf-



C2H2(634)



CRX
Exemplary diseases include dominant cone-rod



dystrophy. Repair mutation.


FOCX2
Exemplary diseases include lymphedema-distichiasis.



Repair mutation.


FOXP2
Exemplary diseases include developmental verbal



dyspraxia. Repair mutation.


FOXP3
Exemplary diseases include autoimmune diseases.



Repair mutation.


GAT4
Exemplary diseases include congenital heart defects.



Repair mutation.


HNFl
Exemplary diseases include mature onset diabetes of


through
the young (MODY), hepatic adenomas and renal


HNF6
cysts. Repair mutation.


LHX3
Exemplary diseases include Pituitary disease. Repair



mutation.


MECP2
Exemplary diseases include Rett syndrome. Repair



mutation.


MEF2A
Exemplary diseases include Coronary artery disease.



Repair mutation.


NARA2
Exemplary diseases include Parkinson disease.



Repair mutation.


NF-KB
Exemplary diseases include autoimmune arthritis,


Activation
asthma, septic shock, lung fibrosis, glomerulonephritis,



atherosclerosis, and AIDS. Repair mutation.


NF-KB
Exemplary diseases include apoptosis, inappropriate


Inhibition
immune cell development, and delayed cell growth.



Repair mutation.


NIKX2-5
Exemplary diseases include cardiac malformations and



atrioventricular conduction abnormalities.


NOTCHl
Exemplary diseases include aortic valve abnormalities.









Modulators of Alternative Splicing


In an embodiment, the modulator of gene expression modulates splicing. For example, a modulator can modulate exon skipping or cassette exon, mutually exclusive exons, alternative donor site, alternative acceptor site, intron retention, or a combination thereof. In an embodiment, the modulator is selected from or modulates one or more general or alternative splicing factors, e.g., ASF1. In an embodiment, the modulator modulates alternative splicing (e.g., influences splice site selection) in a concentration-dependent manner.


Modulators of Post-Transcriptional Modification


In an embodiment, the modulator of gene expression modulates post-transcriptional modification. For example, the modulators described herein can promote or inhibit 5′ capping, 3′ polyadenylation, and RNA splicing. In an embodiment, the modulator is selected from, or modulates, one or more factors involved in 5′ capping, e.g., phosphatase and guanosyl transferase. In an embodiment, the modulator is selected from, or modulates, one or more factors involved in 3′ polyadenylation, e.g., polyadenylate polymerase, cleavage and polyadenylation specificity factor (CPSF), and poly(A) binding proteins. In an embodiment, the modulator is selected from, or modulates, one or more factors involved in RNA splicing, e.g., general or alternative splicing factors.


Exemplary endogenous or exogenous modulators of post-transcriptional modification are described herein, e.g., in Table VI-6.









TABLE VI-6







POST-TRANSCRIPTIONAL CONTROL MODULATORS


 mRNA processing


  Polyadenylation


    PARN: polyadenylation specific ribonuclease


    PAN: PolyA nuclease


    CPSF: cleavage/polyadenylation specificity factor


    CstF: cleavage stimulation factor


    PAP: polyadenylate polymerase


    PABP: polyadenylate binding protein


    PAB2: polyadenylate binding protein 2


    CPI: cleavage factor I


    CHI: cleavage factor II


  Capping/Methylation of S'end


    RNA triposphatase


    RNA gluanyltransferase


    RNA mehyltransferase


    SAM synthase


    ubiquitin-conjugating enzyme E2R1


  Splicing


    SR proteins SERS1-SFR1 1 which, when bound to


    exons, tend to promote


    ImRNP proteins: coded by the following


    genes: HNRNPAO, HNRNPA1, HNRNPA1L1,


    HNRNPA1L2, HNRNPA3, HNRNPA2B1,


    HNRNPAB, HNRNPB1, HNRNPC, HNRNPCL1,


    HNRNPD, HNRPDL, HNRNPF, HNRNPH1,


    HNRNPH2, HNRNPH3, HNRNPK, HNRNPL,


    HNRPLL, HNRNPM, HNRNPR, HNRNPU,


    HNRNPUL1, HNRNPUL2, HNRNPUL3


  Editing protein


    ADAR


  Nuclear export proteins


    Mex67


    Mtr2


    Nab2


    DEAD-box helicase (“DEAD” disclosed as SEQ ID


    NO: 40)


TRANSLATION


 Initiation


   eIF4A, eIF4B, eIF4E, and eIF4G: Eukaryotic initiation factors


   GEF: Guanine exchange factor


   GCN2, PKR, HRI and PERK: Kinases involved in phosphorylating


   some of the initiation factors


 Elongation


   eEF1 and eEF2: elongation factors


   GCN: kinase


 Termination


   eRF3: translation termination factor


POST-TRANSLATIONAL CONTROL


 mRNA Degradation


   ARE-specific binding proteins


   EXRNI: exonuclease


   DCP1, DCP2: Decapping enzymes


   RCK/p54, CPEB, eIF4E: Translation repression


   microRNAs and siRNAs: Probably regulate 30% of all genes


   DICER


   Ago proteins


   Nonsense-mediated mRNA decay proteins


    UPF3A


    UPF3B


    eIF4A3


    MLN51


    Y14/MAGOH


    MG-1


    SMG-5


    SMG-6


    SMG-7


 mRNA Modification


   Enzymes carry the following functions


    Phosphorylation


    N-linked glycosylation


    Acetylation


    Amidation


    Hydroxylation


    Methylation


    0-linked glycosylation


    Ubiquitylation









Inhibitors


In an embodiment a payload comprises an inhibitor of a payload described above, e.g., an inhibitor of an enzyme transcription factor. In an embodiment a payload comprises an inhibitor of any of the aforementioned payload molecules, processes, activities or mechanisms. In an embodiment, the inhibitor is an antibody molecule (e.g., a full antibody or antigen binding fragment thereof) specific for one of the payload molecules described herein. In an embodiment the inhibitor is a small molecule compound. In an embodiment, the inhibitor is a nucleic acid (e.g., siRNA, shRNA, ribozyme, antisense-oligonucleotide, and aptamer). For example, the payload is an inhibitor of a target, e.g., a transcription factor, a post-translational modification enzyme, a post-transcriptional modification enzyme, etc., or a nucleic acid sequence encoding any of the foregoing.


Orthologs


If a non-human gene or protein is recited herein it is understood that the invention also comprises the human counterpart or ortholog and uses thereof.


VIIA. Targets: Cells


Cas9 molecules and gRNA molecules, e.g., a Cas9 molecule/gRNA molecule complex, can be used to manipulate a cell (e.g., an animal cell or a plant cell), e.g., to deliver a payload, or edit a target nucleic acid, in a wide variety of cells. Typically an eiCas9 molecule/gRNA molecule complex is used to deliver a payload and an eaCas9 molecule/gRNA complex is used to edit or alter the structure of a target nucleic acid. Delivery or editing can be performed in vitro, ex vivo, or in vivo.


In an embodiment, a cell is manipulated by editing (e.g., introducing a mutation or correcting) one or more target genes, e.g., as described herein. In an embodiment, a cell is manipulated by delivering a payload comprising one or more modulators (e.g., as described herein) to the cell, e.g., to a target sequence in the genome of the cell. In an embodiment, the expression of one or more target genes (e.g., one or more target genes described herein) is modulated. e.g., in vivo. In an embodiment, the expression of one or more target genes (e.g., one or more target genes described herein) is modulated, e.g., ex vivo.


In an embodiment, the cells are manipulated (e.g., converted or differentiated) from one cell type to another. In an embodiment, a pancreatic cell is manipulated into a beta islet cell. In an embodiment, a fibroblast is manipulated into an iPS cell. In an embodiment, a preadipocyte is manipulated into a brown fat cell. Other exemplary cells include, e.g., muscle cells, neural cells, leukocytes, and lymphocytes.


In an embodiment, the cell is a diseased or mutant-bearing cell. Such cells can be manipulated to treat the disease, e.g., to correct a mutation, or to alter the phenotype of the cell. e.g., to inhibit the growth of a cancer cell. For examples, a cell is associated with one or more diseases or conditions describe herein. In an embodiment, the cell is a cancer stem cell. For example, cancer stem cells can be manipulated by modulating the expression of one or more genes selected from: TWIST (TF), HIP-1a, HER2/neu, Snail (TF), or Wnt.


In an embodiment, the manipulated cell is a normal cell.


In an embodiment, the manipulated cell is a stem cell or progenitor cell (e.g., iPS, embryonic, hematopoietic, adipose, germline, lung, or neural stem or progenitor cells).


In an embodiment, the manipulated cells are suitable for producing a recombinant biological product. For example, the cells can be CHO cells or fibroblasts. In an embodiment, a manipulated cell is a cell that has been engineered to express a protein.


In an embodiment, the cell being manipulated is selected from fibroblasts, monocytic precursors, B cells, exocrine cells, pancreatic progenitors, endocrine progenitors, hepatoblasts, myoblasts, or preadipocytes. In an embodiment, the cell is manipulated (e.g., converted or differentiated) into muscle cells, erythroid-megakaryocytic cells, eosinophils, iPS cells, macrophages, T cells, islet beta-cells, neurons, cardiomyocytes, blood cells, endocrine progenitors, exocrine progenitors, ductal cells, acinar cells, alpha cells, beta cells, delta cells, PP cells, hepatocytes, cholangiocytes, or brown adipocytes.


In an embodiment, the cell is a muscle cell, erythroid-megakaryocytic cell, eosinophil, iPS cell, macrophage, T cell, islet beta-cell, neuron, cardiomyocyte, blood cell, endocrine progenitor, exocrine progenitor, ductal cell, acinar cell, alpha cell, beta cell, delta cell. PP cell, hepatocyte, cholangiocyte, or white or brown adipocyte.


The Cas9 and gRNA molecules described herein can be delivered to a target cell. In an embodiment, the target cell is a normal cell.


In an embodiment, the target cell is a stem cell or progenitor cell (e.g., iPS, embryonic, hematopoietic, adipose, germline, lung, or neural stem or progenitor cells).


In an embodiment, the target cell is a CHO cell.


In an embodiment, the target cell is a fibroblast, monocytic precursor. B cells exocrine cell, pancreatic progenitor, endocrine progenitor, hepatoblast, myoblast, or preadipocyte.


In an embodiment, the target cell is a muscle cell, erythroid-megakaryocytic cell, eosinophil, iPS cell, macrophage, T cell, islet beta-cell, neurons (e.g., a neuron in the brain, e.g., a neuron in the striatum (e.g., a medium spiny neuron), cerebral cortex, precentral gyms, hippocampus (e.g., a neuron in the dentate gyrus or the CA3 region of the hippocampus), temporal cortex, amygdala, frontal cortex, thalamus, cerebellum, medulla, putamen, hypothalamus, tectum, tegmentum or substantia nigra), cardiomyocyte, blood cell, endocrine progenitor, exocrine progenitor, ductal cell, acinar cell, alpha cell, beta cell, delta cell. PP cell, hepatocyte, cholangiocyte, or brown adipocyte.


In an embodiment, the target cell is manipulated ex vivo by editing (e.g., introducing a mutation or correcting) one or more target genes and/or modulating the expression of one or more target genes, and administered to the subject.


Exemplary cells that can be manipulated and exemplary genes that can be modulated are described in Table VI-8.












TABLE VII-8





Cell starting
Differentiated

Exemplary gene(s) to


point
state
Exemplary payload manipulation
modify expression of







fibroblasts
Muscle cells
Deliver Cas9-activators to target
MyoD




activation of transcription factors





required for differentiation in vivo.



Monocytic
Erythroid-
Deliver Cas9-activators to target
GATAl


precursors
megakaryocytic
activation of transcription factors




cells,
required for differentiation in vivo.




eosinophils




fibroblasts
iPS cells
Deliver Cas9-activators to target
Oct4




activation of transcription factors
Sox2




required for differentiation in vivo.
Klf4




Multiplex.
Myc


B cells
Macrophages
Deliver Cas9-activators to target
C/EBPa




activation of transcription factors





required for differentiation in vivo.



B cells
T cells,
Delivery Cas9-repressors OR
Pax5



macrophages
deliver Cas9 endonuclease to





ablate Pax5



Exocrine
Islet-cells
Deliver Cas9-activators to target
Pdxl


cells

activation of transcription factors
Ngn3




required for differentiation in vivo.
MafA




Multiplex.



Fibroblasts
Neurons
Deliver Cas9-activators to target
Ascll




activation of transcription factors
Bm2




required for differentiation in vivo.
Mytll




Multiplex.



fibroblasts
cardiomyocytes
Deliver Cas9-activators to target
Gata4




activation of transcription factors
Mef2c




required for differentiation in vivo.
Tbx5




Multiplex.



Fibroblasts
Blood cells
Deliver Cas9-activators to target
Oct4




activation of transcription factors





required for differentiation in vivo.



Fibroblsts
cardiomyocytes
Deliver Cas9-activators to target
Oct4




activation of transcription factors
Sox2




required for differentiation in vivo.
Klf4




Multiplex.



Pancreatic
Endocrine
Deliver Cas9-activators to target
Ngn3


progenitor
progenitor
activation of transcription factors





required for differentiation in vivo.



Pancreatic
Exocrine
Deliver Cas9-activators to target
P48


progenitor
progenitor
activation of transcription factors





required for differentiation in vivo.



Pancreatic
Duct
Deliver Cas9-activators to target
Hnf6/0C-1


progenitor

activation of transcription factors





required for differentiation in vivo.



Pancreatic
acmar
Deliver Cas9-activators to target
Ptfla


progenitor

activation of transcription factors
Rpbjl




required for differentiation in vivo.





Multiplex.



Endocrine
a cell
Deliver Cas9-activators to target
Foxa2


progenitor

activation of transcription factors
Nkx2.2


(to make

required for differentiation in vivo.
Pax6


glucagon)

Multiplex.
Arx


Endocrine
cell
Deliver Cas9-activators to target
Mafa


progenitor

activation of transcription factors
Pdxl


(to make

required for differentiation in vivo.
Hlx69


insulin)

Multiplex.
Pax4





Pax6





Isll





Nkx2.2





Nkx6.1


Endocrine
8 cell
Deliver Cas9-activators to target
Pax4


progenitor

activation of transcription factors
Pax6


(to make

required for differentiation in vivo.



somatostatin)

Multiplex.



Endocrine
PP cell
Deliver Cas9-activators to target
Nkx2.2


progenitor

activation of transcription factors



(to make

required for differentiation in vivo.



pancreatic





polypeptide)





Hepatoblast
hepatocyte
Deliver Cas9-activators to target
Hnf4




activation of transcription factors





required for differentiation in vivo.



Hepatoblast
Cholangiocyte
Deliver Cas9-activators to target
Hnf6/0C-1




activation of transcription factors





required for differentiation in vivo.



Myoblasts
Brown
Deliver Cas9-activators to target
PRDM16



adipocyte
activation of transcription factors
C/EBP




required for differentiation in vivo.
PGCla




Multiplex.
PPARy


preadipocytes
Brown
Deliver Cas9-activators to target
PRDM16



adipocyte
activation of transcription factors
C/EBP




required for differentiation in vivo.





Multiplex.
















TABLE VII-9





Exemplary cells for manipulation







Pancreatic cells, e.g., beta cells


Muscle cells


Adipocytes


Pre-adipocytes


Neural cells


Blood cells


Leukocytes


Lymphocyes


B cells


T cells
















TABLE VII-10





Exemplary stem cells for manipulation


embryonic stem cells







non-embryonic stem cells


hematopoietic stem cells


adipose stem cells


germline stem cells


lung stem cells


neural stem cells
















TABLE VII-11





Exemplary cancer cells for manipulation







lung cancer cells


breast cancer cells


skin cancer cells


brain cancer cells,


pancreatic cancer cells


hematopoietic cancer cells


liver cancer cells


kidney cancer cells


ovarian cancer cells
















TABLE VII-12





Exemplary non-human cells for manipulation







Plant cells, e.g., crop cells, e.g., corn, wheat, soybean, citrus


or vegetable cells


Animal cells, e.g., a cow, pig, horse, goat, dog or cat cell









Exemplary endogenous or exogenous modulators of cancer stem cells (CSCs) are described herein, e.g., in Table VII-13.









TABLE VII-13







TWIST 1 (TF)


HIP-la (TF)


HER2/neu


Snail (TF)


Wnt


TGF


FGF


EGF


HGF


STAT3 (TF)


Notch


P63 (TF)


PI3K)/AKT


Hedgehog


NF-KB (TF)


ATF2 (TF)


miR-200 and miR-34


P53 (TF)


E-cadherin


Transcription factors that inhibit E-cadherin directly


ZEBl


ZEB2


E47


KLF8


Transcription factors that inhibit E-cadherin directly


TCF4


SIXJ


FOXC2


G-CSF and CD34 in AML


PML and FOXO in CML


CD133 in glioblastoma multiforme, osteosarcoma, Ewing's sarcoma,


endometrial, hepatocellular, colon and lung carcinomas and ovarian


and pancreatic adenocarcinoma


CD44 in head and neck cancer, prostate, gastric and colorectal


carcinoma stem cells


CD34 in leukemia


CD38 in leukemia


IL3Ra in leukemia


EpCAM in colon carcinoma and pancreatic adenocarcinoma stem


cells


ALDH in melanoma, colorectal, breast, prostate and squamous cell


carcinomas, pancreatic adenocarcinoma, and osteosarcoma


MAP2 in melanoma


a6-integrin in glioblastoma


SSEA-1 ingliobalstoma


CD24 in breast cancer and other tumors









Cas9 molecules and gRNA molecules, e.g., a Cas9 molecule/gRNA molecule complex, can be used to manipulate a cell (e.g., a cell described herein). e.g., to deliver a payload, or edit a target nucleic acid, e.g., to increase cell engraftment, e.g., to achieve stable engraftment of cells into a native microenvironment. The engrafting cells, the cells in the native microenvironment, or both, can be manipulated. Typically an eiCas9 molecule/gRNA molecule complex is used to deliver a payload and an eaCas9 molecule/gRNA complex is used to edit or alter the structure of a target nucleic acid.


For example, increased efficiency of engraftment of cells can be achieved by: increasing the expression of one or more of the genes described herein, e.g., homing genes, adhesion genes, survival genes, proliferative genes, immune evasion genes, and/or cell protection genes, and/or decreasing the expression of one or more of the genes described herein, e.g., quiescence genes, death/apoptosis genes, and/or immune recognition genes.


In an embodiment, the gene encodes a homing receptor or an adhesion molecule, e.g., that is involved in directing cell migration towards a tissue in association with a tissue-expressed ligand or region rich in soluble cytokine. In an embodiment, the homing receptor or adhesion molecule is expressed on leukocytes, e.g., lymphocytes or hematopoietic stem cells. In an embodiment, the tissue is bone marrow, e.g., extracellular matrix or stromal cells. In an embodiment, the homing receptor or adhesion molecule is C—X—C chemokine receptor type 4 (CXCR4, also known as fusin or CD184). For example, the expression of CXCR4 on hematopoietic stem cells is upregulated. In an embodiment, the ligand is stromal-derived-factor-1 (SDF-1, also known as CXCL12). In an embodiment, the homing receptor or adhesion molecule is CD34. In an embodiment, the ligand is addressin (also known as mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1)).


In an embodiment, the gene encodes a receptor, e.g., expressed on a stem cell or progenitor cell, that binds to a ligand, e.g., a chemokine or cytokine. For example, the receptor can be associated with sternness of the cell and/or attracting the cell to a desired microenvironment. In an embodiment, the receptor is expressed on a hematopoietic stem cell. In an embodiment, the receptor is expressed on a neural stem cell. In an embodiment, the receptor is mast/stem cell growth factor receptor (SCFR, also known as proto-oncogene c-Kit or tyrosine-protein kinase Kit or CD117). In an embodiment, the ligand is stem cell factor (SCP, also known as steel factor or c-kit ligand). In an embodiment, the receptor is myeloproliferative leukemia virus oncogene (MPL, also known as CD110). In an embodiment, the ligand is thrombopoietin (TPO).


In an embodiment, the gene encodes a marker, e.g., that promotes survival or proliferation of the cells expressing that marker, or allows the cells expressing that marker to evade an immune response or to be protected from an adverse environment, e.g., that leads to cell death. For example, cells expressing CD47 (also known as integrin associated protein (IAP) can avoid phagocytosis, e.g., during cell migration. As another example, cells that express BCL2 can be protected from apoptosis. In an embodiment, the cell is a blood cell, e.g., an erythrocyte or leukocyte. In an embodiment, the cell is a hematopoietic stem cell or progenitor cell.


In an embodiment, the expression of one or more of CXCR4, SDF1, CD117, MPL, CD47, or BCL2, in a stem cell or progenitor cell, e.g., a hematopoietic stem cell or progenitor cell, is upregulated.


Cas9 molecules and gRNA molecules, e.g., a Cas9 molecule/gRNA molecule complex, can be used to manipulate a cell (e.g., a cell described herein), e.g., to deliver a payload, or edit a target nucleic acid, e.g., to manipulate (e.g., dictate) the fate of a targeted cell, e.g., to better target specific cell type of interest and/or as a suicide mechanism. Typically an eiCas9 molecule/gRNA molecule complex is used to deliver a payload and/or an eaCas9 molecule/gRNA complex is used to edit or alter the structure of a target nucleic acid. Exemplary genes that can be modulated include, e.g., one or more of chemotherapy resistance genes, chemotherapy sensitivity genes, antibiotic resistance genes, antibiotic sensitivity genes, and cell surface receptor genes, e.g., as described herein.


In an embodiment, a chemotherapy resistance gene, a chemotherapy sensitivity gene, an antibiotic resistance gene, and/or an antibiotic sensitivity gene is modulated, e.g., such that modified or undesirable cells (e.g., modified or undesirable hematopoietic stem cells (HSCs), e.g., in bone marrow) can be reduced or removed, e.g., by chemotherapeutic or antibiotic treatment.


For example, genes or gene products that modulate (e.g., increase) chemotherapy resistance or antibiotic resistance can be delivered into the cells. Cells modified by the chemotherapy or antibiotic resistance gene or gene product can have a higher (e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, or 100 fold higher) survival rate than cells without such modification after chemotherapeutic or antibiotic treatment. In an embodiment, the chemotherapeutic or antibiotic treatment is performed in vivo. In an embodiment, the chemotherapeutic or antibiotic treatment is performed in vitro or ex vivo. In an embodiment, the chemotherapy resistance gene is a gene encoding 06-alkylguanine DNA alkyltransferase (MGMT). In an embodiment, the chemotherapy comprises temozolomide.


As another example, genes or gene products that modulate (e.g., increase) chemotherapy sensitivity or antibiotic sensitivity can be delivered into the cells. The genes or gene products that confer chemotherapy sensitivity or antibiotic sensitivity can be used as suicide signals, e.g., causing apoptosis of the cells. Cells modified by the chemotherapy or antibiotic sensitivity gene or gene product can have a lower (e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, or 100 fold lower) survival rate than cells without such modification after chemotherapeutic or antibiotic treatment. In an embodiment, the chemotherapeutic or antibiotic treatment is performed in vivo. In an embodiment, the chemotherapeutic or antibiotic treatment is performed in vitro or ex vivo.


The method described herein can be used to select or enrich cells that have a modified or desired phenotype, e.g., chemotherapy resistance and/or antibiotic resistance. The method described herein can also be used to remove or reduce the number of cells that have a modified or undesired phenotype, e.g., chemotherapy sensitivity and/or antibiotic sensitivity. For example, cells that exhibit an undesired effect, e.g., an off-target effect or a cancer phenotype, e.g., caused by editing of a nucleic acid in an undesired genomic location or cell type, can be removed.


In an embodiment, a cell surface receptor gene is modulated (e.g., the expression of the cell surface receptor is increased or decreased), such that a therapeutic agent (e.g., a therapeutic antibody) can be used to target a cell (e.g., to kill the cell) that has increased or decreased expression of the cell surface receptor. In an embodiment, the cell surface receptor is CD20. In an embodiment, the therapeutic antibody is Rituximab.


In an embodiment, the cell surface receptor is selected from, e.g., CD52, VEGFR, CD30, EGFR, CD33, or ErbB2. In an embodiment, the therapeutic antibody is selected from, e.g., Alemtuzumab, Rituximab, Cetuximab, Panitumumab, Gentuzaumab, and Trastuzumab. In an embodiment, the cell surface receptor is CD52 and the therapeutic antibody is Alemtuzumab. In an embodiment, the gene encodes VEGF and the therapeutic antibody is Rituximab. In an embodiment, the cell surface receptor is EGFR and the therapeutic antibody is Cetuximab or Panitumumab. In an embodiment, the cell surface receptor is CD33 and the therapeutic antibody is Gentuzaumab. In an embodiment, the cell surface receptor is ErbB2 and the therapeutic antibody is Trastuzumab.


In an embodiment, the expression or activity of the Cas9 molecule and/or the gRNA molecule is induced or repressed, e.g., when the cell is treated with a drug, e.g., an antibiotic, e.g., in vivo. For example, the induction or repression of the expression or activity of the Cas9 molecule and/or the gRNA molecule can be used to reduce toxicity and/or off-target effects, e.g., in certain tissues. In an embodiment, the expression of the Cas9 molecule, the gRNA molecule, or both, is driven by an inducible promoter. In an embodiment, binding of a drug (e.g., an antibiotic) to the Cas9 molecule and/or the gRNA molecule activates or inhibits the activity of the Cas9 molecule and/or the gRNA molecule. In an embodiment, the drug (e.g., antibiotic) is administered locally. In an embodiment, the cell treated with the drug (e.g., antibiotic) is located in the eye, ear, nose, mouth, or skin.


Cas9 molecules and gRNA molecules, e.g., a Cas9 molecule/gRNA molecule complex, can be used to manipulate a cell (e.g., a cell described herein), e.g., to deliver a payload, or edit a target nucleic acid. e.g., in directed enzyme prodrug therapy (DEPT). Typically an eiCas9 molecule/gRNA molecule complex is used to deliver a payload and an eaCas9 molecule/gRNA complex is used to edit or alter the structure of a target nucleic acid.


Directed enzyme prodrug therapy (DEPT) uses enzymes artificially introduced into the body to convert prodrugs, which have no or poor biological activity, to the active form in the desired location within the body. For example, directed enzyme prodrug therapy can be used to reduce the systemic toxicity of a drug, by achieving high levels of the active drug only at the desired site.


In an embodiment, an enzyme required for prodrug conversion or a gene encoding such an enzyme is delivered to a target cell, e.g., a cancer cell. For example, the enzymes or genes can be delivered by a method described herein. In an embodiment, the gene encoding the enzyme required for prodrug conversion is delivered by a viral vector.


Cas9 molecules and gRNA molecules, e.g., a Cas9 molecule/gRNA molecule complex, can be used to manipulate a cell (e.g., a cell described herein), e.g., to deliver a payload, or edit a target nucleic acid, e.g., to improve immunotherapy, e.g. cancer immunotherapy. Typically an eiCas9 molecule/gRNA molecule complex is used to deliver a payload and an eaCas9 molecule/gRNA complex is used to edit or alter the structure of a target nucleic acid. Exemplary genes that can be modulated include, e.g., one or more genes described herein, e.g., PD-LI and/or PD-L2 genes.


VIIB. Targets: Pathways and Genes


Cas9 molecules and gRNA molecules, e.g., a Cas9 molecule/gRNA molecule complex, can be used to manipulate one, two, three or more, elements or a pathway, e.g., by targeting sequences that encode an RNA or protein of a pathway, or sequences that control the expression of an RNA or protein of a pathway. In an embodiment, an element of a first pathway and an element of a second pathway are manipulated. In an embodiment, manipulation comprises delivery of a payload to, or editing, a target nucleic acid. Typically an eiCas9 molecule/gRNA molecule complex is used to deliver a payload and an eaCas9 molecule/gRNA complex is used to edit or alter the structure of a target nucleic acid. Delivery or editing can be performed in vitro, ex vivo, or in vivo.


An element of a pathway can be up or down regulated, e.g., the expression of a gene encoding a protein of a pathway can be increased or decreased. The increase or decrease can be effected by delivery of a payload (e.g., a transcription factor or inhibitor of a transcription factor) or by editing a target nucleic acid (e.g., the use of a template nucleic acid to alter a sequence, e.g., correct or introduce a mutation, in e.g., a control or coding region).


Exemplary pathways comprise pathways associated with: cell proliferation; cell cycle; carbon metabolism; energy metabolism; glycolysis, anerobic respiration, anerobic respiration; transmembrane signal transduction, angiogenesis, DNA replication or repair, or pain.


Exemplary pathways and genes are discussed herein. It will be understood that a pathway or gene can be associated with one or more aspect of cell or organismal function, e.g., a pathway or gene can be involved in both cancer and energy metabolism. Manipulation of a pathway or gene is not limited to the exemplary cell or organismal function listed below. In an embodiment a pathway is associated with one or more diseases or conditions.


In an embodiment, the pathway is associated with cancer, e.g., associated with proliferation (e.g., RAF pathway), evading growth repressors, resisting cell death, enabling replicative immortality/aging, inducing angiogenesis, activating invasion and metastasis, energy metabolism and evading, cancer stem cells, cytokine-receptor interactions, or tumor suppressors. In an embodiment, the pathway is associated with cell cycle control. In an embodiment, the pathway is associated with angiogenesis.


Pathways and genes associated with cancer are described herein, e.g., include the following:









TABLE VII-14







Target Genes from Selected Pathways













CRISPR


Protein/Gene
Pathway
Disease
Regulation











Cancer











PI3K
Proliferation

Down


B-Raf
Proliferation
66% of all melanoma cancers
Down




have a single substitution in





codon 599



AKT
Proliferation

Down


PTEN
Proliferation
Germline mutations leading to
Down




a predisposition to breast and





thyroid cancer





Mutations found in sporadic





brain, breast and prostate



mTOR
Proliferation

Down


JUN
Proliferation

Down


FOS
Proliferation

Down


ERK
Proliferation

Down


MEK
Proliferation

Down


TGF-b
Proliferation

Down


Myc
Proliferation

Down


K-Ras
Proliferation
Mutated in lung cancer (10%
Down




of all Asians and 30% of all





Caucasians)



Src
Proliferation

Down


PYK2
Proliferation

Down


PAK
Proliferation

Down


PAK
Proliferation

Down


PKA
Proliferation

Down


RAC
Proliferation

Down


ALK
Proliferation
Mutated in a subset (2-7%) of





lung cancers



Rb
Evading growth

Up



suppressors/





pro-apoptotic




P53
Evading growth
Mutation in colon, lung,
Up



suppressors/
esophagus, breast, liver, brain




pro-apoptotic
reticuloendothelial tissues, and





hemopoietic tissues



APC
Evading growth
Mutations found in colon and




suppressors/
intestine




pro-apoptotic




CDK4/6
Evading growth

Up



suppressors/





pro-apoptotic




INK4B
Evading growth

Up



suppressors/





pro-apoptotic




CDK2
Evading growth

Up



suppressors/





pro-apoptotic




WNT
Evading growth

Up



suppressors/





pro-apoptotic




WAFl
Evading growth

Up



suppressors/





pro-apoptotic




Frizzled
Evading growth

Up



suppressors/





pro-apoptotic




VHL
Evading growth
Mutated in all clear cell renal
Up



suppressors/
carcinomas




pro-apoptotic




Fas ligand
Resisting cell death/

Down



anti-apoptotic




Fas receptor
Resisting cell death/

Down



anti-apoptotic




Caspase 8
Resisting cell death/

Down



anti-apoptotic




Caspase 9
Resisting cell death/

Down



anti-apoptotic




Bcl-2
Resisting cell death/
Correct notation large deletion
Down



anti-apoptotic
in follicular lymphoma, breast





prostate CLL, melanoma



Bcl-xL
Resisting cell death/

Down



anti-apoptotic




Bcl-w
Resisting cell death/

Down



anti-apoptotic




Mcl-1
Resisting cell death/

Down



anti-apoptotic




Bax
Resisting cell death/

Down



anti-apoptotic




Bak
Resisting cell death/

Down



anti-apoptotic




IGF-1
Resisting cell death/

Down



anti-apoptotic




Puma
Resisting cell death/

Down



anti-apoptotic




Bim
Resisting cell death/

Down



anti-apoptotic




Beclin-1
Resisting cell death/

Down



anti-apoptotic




TGF-b
Enabling replicative





immortality/aging




Telomerase/
Enabling replicative

Down


TERT
immortality/aging




ATAD2
Enabling replicative





immortality/aging




DAF-2
Enabling replicative





immortality/aging




SRT
Enabling replicative





immortality/aging




Eph-A/B
Inducing angiogenesis

Down


Robo
Inducing angiogenesis

Down


Neuropilin
Inducing angiogenesis

Down


Notch
Inducing angiogenesis

Down


Endostatin
Inducing angiogenesis

Down


Angiostatin
Inducing angiogenesis

Down


FGF family
Inducing angiogenesis

Down


Extracellular
Inducing angiogenesis

Down


matrix-degrading





proteases (e.g.,





MMP-2 &





MMP-9)





VEGF-A
Inducing angiogenesis

Down


TSP-1
Inducing angiogenesis

Down


VEGFR-1
Inducing angiogenesis

Down


VEGFR-2
Inducing angiogenesis

Down


VEGFR-3
Inducing angiogenesis

Down


NF2
Activating invasion

Down



and metastasis




LKBl
Activating invasion
Up-regulated in multiple
Down



and metastasis
cancer, including intestine



Snail
Activating invasion

Down



and metastasis




Slug
Activating invasion

Down



and metastasis




Twist
Activating invasion

Down



and metastasis




Zeb1/2
Activating invasion

Down



and metastasis




CCLR5
Activating invasion

Down



and metastasis




cysteine cathepsin
Activating invasion

Down


protease family
and metastasis




Extracellular
Activating invasion

Down


matrix-degrading
and metastasis




proteases (e.g.,





MMP-2 &





MMP-9)





EGF
Activating invasion and

Down



metastasis




CSF-1
Activating invasion and





metastasis




PP2
Energy metabolism

Down


eIF4E
Energy metabolism

Down


RSK
Energy metabolism

Down


PIK3CA
Energy metabolism
Mutated in many breast,
Down




bladder cancers and





hepatocellular carcinoma



BAPl
Energy metabolism
Mutated in renal cell
Down




carcinoma



TWIST (TF)
Cancer Stem Cells

Down


HIP-la
Cancer Stem Cells
Over expressed in renal cell
Down




carcinoma



HER2/neu
Cancer Stem Cells

Down


Snail (TF)
Cancer Stem Cells

Down


Wnt
Cancer Stem Cells

Down


EPCAM
Cancer Stem Cells
Overexpressed in breast, colon,
Down




uterus and other cancers



EGF
Cytokine-receptor

Down



interactions




TGFa
Cytokine-receptor

Down



interactions




PDGF
Cytokine-receptor

Down


IGF-1
interactions




KILTLG





FLT3LG
Cytokine-receptor

Down



interactions




HGF
Cytokine-receptor

Down



interactions




FGF
Cytokine-receptor

Down



interactions




EGFR
Cytokine-receptor
Mutated in lung cancer (40%
Down



interactions
of all Asians and 10-15% of





all Caucasians)



ERBB2
Cytokine-receptor

Down



interactions




PDGFR
Cytokine-receptor

Down



interactions




IGFR
Cytokine-receptor

Down



interactions




c-KIT
Cytokine-receptor

Down



interactions




FLT3
Cytokine-receptor

Down



interactions




MET
Cytokine-receptor

Down



interactions




FGFR
Cytokine-receptor
Mutations in bladder cancer
Down



interactions










DNA damage and genomic instability











DNMTl
Methyl transferases




DNMT2
Methyl transferases




DNMT3a
Methyl transferases




DNMT3b
Methyl transferases




H3K9Me3
Histone methylation




H3K27Me
Histone methylation




Lsh
Helicase activity




BLM
Helicase activity
Bloom's syndrome > Cancer
Correct


WRN
Helicase activity
Werner's syndrome > Cancer
Correct


RTS
Helicase activity
Rothmund-Thompson >
Correct




Cancer



XPA through
Nucleotide excision
Xeroderm pigmentosa



XPG





XPB
Nucleotide excision
Cockayne's syndrome




repa1r




XAB2
Nucleotide excision





repa1r




XPD
Nucleotide excision
Cockayne's syndrome




repa1r




TFIIH
Nucleotide excision





repa1r




RFC
Nucleotide excision





repa1r




PCNA
Nucleotide excision





repa1r




LIG 1
Nucleotide excision





repa1r




Flap
Nucleotide excision




endonueclease 1
repa1r




MNAT
Nucleotide excision





repa1r




MMS19
Nucleotide excision





repa1r




RAD23A
Nucleotide excision





repa1r




RAD23B
Nucleotide excision





repa1r




RPAl
Nucleotide excision





repa1r




RPA2
Nucleotide excision





repa1r




CCNH
Nucleotide excision





repa1r




CDK7
Nucleotide excision





repa1r




CETN2
Nucleotide excision





repa1r




DDBl
Nucleotide excision





repa1r




DDB2
Nucleotide excision





repa1r




ERCCl
Nucleotide excision





repa1r




ATM
Recombinational repair




NBN
Recombinational repair




BRCAl
Recombinational repair
Breast, ovarian and pancreatic
Correct




cancer susceptibility
or Up


BRCA2
Recombinational repair
Breast cancer and ovarian
Correct




susceptibility
or UP


RAD51
Recombinational repair




RAD52
Recombinational repair




WRN
Recombinational repair




BLM
Recombinational repair




FANGB
Recombinational repair




MLHl
Mismatch repair
Multiple (including colon and





uterus)



MLH2
Mismatch repair
Multiple (including colon and





uterus)



MSH2
Mismatch repair




MSH3
Mismatch repair




MSH4
Mismatch repair




MSH5
Mismatch repair




MSH6
Mismatch repair
Multiple (including colon and





uterus)



PMSl
Mismatch repair




PMS2
Mismatch repair
Multiple (including colon and





uterus)



PMS2L3
Mismatch repair










Aging











DAF-2





IGF-1





SRTl
















TABLE VII-15





Genes Mutated in Common Cancers
















Bladder
FGFR3, RBl, HRAS, KRAS, TP53, TSCl, FGFR3


Breast and Ovarian
BRCA, BRCA 2, BARD1, BRIPl, CHEK2, MREl1A, NBN,



PALB2, PTEN, RAD50, RAD50, RAD51C, RAD51D,



PPMID, TP53, BRIP1, RAD54L, SLC22A1L, PIK3CA,



RBlCCl,


Cervical
FGFR3


Colon and Rectal
PT53, STKl 1, PTEN, BMPRlA, SMAD, MLHl, MSH2,



MSH6, PMS, EPCAM, AKTl, APC, MYH, PTPRJ, AXIN2


Endometrial/Uterine
MLHl, MSH2, MSH6, PMS, EPCAM


Esophageal
DLECl, TGFBR2, RNF6, LZTlSl, WWOX


Hepatocellular carcinoma
PDGFRL, CTNNBl, TP53, MET, CASP8, PIK3CA


Renal
VHL, PBRMQ, BAPl, SETD2, HIFl-a


Lung
KRAS, EGFR, ALK, BRAF, ERBB2, FLCN, D1RC2,



RNF139, OGGl, PRCC, TFE, MET, PPP2R1B, RASSFl,



SLC22A1L


Melanoma
BRAF, CDKA, CDKN2A, CDKN2B, CDKND, MClR, TERT,



ATFl, CREBl, EWSRl


Non-Hodgkin Lymphoma
CASPlO, EGFR, IRFl, PIK3CA


Osteosarcoma
CKEK2, LOJ18CR1, RBl


Ovarian
PRKN, AKTl


Pancreatic
KRAS, BRCA2, CDKN2A, MANF, PALB2, SMAD4, TP53,



IPFl


Prostate
MLHl, MSH2, MSH6, and PMS2, BRCA 1, HOXB13,



CHEK2, ELAC2, EPHB2, SDR5A2, PRKARlA, PMCl


Papillary and Follicular
BRAF, NARAS, ERCl, FOXEl, GOLCA5, NCOA4, NKX2-1,


Thyroid
PMCl, RET, TFG, TPR, TRIM24, TRIM27, TRIM33


Erwing Sarcoma
ERG, ETVl, ETV4, EWSRl, FLil


Leukemia
BRC, AMCR2, GMPS, JAK2, AFlO, ARFGEF12, CEBPA,



FLT3, KIT, LPP, MLFl, NPMl, NSD1, NUP214, PICALM,



RUNXl, SH3GL1, WHSClLl, ETV6, RARA, BCR,



ARHGAP26, NFl, PTPNll, GATAl









Any of the following cancer associated genes provided in Table VII-16 can be targeted.









TABLE VII-16





Exemplary Target Genes Associated With Cancer















ABLI, ABL2, ACSL3, AF15Q14, AFIQ, AF3p21, AF5q31, AKAP9, AKTI, AKT2, ALDH2, ALK,


AL017, APC, ARHGEF12, ARHH, ARIDIA, ARID2, ARNT, ASPSCRI, ASXLI, ATFI, ATIC,


ATM, ATRX, AXINI, BAPI, BCLIO, BCLIIA, BCLIIB, BCL2, BCL3, BCL5, BCL6, BCL7A,


BCL9, BCOR, BCR, BHD, BIRC3, BLM, BMPRIA, BRAF, BRCAI, BRCA2, BRD3, BRD4, BRIPI,


BTGI, BUBIB, C12orf9, C15orf21, C15orf55, C16orf75, C2orf44, CAMTAI, CANTI, CARD11,


CARS, CBFA2Tl, CBFA2T3, CBFB, CBL, CBLB, CBLC, CCDC6, CCNBIIPI, CCND1, CCND2,


CCND3, CCNEI, CD273, CD274, CD74, CD79A, CD79B, CDHI, CDHll, CDK12, CDK4, CDK6,


CDKN2A, CDKN2a(pl4), CDKN2C, CDX2, CEBPA, CEPI, CHCHD7, CHEK2, CHIC2, CHNI, CIC,


CIITA, CLTC, CLTCLI, CMKORI, CNOT3, COLIAI, COPEB, COX6C, CREBI, CREB3L1,


CREB3L2, CREBBP, CRLF2, CRTC3, CTNNBI, CYLD, D10S170, DAXX, DDB2, DDIT3, DDXIO,


DDXS, DDX6, DEK, DICER!, DNM2, DNMT3A, DUX4, EBFI, ECT2L, EGFR, EIF4A2, ELF4,


ELK4, ELKS, ELL, ELN, EML4, EP300, EPS15, ERBB2, ERCC2, ERCC3, ERCC4, ERCC5, ERG,


ETVI, ETV4, ETV5, ETV6, EVII, EWSRI, EXTI, EXT2, EZH2, EZR, FACL6, FAM22A, FAM22B,


FAM46C, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FBXOll, FBXW7, FCGR2B,


FEV, FGFRI, FGFRIOP, FGFR2, FGFR3, FH, FHIT, FIPILI, FLII, FLJ27352, FLT3, FNBPI,


FOXL2, FOXOIA, FOX03A, FOXPI, FSTL3, FUBPI, FUS, FVTI, GAS7, GATAI, GATA2,


GATA3, GMPS, GNAll, GNAQ, GNAS, GOLGA5, GOPC, GPC3, GPHN, GRAF, H3F3A,


HCMOGT-1, HEAB, HERPUD1, HEYI, HIPI, HISTIH3B, HISTIH4I, HLF, HLXB9, HMGAI,


HMGA2, HNRNPA2B1, HOOK3, HOXAll, HOXA13, HOXA9, HOXCll, HOXC13, HOXD11,


HOXD13, HRAS, HRPT2, HSPCA, HSPCB, IDHI, IDH2, IGH@, IGK@, IGL@, IKZFI, IL2, IL21R,


IL6ST, IL7R, IRF4, IRTAI, ITK, JAKI, JAK2, JAK3, JAZFI, JUN, KCNJS, KDMSA, KDMSC,


KDM6A, KDR, KIAA1549, KIF5B, KIT, KLF4, KLK2, KRAS, KTNI, LAF4, LASPI, LCK, LCPI,


LCX, LHFP, LIFR, LMOI, LM02, LPP, LRIG3, LYLI, MADH4, MAP, MAFB, MALTI, MAML2,


MAP2Kl, MAP2K2, MAP2K4, MAX, MDM2, MDM4, MDSI, MDS2, MECTI, MED12, MENI,


MET, MITF, MKLI, MLFI, MLHI, MLL, MLL2, MLL3, MLLTI, MLLTIO, MLLT2, MLLT3,


MLLT4, MLLT6, MLLT7, MNI, MPL, MSF, MSH2, MSH6, MSI2, MSN, MTCPI, MUCI, MUTYH,


MYB, MYC, MYCLI, MYCN, MYD88, MYHll, MYH9, MYST4, NACA, NBSI, NCOA1, NCOA2,


NCOA4, NDRGI, NFI, NF2, NFE2L2, NFIB, NFKB2, NIN, NKX2-1, NONO, NOTCH!, NOTCH2,


NPMI, NR4A3, NRAS, NSD1, NT5C2, NTRKI, NTRK3, NUMAI, NUP214, NUP98, OLIG2, OMD,


P2RY8, PAFAHIB2, PALB2, PAX3, PAX5, PAX7, PAX8, PBRMI, PBXI, PCMI, PCSK7,


PDE4DIP, PDGFB, PDGFRA, PDGFRB, PERI, PHF6, PHOX2B, PICALM, PIK3CA, PIK3RI, PIMI,


PLAGI, PML, PMSI, PMS2, PMXI, PNUTLI, POTI, POU2AFI, POU5FI, PPARG, PPP2RIA,


PRCC, PRDMI, PRDMI6, PRFI, PRKARIA, PR01073, PSIP2, PTCH, PTEN, PTPNll, RAB5EP,


RACI, RAD5IL1, RAFI, RALGDS, RANBPI7, RAPIGDSI, RARA, RBI, RBMI5, RECQL4, REL,


RET, RNF43, ROSI, RPLIO, RPL22, RPL5, RPNI, RUNDC2A, RUNXI, RUNXBP2, SBDS, SDC4,


SDHS, SDHB, SDHC, SDHD, SEPT6, SET, SETBPI, SETD2, SF3BI, SFPQ, SFRS3, SH2B3,


SH3GL1, SIL, SLC34A2, SLC45A3, SMARCA4, SMARCBI, SMARCEI, SMO, SOCSI, SOX2,


SRGAP3, SRSF2, SSI8, SSI8Ll, SSH3BPI, SSXI, SSX2, SSX4, STAT3, STKll, STL, SUFU,


SUZI2, SYK, TAFI5, TALI, TAL2, TCEAI, TCFI, TCFI2, TCF3, TCF7L2, TCLIA, TCL6, TERT,


TET2, TFE3, TFEB, TFG, TFPT, TFRC, THRAP3, TIFI, TLXI, TLX3, TMPRSS2, TNFAIP3,


TNFRSFI4, TNFRSFI7, TNFRSF6, TOPI, TP53, TPM3, TPM4, TPR, TRA@, TRAF7, TRB@,


TRD@, TRIM27, TRIM33, TRIPll, TSCI, TSC2, TSHR, TTL, U2AFI, USP6, VHL, VTIIA, WAS,


WHSCI, WHSCILI, WIFI, WRN, WTI, WTX, WWTRI, XPA, XPC, XPOI, YWHAE, ZNFI45,


ZNFI98, ZNF278, ZNF33I, ZNF384, ZNF52I, ZNF9, or ZRSR2









Exemplary pathways and genes associated with energy metabolism are provided in Table VII-17. Exemplary metabolic targets disclosed herein may be modulated using CRISPR/Cas9 as described herein. Modulation may be used to knockdown a gene of interest, correct a defect or mutation in the gene, or to activate a gene of interest.









TABLE VII-17







Exemplary Metabolic Target List










Target
How to Modulate







ACAT, acyl-CoA:cholesterol
Knockdown



acyltransferase




AGPAT2, 1-acyl-glcero-3-phosphate
Knockdown



acyltransferase2




DGAT, diacylglycerol
Knockdown



acyltransferase




GL, gastric lipase
Knockdown



PL, pancreatic lipase
Knockdown



sPLA2, secretory phospholipase A2
Knockdown



ACC, acetyl-CoA carboxylase
Knockdown



CPT, camitine palmitoyl transferase
Knockdown



FAS, fatty-acid synthase
Knockdown



MTP, microsomal triglyceride-
Knockdown



transfer protein




Insulin receptor
Correct defects




or activate



SU receptor/K + ATP channel
Activate with




mutation



a-glucosidase
Knockdown



PPARy
Activate with




mutation



Glycogen phosphorylase
Knockdown



Fructose-I, 6-bisphosphatase
Knockdown



glucose-6-phosphatase
Knockdown



PTP-1B
Knockdown



SHIP-2
Knockdown



GSK-3
Knockdown



lkB kinase
Knockdown



PKCq
Knockdown



GLP1R
Correct mutation



GIPR
Correct mutation



GPR40
Correct mutation



GPR119
Correct mutation



GPR41
Correct mutation



GPR43
Correct mutation



GPR120
Correct mutation



GCGR
Correct mutation



PACl
Correct mutation



VPAC2
Correct mutation



Y1
Knockdown



GHSR
Knockdown



CCKAR
Correct mutation



b2
Correct mutation



a2
Knockdown



MT1
Knockdown



M3
Correct mutation



CB1
Knockdown



P2Y
Correct mutation



H3
Inhibit



MCH-R1
Correct mutation



MCH-R2
Correct mutation



Ghrelin R
Inhibit



FASN
Inhibit



Bombesin-R3
Inhibit



CCK-A Receptor
Correct mutation



Seratonin System
Correct mutation



CBI Cannabinoid Receptors
Inhibit



Dopaminergic System
Correct mutation



Enterostatin
Mutate to super




agonist



CNTF
Mutate to super




agonist



CNTF-R
Correct mutation



SOCS-3
Knockdown



46a
Knockdown



PrPP Receptors
Correct mutation



Amylin
Mutate to super




agonist



CRH System
Mutate to super




agonist



Galanin Receptors
Knockdown



Orexin Receptors
Knockdown



Noradrenalin System
Mutate to super




agonist



CART
Mutate to super




agonist



FATP4
Knockdown



Pancreatic Lipase
Knockdown



ACRP30
Super agonist




mutations



Thyroid Hormone
Correct mutation



B-3 Adrenergic Receptor
Correct mutation



UCPs
Upregulate



PTP-1B
Knockdown



MC3
Correct mutation



ACC2
Knockdown



Perilipin
Knockdown



HMGIC
Knockdown



llBHSD-1
Knockdown



Glucagon R
Knockdown



Glucocoricoid R
Knockdown



llbeta-HSD I
Knockdown



PGC-1
Correct mutation



DPPP-IV
Knockdown



GLP
Mutate to super




agonist



GIP
Mutate to super




agonist



GLP-IR
Correct mutation



AMP Kinase
Correct mutation



IKK-b
Knockdown



PPARa/g
Knockdown



INS-R
Knockdown



SOLT
Knockdown



a-glucosidase
Knockdown



HMGCR
Knockdown



PCSK9
Knockdown



ApoB-100
Knockdown



Leptin
Mutate to super




agonist



Leptin Receptor
Mutate to




constitutively




active receptor



MC4R
Mutate to




constitutively




active receptor



VOMC
Mutate MSH




region to




super agonist



AGRP
Knockdown



IVPY Receptors
Introduce




constitutively




active mutations



5HT2C
Introduce




constitutively




active mutations



GLP-1
Mutate to super




agonist



GLP-1 Receptor
Mutate to




constitutively




active receptor










In an embodiment, the pathways and genes described herein, e.g., in Table VII-17, are also associated with diabetes, obesity, and/or cholesterol and lipids.


Exemplary pathways and genes associated with the cell cycle are provided in Table VII-18.









TABLE VII-18





CELL CYCLE PATHWAYS and REPRESENTATIVE GENES

















DNA Damage
Mismatch repair
Apoptosis





ATM
PMS2
Fas-L


MRE11
MLH1
FasR


NBS1
MSH6
Trail-L


RAD50
MSH2
Trail-R


53BP1
RFC
TNF-a


P53
PCNA
TNF-R1


CHKE
MSH3
FADD


E2Fl
MutS homolog
TRADD


PML
MutL homolog
RIPI


FANCD2
Exonuclease
MyD88


SMC1
DNA Polymerase
IRAK


BLM1
delta (POLD1,
NIL


BRCA1
POLD2, POLD3,
IKK


H2AX
and POLD4-genes




encoding subunits)



ATR
Topoisomerase 1
NF-K


RPA
Topoisomerase 2
IKBa


ATRIP
RNAseH1
IAP


RAD9
Ligase 1
Caspase 3


RAD1
DNA polymerase 1
Caspase 6


HUS
DNA polymerase 3
Caspase 7


RAD17
Primase
Caspase 8


RFC
Helicase
Caspase 10


CHK1
Single-strand binding
HDAC1


TLK1
proteins
HDAC2


CDC25

Cytochrome C




Bx1-xL




STAT3




STATS




DFF45




Vcl-2




ENDO-G




PI3K




Akt




Calpain




Bad




Bax





Ubiquitin-mediated proteolysis
Hypoxia
Cell Proliferation














E1
HERCl
TRAF6
HIP-1a
MAPK


E2
UBE2Q
MEKK1
HIP-1
MAPKK


E3
UBE2R
COP1
Refl
MAPKKK


UBLE1A
UBE2S
PIPH2
HSP90
c-Met


UBLE1B
UBE2U
cIAP
VEGP
HGP


UBLEIC
UBE2W
PIAS
PAS
ERKS1/2


UBE2A
UBE2Z
SYVN
ARNT
ATK


UBE2B
APCLLCN
NHLRC1
VHL
PKCs


UBE2C
UBE1
AIRE
HLP
Paxilin


UBE2A
E6AP
MGRN1
EPP
PAK


UBE2E
UBE3B
BRCA1
VDU2
Adducin


UBE2P
Smmf
PANCL
SUMORESUME
PYK1


UBE2G1
Itch
MID1
SENP1
RB


UBE2G2
HERC2
Cdc20
Calcineurin A
RB1


UBE2I
HERC3
Cdh1
RACKI
Raf-1


UBE2J1
HERC4
Ape1
PTB
A-Raf


UBE2J2
UBE4A
Apc2
Hur
B-raf


UBE2L3
UBE4B
Apc3
PHD2
MEK1/2


UBE2L6
CHIP
Apc4
SSAT2
ERK1/2


UBE2M
CYC4
Apc5
SSAT1
Ets


UBE2N
PPR19
Apc6
CSK3
Elk1


UBE20
UIP5
Apc7
CBP
SAP1


WWPI
Mdm2
Apc8
POX04
cPLA2


WWP2
Parkin
Apc9
FIH-1



TRIP12
Trim32
Apc1O




NEED4
Trim37
Apc11




ARP-BP1
SIAH-1
Apc12




EDD1
PML











Cell survival
Cell cycle arrest





SMAD1
P21


SMAD5
BAX


SAMD8
MDR


LEF1
DRAIL IGFBP3


TCF3
GADD45


TCF4



P300



HAT1



PI3K



Akt



GF1









Exemplary cell cycle genes characterized by their function are provided in Table VII-19.









TABLE V11-19





CELL CYCLE GENES

















Translation initiation factors
Cyclins
Cyclin-dependent Kinases (OKs)





E2F1
CCNA1, CCNA2, CCNB1,
CDK1, CDK2, CDK3, CDK5,


E2F2
CCNB2, CCNB3, CCNC,
CDK6, CDK7, CDK8, CDK9,


E2F3
CCND1, CCND2, CCND3,
CDK11,


E2F4
CCNE1, CCNE2, CCNF,



E2F5
CCNG1, CCNG2, CCNH,



E2F6
CCNI, CCNI2, , CCNO,



E2F8
CCNT1, CCNT2, CCNY,




CCNYL1, CCNYL2,




CCNYL3






CDK inhibitory proteins
CDK regulators (both


Cyclin regulators
(CDK1s)
positive and negative)





c-Jun
INK4 family
RINGO/Speedy family


c-Fos
P15
P53



P16
MDM2



P18
RB



P19
CHK1



CIP/KIP family
CHk2



P21
ATM



P27
ATR



P57
CDC2




HDAC1




HDAC2









Exemplary pathways and genes associated with the angiogenesis are described provided in Table VII-20.









TABLE VH-20







ANGIOGENESIS PATHWAY GENES












Extracellular
Cell surface
Signal
Transcription



ligands
receptors
transduction
factors







PLGF
VEGFR1
PLC’)’
c-FOS



VEGF
VEGFR2
SHC
E2F7



VEGFB
VEGFR3
PI3K




VEGFC
Nrpl
PIP3




VEGFD

IP3






DAG






GRB2






SOS






Akt






PKB






PKC






Ras






RAFl






DAG






eNOS






NO






ERK1






ERK2






cPLA2






MEK1






MEK2










Exemplary pathways and genes associated with the mitochondrial function are provided in Table VII-24.









TABLE VII-241





Pathways and genes associated with mitochondrial function


















B-oxidation
TCA Cycle
Mitochondrial apoptosis
Valine oxidation pathway





acyl CoA
Citrate synthase

Transaminase


dehydrogenase
Aconitase

BCKADH complex


enoyl CoA hydratase
Isocitrate dehydrogenase

ACAD-8


3-hydroxyacyl-CoA
Alpha-ketoglutarate

Crotonoase


dehydrogenase-
dehydrogenase

HIBCH


ketothiolase
Succinyl-CoA synthetase

HIBADH



Succinate dehydrogenase

MMSDH



Fumarase

Atninotransferase



Malate dehydrogenase

Hydratase





Deacylase





Dehydrogenase





Carboxylase





Mutase












Fatty acid oxidation

Isoleucine


disorders (enzyme
Leucine Oxidation
oxidation


deficiencies)
Pathway
pathway





OCTN2
Aminotransferase
Aminotransferase


FATP1-6
Branched chain
Branched chain


CPT-1
aminotransferase 2,
aminotransferase 2,


CACT
mitochondrial
mitochondrial


CPT-II
Isobutytyl-CoA
2-methylbutytyl-CoA


SCAD
dehydrogenase
Dehydrogenase


MCAD
(Branched Chain
(Branched Chain


VLCAD
Keto Acid
Keto Acid


ETF-DH
Dehydrogase
Dehydrogenase


Alpha-ETF
Complex)
Complex)


Beta-ETF
Hydratase
Hydratase


SCHAD
HMG-CoA lyase
2-methyl-3-OH-butyryl-CoA


LCHAD

dehydrogenase


MTP

3-Oxothiolase


LKAT




DECR1




HMGCS2




HMGCL










Additional mitchondrial genes and related diseases caused by mutations











Mt-ND1
Leber's hereditary optic neuropathy


Mt-ND4
Leber's hereditary optic neuropathy


Mt-ND6
Leber's hereditary optic neuropathy


OPA1
Autosomal dominant optic atrophy


CMT2A
Charcot-Marie-Toothhereditary neuropathy type 2A


mt-TK
Myoclonic epilepsy with ragged red fibres


Mitochondrial
Related diseases


Respiratory chain



Genes



NADH CoQ
Alpers, Alzheimer's, Parkinsonism, Cardiomyopathy, Deficiency (Barth


Reductase
and/or Lethal infantile), Encephalopathy, infantile CNS, Leber's, Leigh,



Longevity, MELAS, MERRF, Myopathy ± CNS, PEO, Spinal cord disorders


Succinate-CoQ
Kearns-Sayre, Leigh's Myopathy (e.g.,


Reductase
Infantile ± CNS). Paraganglioma,



Pheochromocytoma


CoQ-Cytochrome
Cardiomyopathy, Fatal infantile, GRACILE, Lehers,


C Reductase
Myopathy (e.g., ± CNS, PEO)


Cytochrome C
Alper's, Ataxia, Deafness, Leber's, Leigh's, Myopathy (e.g., Infantile


Oxidase
(e.g., Fatal, Benign), Adult), Rhabdomyolysis, PEO, KSS, MNGIE, MERRF,



MELAS


ATP Synthase
Cardiomyopathy, Encephalopathy, Leber's, Leigh, Multisystem, NARP










Complex I (NADH-Ubiquinone Oxidoreductase)










Nuclear


Subunits involved


encoded
Mitochondral DNA
Supernumerary
in regulation of


proteins
encoded proteins
subunits
Complex I activity





NDUFS1: Childhood
ND1
NDLIFAB 1 (SDAP):
(AQDQ)


encephalopathy: Most
ND2
Carrier of fatty acid chain
NDUFS4 Functions:


common Complex I
ND3
NDUFA
Increased Complex I


mutations (3%)
ND4
(MWFE)
activity with


NDUFS2:
ND4L
Primarily expressed
phosphorylation


Cardiomyopathy +
ND5
in heart & skeletal muscle
Disorders:


Encephalomyopathy
ND6
Disorders:
Multisystem


NDUFS3: Leigh

Encephalopathies
childhood


NDUFS7: Leigh

NDUFA2:
encephalopathy


NDUFS8: Leigh

Encephalopathy &
with Complex I


NDUFV1: Childhood

Cardiomyopathy
deficiency, Leigh


encephalopathy

NDUFA9: Leigh syndrome
syndrome


NDUFV2: Encephalopathy +

NDUFA10: Leigh syndrome
NDUFA12: Leigh syndrome


Cardiomyopathy

NDUFA11 Disorder:
NDUFB9: Hypotonia


ELAC2: Cardiomyopathy,

Encephalopathy &
NDUFS6: Lethal infantile


Hypertrophic

Cardiomyopathy
Mitochondrial Disease











Proteins involved in



Complex I assembly
Other





NDUFAF1:
NDUFA13: Thyroid carcinoma


Cardiomyopathy +
(Hurthle cell)


Encephalopathy
NDUFB3: Severe


NDUFAF2 (NDUFA12L):
lethal mitochondrial


Childhood encephalopathy;
complex I deficiency


Usually null mutations
MTHFR deficiency


NDUFAF3: Lethal neonatal
MGMEI: PEO + Myopathy


encephalopathy



NDUFAF4: Encephalopathy



C6ORF66: Encephalopathy



C8orf38: Leigh syndrome



C20orf7: Lethal neonatal



NUBPL: Encephalomyopathy



ACAD9: Fatigue & Exercise



intolerance; Most missense



mutations



FOXRED1: Leigh syndrome



Ecsit



AIF (AIFM1; PDCD8)



IndI










Complex I (NA DII-Ubiquinone Oxidoreductase)








Flavoprotein: FAD (SDHA; Fp)
Mutations cause Leigh syndrome with



Complex II deficiency



Late onset neurodegenerative disorder)


Iron-Sulfur protein: SDHB (Ip)
Mutations cause Reduced tumor suppression



Neoplasms: Pheochromocytoma &



Paraganglioma


SDHC: SDHD (cytochrome C subunits)
Mutations lead to paraganglioma










Complex III (Cytochrome reductase)








Cytochrome c1 (CYC1)



Rieske FeS protein (UQCRFS1)



Ubiquinol-cytochrome c reductase core
May mediate formation of complex between


protein I (UQCRC1; QCR; Subunit 1)
cytochromes c and c1


Ubiquinol-cytochrome c reductase core
Required for assembly of complex III


protein II (UQCRC2; QCR2; Subunit 2)



UQCRH (Subunit 6)
May mediate formation of complex between



cytochromes c and c1



Ubiquinone-binding protein (UQBC;

Redox-linked proton pumping


UQPC; UQCRB; UQBP; Subunit 7)




UQCRQ (Subunit 8)

Binds to ubiquinone


Ubiquinol-cytochrome C reductase
Interacts with cytochrome c1


complex, 7.2-KD Subunit (UCRC;



UQCR 10; Subunit 9)



UQCR (UQCR11: Subunit 10)
function as iron-sulfur protein binding factor


Cleavage product of UQCRFS1



(Cytochrome b-c1 complex subunit 11)










Inner membrane proteins and related disorders


ABCB7: Ataxia + Anemia


ACADVL: Myopathy


ADCK3: SACR9


AGK: Sengers


ATP5A1: Encephalopathy, neonatal


ATP5E: Retardation + Neuropathy


BRP44L: Encephalopathy


c12orf62: Encephalocardiomyopathy


Cardiolipin: Barth


COX4I2: Pancreas + Anemia


COX6B1: Encephalomyopathy


CPT2: Myopathy


CRAT: Encephalomyopathy


CYC1: Hyperglycemia & Encephalopathy


CYCS


CYP11A1


CYP11B1


CYP11B2


CYP24A1


CYP27A1: Cerebrotendinous Xanthomatosis


CYP27B1


DHODH


DNAJC19: Cardiac + Ataxia


FASTKD2: Encephalomyopathy


GPD2


HADHA: Multisystem; Myopathy


HADHB: Encephalomyopathy


HCCS: MIDAS


L2HGDH: Encephalopathy


MMAA


MPV17: Hepatocerebral


NDUFA1: Encephalopathy


NDUFA2: Leigh + Cardiac


NDLTA4: Leigh


NDUFA9: Leigh


NDUIPAlO: Leigh


NDUFAl 1: Encephalocardiomyopathy


NDUFA12: Leigh


NDUFA13


NDUFB3: Lethal infantile


NDUFB9: Encephalopathy


NDUFV1: Encephalopathy


NDUFV2: Encephalopathy + Cardiac


NDUSl: Leukodystrophy


NDUFS2: Encephalopathy + Cardiac


NDUFS3: Dystonia


NDUFS4: Encephalopathy


NDUFS6: Lethal infantile


NDUFS7: Encephalopathy


NDUFS8: CNS + Cardiac


OPA1: Optic atrophy


OPA3: Optic atrophy


PDSS1: Coenzyme Q10 deficiency


SDHA: Leigh; Cardiac; Paraganglioma


SDHB: Paraganglioma


SDHC: Paraganglioma


SDHD: Paraganglioma


SLC25A carriers


SLC25A1: Epileptic encephalopathy


SLC25A3: Cardiac; Exercise intolerance


SLC25A4: PEOA2


SLC25A12: Hypomyelination


SLC25A13: Citrullinemia


SLC25A15: HHH


SLC25A19: Microcephaly


SLC25A20: Encephalocardiomyopathy


SLC25A22: Myoclonic epilepsy


SLC25A38: Anemia


Paraplegin: SPG7


TIMM8A: Deaf-Dystonia-Dementia


UCP1


UCP2


UCP3


UQCRB: Hypoglycemia, Hepatic


UQCRC2: Episodic metabolic encephalopathy


UQCRQ: Encephalopathy









Pathways and genes associated with DNA damage and genomic instability include the following methyl transferases, histone methylation, helicase activity, nucleotide excision repair, recombinational repair, or mismatch repair provided in Table VII-21. See also Table VII-22.









TABLE VII-21





PATHWAYS and GENES ASSOCIATED with DNA


DAMAGE and GENOMIC INSTABILITY


















Double-stranded
Replication
DNA
Non-Homologous


Breaks
Stress
Methylation
End-Joining





ATM
ATR
DNMT1
Ku70


RAD50
RAD17
DNMT2
Ku80


MRE119
ATRIP
DNMT3A
DNA


NBS1
RAD9
DNMT3B
PKc


CRCA1
RPA
DNMT3L
XRCC4


H2AX
CHK1
MeCP2
DNA ligase 4


53BP1
BLM
MBD2
XLF


MDCl
H2AX

Rad50


SMCl
53BP1

Artemis


P53
P53

Rad27





TdT





Base-
Nucleotide-




Excision
Excision
Homologous
Mismatch


repair
Repair
Recombination
repair





APE1
UvrA
RecA
PMS2


APE2
UvrB
SSB
MLH1


NEIL1
UvrC
Mrell
MSH6


NEIL2
XPC
Rad50
MSH2


NEIL3
Rad23B
Nbs1
RFC


XRCC1
CEN2
CtlP
PCNA


PNKP
DDB1
RPA
MSH3


Tdpl
XPE
Rad51
MutS


APTX
CSA,
Rad52
MutL


DNA
CSB
Rad54
Exonuclease


polymerase
TFIIH
BRCA1
Topoisomerase 1


DNA
XPB
BRCA2
Topoisomerase 2


polymerase 8
XPD
Exol
RNAseH1


DNA
XPA
BLM
Ligase 1


polymerase £
RPA
Topilla
DNA


PCNA
XPG
GEN1
polymerase 1


FEN1
ERCCl
Yen1
DNA


RFC
XPF
Slx1
polymerase 3


PARP1
DNA
Slx4
Primase


Lig1
polymerase 8
Mus8
Helicase


Lig3
DNA
Eme1
SSBs


UNG
polymerase £
Dssl



MUTY





SMUG





MBD4










Histone Methylation








ASHIL
SETD4


DOT1L
SETD5


EHMT1
SETD6


EHMT2
SETD7


EZH1
SETD8


EZH2
SETD9


MLL
SETDB1


MLL2
SETDB2


MLL3
SETMAR


MLL4
SMYD1


MLL5
SMYD2


NSD1
SMYD3


PRDM2
SMYD4


SET
SMYD5


SETBP1
SUV39H1


SETD1A
SUV39H2


SETD1B
SLIV420H1


SETD2
SUV420H2


SETD3
















TABLE VII-22





Selected Transcription Factors Transcription factors


















NIKX2-5
Cardiac malformations and




atrioventricular conduction




abnormalities



MECP2
Rett syndrome



HNF1 through
Mature onset diabetes of the



HNF6
young (MODY), hepatic




adenomas and renal cysts



FOXP2
Developmental verbal dyspraxia



FOXP3
Autoimmune diseases



NOTCH1
Aortic valve abnormalities



MEF2A
Coronary artery disease



CRX
Dominant cone-rod dystrophy



FOCX2
Lymphedema-distichiasis



NF-KB
Autoimmune arthritis, asthma,



Activation
septic shock, lung fibrosis,




glomerulonephritis,




atherosclerosis, and AIDS



NF-KB
Apoptosis, inappropriate immune cell



Inhibition
development, and delayed cell growth



NARA2
Parkinson disease



LHX3
Pituitary disease



GAT4
Congenital heart defects



P53, APC
Cancer



CTCF
Epigenetics and cell growth regulation



EGR2
Congenital hypomyelinating




neuropathy (CHN) and Charcot-




Marie-Tooth type 1 (CMT1)



STAT family
Cancer and immunosuppression



NF-AT family
Cancer and inflammation



AP-1 family
Cancer and inflammation










A gene including receptors and ionophores relevant to pain in this table can be targeted, by editing or payload delivery. Pathways and genes associated with pain are described herein, e.g., include the following those in Table VII-23.













TABLE VII-23






Part of






nervous





Type of pain
system
Target
Area
How to affect







nociceptive
central
5-HT
central inhibition



nociceptive
central
5HT1A
central inhibition
agonists (activation) serve






as analgesic,






antidepressants, anxiolytics,






psychosis


nociceptive
central
5HT1A
central inhibition
antagonists can work as






antidepressants, nootropics


nociceptive
central
5HT1B
central inhibition
m1grames


nociceptive
central
5HT1D
central inhibition
m1grames


nociceptive
central
5HT1E
central inhibition



nociceptive
central
5HT1F
central inhibition
agonists-psychedelics


nociceptive
central
5HT1F
central inhibition
antagonists-atypical






antipsychotics, NaSSAsm






treatig sertonin syndrome,






sleeping aid


nociceptive
central
5HT2A
central inhibition
agonists-psychadelics


nociceptive
central
5HT2A
central inhibition
antagonists-atypical






antipsychotics, NaSSAs,






treating seratonin syndrome,






sleeping aid


nociceptive
central
5HT2B
central inhibition
m1grames


nociceptive
central
5HT2C
central inhibition
antidepressant, orexigenic,






anorectic, antipsychotic


nociceptive
central
5HT3
central inhibition
antiemetic


nociceptive
central
5HT4
central inhibition
gastroproknetics


nociceptive
central
5HT5A
central inhibition



nociceptive
central
5HT5B
central inhibition



nociceptive
central
5HT6
central inhibition
antidepressant (antagonists






and agonists), anxiolytic






(antagonists and agonists),






nootropic (antagonists),






anorectic (antagonists)


nociceptive
central
5HT7
central inhibition
antidepressant (antagonists),






anxiolytics (antagonists),






nootropic (antagonists)


nociceptive
central
CBl
central inhibition



nociceptive
central
GABA
central inhibition



nociceptive
central
GABAA-$
central inhibition



nociceptive
central
GABAB-R
central inhibition



nociceptive
central
Glucine-R
central inhibition



nociceptive
central
NE
central inhibition



nociceptive
central
Opiod
central inhibition





receptors




nociceptive
central
c-fos
gene expression



nociceptive
central
C-JUn
gene expression



nociceptive
central
CREB
gene expression



nociceptive
central
DREAM
gene expression



nociceptive
peripheral
K+ channel
membrane






excitability of






primary afferents



nociceptive
peripheral
Nav1.8
membrane






excitability of






primary afferents



nociceptive
peripheral
Nav1.9
membrane






excitability of






primary afferents



nociceptive
peripheral
CaMKIV
peripheral






sensitization



nociceptive
peripheral
COX2
peripheral






sensitization



nociceptive
peripheral
cPLA2
peripheral






sensitization



nociceptive
peripheral
EP1
peripheral






sensitization



nociceptive
peripheral
EP3
peripheral






sensitization



nociceptive
peripheral
EP4
peripheral






sensitization



nociceptive
peripheral
ERK1/2
peripheral






sensitization



nociceptive
peripheral
IL-1beta
peripheral






sensitization



nociceptive
peripheral
JNK
peripheral






sensitization



nociceptive
peripheral
Nav1.8
peripheral






sensitization



nociceptive
peripheral
NGF
peripheral






sensitization



nociceptive
peripheral
p38
peripheral






sensitization



nociceptive
peripheral
PKA
peripheral






sensitization



nociceptive
peripheral
PKC
peripheral





isoforms
sensitization



nociceptive
peripheral
TNFalpha
peripheral






sensitization



nociceptive
peripheral
TrkA
peripheral






sensitization



nociceptive
peripheral
TRPV1
peripheral






sensitization



nociceptive
central
AMPA/
postsynaptic





kainate-R
transmission



nociceptive
central
K+ channels
postsynaptic






transmission



nociceptive
central
mGlu-$
postsynaptic






transmission



nociceptive
central
Nav1.3
postsynaptic






transmission



nociceptive
central
NKI
postsynaptic






transmission



nociceptive
central
NMDA-R
postsynaptic






transmission



nociceptive
peripheral
Adenosine-
presynaptic





R
transmission



nociceptive
peripheral
mGluR
presynaptic






transmission



nociceptive
peripheral
VGCC
presynaptic






transmission



nociceptive
central
ERK
signal






transduction



nociceptive
central
JNK
signal






transduction



nociceptive
central
p38
signal






transduction



nociceptive
central
PKA
signal






transduction



nociceptive
central
PKC
signal





isoforms
transduction



nociceptive
peripheral
ASIC
transduction



nociceptive
peripheral
BK1
transduction



nociceptive
peripheral
BK2
transduction



nociceptive
peripheral
DRASIC
transduction



nociceptive
peripheral
MDEG
transduction



nociceptive
peripheral
P2X3
transduction



nociceptive
peripheral
TREK-1
transduction



nociceptive
peripheral
TRPM8
transduction



nociceptive
peripheral
TRPV1
transduction



nociceptive
peripheral
TRPV2
transduction



nociceptive
peripheral
TRPV3
transduction



neuropathic






pam






Inflammatory

histamine




pam






Inflammatory

ATP




pam






Inflammatory

bradykinin




pam






Inflammatory

CB2




pam






Inflammatory

Endothelins




pam






Inflammatory

H+




pam






Inflammatory

Interleukins




pam






Inflammatory

NGF




pam






Inflammatory

prostaglandins




pam






Inflammatory

serotonin




pam






Inflammatory

TNFalpha




pam










VIII. Targets: Disorders Associated with Disease Causing Organisms


Cas9 molecules, typically eiCas9 molecules or eaCas9 molecules, and gRNA molecules, e.g., an eiCas9 molecule/gRNA molecule complex, e.g., an eaCas9 molecule/gRNA molecule complex, can be used to treat or control diseases associated with disease causing organisms, e.g., to treat infectious diseases. In an embodiment, the infectious disease is treated by editing (e.g., correcting) one or more target genes, e.g., of the organism or of the subject. In an embodiment, the infectious disease is treated by delivering one or more payloads (e.g., as described herein) to the cell of a disease causing organism or to an infected cell of the subject, e.g., to a target gene. In an embodiment, the target gene is in the infectious pathogen. Exemplary infectious pathogens include, e.g., viruses, bacteria, fungi, protozoa, or multicellular parasites.


In an embodiment, the target gene is in the host cell. For example, modulation of a target gene in the host cell can result in resistance to the infectious pathogen. Host genes involved in any stage of the life cycle of the infectious pathogen (e.g., entry, replication, latency) can be modulated. In an embodiment, the target gene encodes a cellular receptor or co-receptor for the infectious pathogen. In an embodiment, the infectious pathogen is a virus, e.g., a virus described herein, e.g., HIV. In an embodiment, the target gene encodes a co-receptor for HIV. e.g., CCR5 or CXCR4.


Exemplary infectious diseases that can be treated by the molecules and methods described herein, include. e.g., AIDS, Hepatitis A, Hepatitis B, Hepatitis C, Herpes simplex, HPV infection, or Influenza.


Exemplary targets are provided in Table VII-1. The disease and causative organism are provided.










TABLE VIII-1





DISEASE
SOURCE OF DISEASE







Acinetobacter infections

Acinetobacter baumannii



Actinomycosis

Actinomyces israelii, Actinomyces





gerencseriae and Propionibacterium





propwmcus



African sleeping sickness

Trypanosoma brucei



(African trypanosomiasis)



AIDS (Acquired
HIV (Human immunodeficiency virus)


immunodeficiency syndrome)



Amebiasis

Entamoeba histolytica



Anaplasmosis

Anaplasma genus



Anthrax

Bacillus anthracis




Arcanobacterium haemolyticum


Arcanobacterium haemolyticum



infection



Argentine hemorrhagic fever
Junin virus


Ascariasis

Ascaris lumbricoides



Aspergillosis

Aspergillus genus



Astrovirus infection
Astroviridae family


Babesiosis
Babesia genus



Bacillus cereus infection


Bacillus cereus



Bacterial pneumonia
multiple bacteria


Bacterial vaginosis (BV)
multiple bacteria



Bacteroides infection


Bacteroides genus



Balantidiasis

Balantidium coli



Baylisascaris infection
Baylisascaris genus


BK virus infection
BK virus


Black piedra

Piedraia hortae



Blastocystis hominis infection

Blastocystis hominis



Blastomycosis

Blastomyces dermatitidis



Bolivian hemorrhagic fever
Machupo virus


Borrelia infection

Borrelia genus



Botulism (and Infant botulism)

Clostridium botulinum; Note:




Botulism is not an infection by




Clostridium botulinum but caused




by the intake of botulinum toxin.


Brazilian hemorrhagic fever
Sabia


Brucellosis

Brucella genus



Bubonic plague
the bacterial family Enterobacteriaceae


Burkholderia infection
usually Burkholderia cepacia and other




Burkholderia species



Buruli ulcer

Mycobacterium ulcerans



Calicivirus infection (Norovirus
Caliciviridae family


and Sapovirus)



Campylobacteriosis
Campylobacter genus


Candidiasis (Moniliasis; Thrush)
usually Candida albicans and other




Candida species



Cat-scratch disease

Bartonella henselae



Cellulitis
usually Group A Streptococcus and




Staphylococcus



Chagas Disease (American

Trypanosoma cruzi



trypanosomiasis)



Chancroid

Haemophilus ducreyi



Chickenpox
Varicella zoster virus (VZV)



Chlamydia


Chlamydia trachomatis




Chlamydophila pneumoniae


Chlamydophila pneumoniae



infection (Taiwan acute



respiratory agent or TWAR)



Cholera

Vibrio cholerae



Chromoblastomycosis
usually Fonsecaea pedrosoi


Clonorchiasis

Clonorchis sinensis




Clostridium difficile infection


Clostridium difficile



Coccidioidomycosis

Coccidioides immitis and Coccidioides





posadasii



Colorado tick fever (CTF)
Colorado tick fever virus (CTFV)


Common cold (Acute viral
usually rhinoviruses and coronaviruses.


rhinopharyngitis; Acute coryza)



Creutzfeldt-Jakob disease (CJD)
PRNP


Crimean-Congo hemorrhagic
Crimean-Congo hemorrhagic fever virus


fever (CCHF)



Cryptococcosis

Cryptococcus neoformans



Cryptosporidiosis

Cryptosporidium genus



Cutaneous larva migrans (CLM)
usually Ancylostoma braziliense;



multiple other parasites


Cyclosporiasis

Cyclospora cayetanensis



Cysticercosis

Taenia solium



Cytomegalovirus infection
Cytomegalovirus


Dengue fever
Dengue viruses (DEN-1, DEN-2,



DEN-3 and DEN-4)-Flaviviruses


Dientamoebiasis
Dientamoeba fragilis


Diphtheria

Corynebacterium diphtheriae



Diphyllobothriasis
Diphyllobothrium


Dracunculiasis

Dracunculus medinensis



Ebola hemorrhagic fever
Ebolavirus (EBOV)


Echinococcosis
Echinococcus genus


Ehrlichiosis

Ehrlichia genus



Enterobiasis (Pinworm infection)

Enterobius vermicularis




Enterococcus infection


Enterococcus genus



Enterovirus infection
Enterovirus genus


Epidemic typhus

Rickettsia prowazekii



Erythema infectiosum (Fifth
Parvovirus B19


disease)



Exanthem subitum (Sixth
Human herpesvirus 6 (HHV-6) and


disease)
Human herpesvirus 7 (HHV-7)


Fasciolopsiasis
Fasciolopsis buski


Fasciolosis

Fasciola hepatica and Fasciola gigantica



Fatal familial insomnia (FFI)
PRNP


Filariasis
Filarioidea superfamily


Food poisoning by Clostridium

Clostridium pe, fringens



perfringens



Free-living amebic infection
multiple



Fusobacterium infection


Fusobacterium genus



Gas gangrene (Clostridial
usually Clostridium perfringens; other


myonecrosis)

Clostridium species



Geotrichosis

Geotrichum candidum



Gerstmann-Straussier-Scheinker
PRNP


syndrome (GSS)



Giardiasis

Giardia intestinalis



Glanders

Burkholderia mallei



Gnathostomiasis

Gnathostoma spinigerum and





Gnathostoma
hispidum



Gonorrhea

Neisseria gonorrhoeae



Granuloma inguinale

Klebsiella granulomatis



(Donovanosis)



Group A streptococcal infection

Streptococcus pyogenes



Group B streptococcal infection

Streptococcus agalactiae




Haemophilus influenzae


Haemophilus influenzae



infection



Hand, foot and mouth disease
Enteroviruses, mainly Coxsackie A


(HFMD)
virus and Enterovirus 71 (EV71)


Hantavirus Pulmonary Syndrome
Sin Nombre virus


(HPS)




Helicobacter pylori infection


Helicobacter pylori



Hemolytic-uremic syndrome

Escherichia coli 0157: H7, 0111 and



(HUS)
0104: H4


Hemorrhagic fever with renal
Bunyaviridae family


syndrome (HFRS)



Hepatitis A
Hepatitis A Virus


Hepatitis B
Hepatitis B Virus


Hepatitis C
Hepatitis C Virus


Hepatitis D
Hepatitis D Virus


Hepatitis E
Hepatitis E Virus


Herpes simplex
Herpes simplex virus 1 and 2 (HSV-1



and HSV-2)


Histoplasmosis

Histoplasma capsulatum



Hookworm infection

Ancylostoma duodenale and Necator





amencanus



Human bocavirus infection
Human-bocavirus (HBoV)


Human ewingii ehrlichiosis

Ehrlichia ewingii



Human granulocytic

Anaplasma phagocytophilum



anaplasmosis (HGA)



Human metapneumovirus
Human metapneumovirus (hMPV)


infection



Human monocytic ehrlichiosis

Ehrlichia chaffeensis



Human papillomavirus (HPV)
Human papillomavirus (HPV)


infection



Human parainfluenza virus
Human parainfluenza viruses (HPIV)


infection



Hymenolepiasis

Hymenolepis nana and Hymenolepis





diminuta



Epstein-Barr Virus Infectious
Epstein-Barr Virus (EBY)


Mononucleosis (Mono)



Influenza (flu)
ACCGACAUU


Isosporiasis

Isospora belli



Kawasaki disease
unknown; evidence supports that it is



infectious


Keratitis
multiple


Kingella kingae infection
Kingella kingae


Kuru
PRNP


Lassa fever
Lassa virus


Legionellosis (Legionnaires'

Legionella pneumophila



disease)



Legionellosis (Pontiac fever)

Legionella pneumophila



Leishmaniasis
Leishmania genus


Leprosy

Mycobacterium leprae and





Mycobacterium
lepromatosis



Leptospirosis

Leptospira genus



Listeriosis

Listeria monocytogenes



Lyme disease (Lyme borreliosis)
usually Borrelia burgdorferi and other




Borrelia species



Lymphatic filariasis

Wuchereria bancrofti and Brugia malayi



(Elephantiasis)



Lymphocytic choriomeningitis
Lymphocytic choriomeningitis virus



(LCMV)


Malaria
Plasmodium genus


Marburg hemorrhagic fever
Marburg virus


(MHF)



Measles
Measles virus


Melioidosis (Whitmore's disease)

Burkholderia pseudomallei



Meningitis
multiple


Meningococcal disease

Neisseria meningitidis



Metagonimiasis
usually Metagonimus yokagawai


Microsporidiosis

Microsporidia phylum



Molluscum contagiosum (MC)
Molluscum contagiosum virus (MCV)


Monkeypox
Monkeypox virus


Mumps
Mumps virus


Murine typhus (Endemic typhus)
Rickettsia typhi



Mycoplasma pneumonia


Mycoplasma pneumoniae



Mycetoma
numerous species of bacteria



(Actinomycetoma) and fungi



(Eumycetoma)


Myiasis
parasitic dipterous fly larvae


Neonatal conjunctivitis
most commonly Chlamydia trachomatis


(Ophthalmia neonatorum)
and Neisseria gonorrhoeae


(New) Variant Creutzfeldt-Jakob
PRNP


disease (vCJD, nvCJD)



Nocardiosis
usually Nocardia asteroides and other




Nocardia species



Onchocerciasis (River blindness)
Onchocerca volvulus


Paracoccidioidomycosis (South

Paracoccidioides brasiliensis



American blastomycosis)



Paragonimiasis
usually Paragonimus westermani and



other Paragonimus species


Pasteurellosis

Pasteurella genus



Pediculosis capitis (Head lice)

Pediculus humanus capitis



Pediculosis corporis (Body lice)

Pediculus humanus corporis



Pediculosis pubis (Pubic lice,
Phthirus pubis


Crab lice)



Pelvic inflammatory disease
multiple


(PID)



Pertussis (Whooping cough)

Bordetella pertussis



Plague

Yersinia pestis



Pneumococcal infection

Streptococcus pneumoniae



Pneumocystis pneumonia (PCP)

Pneumocystis jirovecii



Pneumonia
multiple


Poliomyelitis
Poliovirus



Prevotella infection


Prevotella genus



Primary amoebic
usually Naegleriafowleri


meningoencephalitis (PAM)



Progressive multifocal
JC virus


leukoencephalopathy



Psittacosis

Chlamydophila psittaci



Q fever

Coxiella bumetii



Rabies
Rabies virus


Rat-bite fever

Streptobacillus moniliformis and





Spirillum
mmus



Respiratory syncytial virus
Respiratory syncytial virus (RSV)


infection



Rhinosporidiosis

Rhinosporidium seeberi



Rhinovirus infection
Rhinovirus


Rickettsial infection

Rickettsia genus



Rickettsialpox

Rickettsia akari



Rift Valley fever (RVF)
Rift Valley fever virus


Rocky Mountain spotted fever

Rickettsia rickettsii



(RMSF)



Rotavirus infection
Rotavirus


Rubella
Rubella virus


Salmonellosis

Salmonella genus



SARS (Severe Acute Respiratory
SARS coronavirus


Syndrome)



Scabies

Sarcoptes scabiei



Schistosomiasis
Schistosoma genus


Sepsis
multiple


Shigellosis (Bacillary dysentery)

Shigella genus



Shingles (Herpes zoster)
Varicella zoster virus (VZV)


Smallpox (Variola)
Variola major or Variola minor


Sporotrichosis

Sporothrix scherickii



Staphylococcal food poisoning

Staphylococcus genus



Staphylococcal infection

Staphylococcus genus



Strongyloidiasis

Strongyloides stercoralis



Subacute sclerosing
Measles virus


panencephalitis



Syphilis

Treponema pallidum



Taeniasis
Taenia genus


Tetanus (Lockjaw)

Clostridium tetani



Tinea barbae (Barber's itch)
usually Trichophyton genus


Tinea capitis (Ringworm of the
usually Trichophyton tonsurans


Scalp)



Tinea corporis (Ringworm of the
usually Trichophyton genus


Body)



Tinea cruris (Jock itch)
usually Epidermophytonfloccosum,




Trichophyton rubrum, and Trichophyton





mentagrophytes



Tinea manuum (Ringworm of

Trichophyton rubrum



the Hand)



Tinea nigra
usually Hortaea wemeckii


Tinea pedis (Athlete's foot)
usually Trichophyton genus


Tinea unguium (Onychomycosis)
usually Trichophyton genus


Tinea versicolor (Pityriasis
Malassezia genus


versicolor)



Toxocariasis (Ocular Larva

Toxocara canis or Toxocara cati



Migrans (OLM))



Toxocariasis (Visceral Larva

Toxocara canis or Toxocara cati



Migrans (VLM))



Toxoplasmosis

Toxoplasma gondii



Trichinellosis

Trichinella spiralis



Trichomoniasis

Trichomonas vaginalis



Trichuriasis (Whipworm

Trichuris trichiura



infection)



Tuberculosis
usually Mycobacterium tuberculosis


Tularemia

Francisella tularensis



Ureaplasma urealyticum

Ureaplasma urealyticum



infection



Valley fever

Coccidioides immitis or Coccidioides





posadasii.



Venezuelan equine encephalitis
Venezuelan equine encephalitis virus


Venezuelan hemorrhagic fever
Guanarito virus


Viral pneumonia
multiple viruses


West Nile Fever
West Nile virus


White piedra (Tinea blanca)

Trichosporon beigelii



Yersinia pseudotuberculosis

Yersinia pseudotuberculosis



infection



Yersiniosis

Yersinia enterocolitica



Yellow fever
Yellow fever virus


Zygomycosis
Mucorales order (Mucormycosis) and



Entomophthorales order



(Entomophthoramycosis)









AIDS/HIV


HIV Genomic Structural Elements


Long terminal repeat (LTR) refers to the DNA sequence flanking the genome of integrated proviruses. It contains important regulatory regions, especially those for transcription initiation and polyadenylation.


Target sequence (TAR) for viral transactivation, the binding site for Tat protein and for cellular proteins; consists of approximately the first 45 nucleotides of the viral mRNAs in HIV-1 (or the first 100 nucleotides in HIV-2 and SIV.) TAR RNA forms a hairpin stem-loop structure with a side bulge; the bulge is necessary for Tat binding and function.


Rev responsive element (RPE) refers to an RNA element encoded within the env region of HIV-1. It consists of approximately 200 nucleotides (positions 7327 to 7530 from the start of transcription in HIV-1, spanning the border of gp120 and gp41). The RRE is necessary for Rev function; it contains a high affinity site for Rev; in all, approximately seven binding sites for Rev exist within the RRE RNA. Other lentiviruses (HIV-2, SIV, visna, CAEV) have similar RRE elements in similar locations within env, while HTLVs have an analogous RNA element (RXRE) serving the same purpose within their LTR; RRE is the binding site for Rev protein, while RXRE is the binding site for Rex protein. RRE (and RXRE) form complex secondary structures, necessary for specific protein binding.


Psi elements (PE) are a set of 4 stem-loop structures preceding and overlapping the Gag start codon which are the sites recognized by the cysteine histidine box, a conserved motif with the canonical sequence CysX2CysX4HisX4Cys (SEQ ID NO: 41), present in the Gag p7 MC protein. The Psi Elements are present in unspliced genomic transcripts but absent from spliced viral mRNAs.


SLIP, an TTTTTT slippery site, followed by a stem-loop structure, is responsible for regulating the −1 ribosomal frameshift out of the Gag reading frame into the Pol reading frame.


Cis-acting repressive sequences (CRS) are postulated to inhibit structural protein expression in the absence of Rev. One such site was mapped within the pol region of HIV-1. The exact function has not been defined; splice sites have been postulated to act as CRS sequences.


Inhibitory/Instability RNA sequences (INS) are found within the structural genes of HIV-1 and of other complex retroviruses. Multiple INS elements exist within the genome and can act independently; one of the best characterized elements spans nucleotides 414 to 631 in the gag region of HIV-1. The INS elements have been defined by functional assays as elements that inhibit expression posttranscriptionally. Mutation of the RNA elements was shown to lead to INS inactivation and up regulation of gene expression.


Genes and Gene Products


Essential for Replication


The genomic region (GAG) encoding the capsid proteins (group specific antigens). The precursor is the p55 myristylated protein, which is processed to p17 (MAtrix), p24 (CApsid), p7 (NucleoCapsid), and p6 proteins, by the viral protease. Gag associates with the plasma membrane where the virus assembly takes place. The 55 kDa Gag precursor is called assemblin to indicate its role in viral assembly.


The genomic region, POL, encoding the viral enzymes protease, reverse transcriptase, RNAse, and integrase. These enzymes are produced as a Gag-Pol precursor polyprotein, which is processed by the viral protease; the Gag-Pol precursor is produced by ribosome frame shifting near the end of gag.


Viral glycoproteins (e.g., ENV) produced as a precursor (gp160) which is processed to give a noncovalent complex of the external glycoprotein gp120 and the transmembrane glycoprotein gp41. The mature gp120-gp41 proteins are bound by non-covalent interactions and are associated as a trimer on the cell surface. A substantial amount of gp120 can be found released in the medium, gp120 contains the binding site for the CD4 receptor, and the seven transmembrane do-main chemokine receptors that serve as co-receptors for HIV-1.


The transactivator (TAT) of HIV gene expression is one of two essential viral regulatory factors (Tat and Rev) for HIV gene expression. Two forms are known, Tat-1 exon (minor form) of 72 amino acids and Tat-2 exon (major form) of 86 amino acids. Low levels of both proteins are found in persistently infected cells. Tat has been localized primarily in the nucleolus/nucleus by immunofluorescence. It acts by binding to the TAR RNA element and activating transcription initiation and elongation from the LTR promoter, preventing the LTR AATAAA polyadenylation signal from causing premature termination of transcription and polyadenylation. It is the first eukaryotic transcription factor known to interact with RNA rather than DNA and may have similarities with prokaryotic anti-termination factors. Extracellular Tat can be found and can be taken up by cells in culture.


The second necessary regulatory factor for HIV expression is REV. A 19 kDa phosphoprotein, localized primarily in the nucleolus/nucleus, Rev acts by binding to RRE and promoting the nuclear export, stabilization and utilization of the un-spliced viral mRNAs containing RRE. Rev is considered the most functionally conserved regulatory protein of lentiviruses. Rev cycles rapidly between the nucleus and the cytoplasm.


Others


Viral infectivity factor (VIP) is a basic protein of typically 23 kDa. Promotes the infectivity but not the production of viral particles. In the absence of Vif the produced viral particles are defective, while the cell-to-cell transmission of virus is not affected significantly. Found in almost all lentiviruses, Vif is a cytoplasmic protein, existing in both a soluble cytosolic form and a membrane-associated form. The latter form of Vif is a peripheral membrane protein that is tightly associated with the cytoplasmic side of cellular membranes. In 2003, it was discovered that Vif prevents the action of the cellular APOBEC-3G protein which deaminates DNA:RNA heteroduplexes in the cytoplasm.


Viral Protein R (VPR) is a 96-amino acid (14 kDa) protein, which is incorporated into the virion. It interacts with the p6 Gag part of the Pr55 Gag precursor. Vpr detected in the cell is localized to the nucleus. Proposed functions for Vpr include the targeting the nuclear import of preintegration complexes, cell growth arrest, transactivation of cellular genes, and induction of cellular differentiation. In HIV-2, SIV-SMM, SIV-RCM, SIV-MND-2 and SIV-DRL the Vpx gene is apparently the result of a Vpr gene duplication event, possibly by recombination.


Viral Protein U (VPU)) is unique to HIV-1, SIVcpz (the closest SIV relative of HIV-1), SIV-GSN, SIV-MUS, SIV-MON and SIV-DEN. There is no similar gene in HIV-2, SIV-SMM or other SIVs. Vpu is a 16 kDa (81-amino acid) type I integral membrane protein with at least two different biological functions: (a) degradation of CD4 in the endoplasmic reticulum, and (b) enhancement of virion release from the plasma membrane of HIV-I-infected cells. Env and Vpu are expressed from a bicistronic mRNA. Vpu probably possesses an N-terminal hydrophobic membrane anchor and a hydrophilic moiety. It is phosphorylated by casein kinase II at positions Ser52 and Ser56. Vpu is involved in Env maturation and is not found in the virion. Vpu has been found to increase susceptibility of HIV-1 infected cells to Fas killing.


NEF is a multifunctional 27-kDa myristylated protein produced by an ORF located at the end of the primate lentiviruses. Other forms of Nef are known, including nonmyristylated variants. Nef is predominantly cytoplasmic and associated with the plasma membrane via the myristyl residue linked to the conserved second amino acid (Gly). Nef has also been identified in the nucleus and found associated with the cytoskeleton in some experiments. One of the first HIV proteins to be produced in infected cells, it is the most immunogenic of the accessory proteins. The nef genes of HIV and SIV are dispensable in vitro, but are essential for efficient viral spread and disease progression in vivo. Nef is necessary for the maintenance of high virus loads and for the development of AIDS in macaques, and viruses with defective Nef have been detected in some HIV-1 infected long term survivors. Nef downregulates CD4, the primary viral receptor, and MHC class I molecules, and these functions map to different parts of the protein. Nef interacts with components of host cell signal transduction and clathrin-dependent protein sorting pathways. It increases viral infectivity. Nef contains PxxP motifs that bind to SH3 domains of a subset of Src kinases and are required for the enhanced growth of HIV but not for the downregulation of CD4.


VPX is a virion protein of 12 kDa found in HIV-2, SIV-SMM, SIV-RCM, SIV-MND-2 and SIV-DRL and not in HIV-1 or other SIVs. This accessory gene is a homolog of HIV-1 vpr, and viruses with Vpx carry both vpr and vpx. Vpx function in relation to Vpr is not fully elucidated; both are incorporated into virions at levels comparable to Gag proteins through interactions with Gag p6. Vpx is necessary for efficient replication of SIV-SMM in PBMCs. Progression to AIDS and death in SIV-infected animals can occur in the absence of Vpr or Vpx. Double mutant virus lacking both vpr and vpx was attenuated, whereas the single mutants were not, suggesting a redundancy in the function of Vpr and Vpx related to virus pathogenicity.


Hepatitis A Viral Target Sequences


5′ untranslated region contains IRES—internal ribosome entry site


P1 Region of Genome—Capsid Proteins

    • VP1
    • VP2
    • VP3
    • VP4


P2 Region of genome

    • 2A
    • 2B
    • 2C


P3 Region of Genome

    • 3A
    • 3B
    • 3C—viral protease
    • 3D—RNA polymerase


Hepatitis B Viral Target Sequences


Precursor Polypeptide encoding all HCV protein is produced and then spliced into functional proteins. The following are the proteins (coding regions) encoded:

    • C—core protein—coding region consists of a Pre-C and Core coding region
    • X—function unclear but suspected to play a role in activation of viral transcription process
    • P—RNA polymerase
    • S—surface antigen—coding region consists of a Pre-S1, Pre-S2 and Surface antigen coding regions


Hepatitis C Viral Target Sequences


Precursor Polypeptide encoding all HCV protein is produced and then spliced into functional proteins. The following are the proteins (coding regions) encoded:

    • RES—non-coding internal ribosome entry site (5′ to polyprotein encoding sequence)
    • 3′ non-coding sequences—
    • C region—encodes p22 a nucleocapsid protein
    • E1 region—encodes gp35 envelope glycoprotein—important in cell entry
    • E2 region—encodes gp70 envelope glycoprotein—important in cell entry
    • NS1—encodes p7—not necessary for replication but critical in viral morphogenesis
    • NS2—encodes p23 a transmembrane protein with protease activity
    • NS3—encodes p70 having both serine protease and RNA helicase activities
    • NS4A—encodes p8 co-factor
    • NS4B—encodes p27 cofactor—important in recruitment of other viral proteins
    • NS5A—encodes p56/58 an interferon resistance protein—important in viral replication
    • NS5B—encodes RNA polymerase


Herpes Simplex Virus Target Sequence















El
Genome replication: ATP-dependent DNA helicase


E2
Genome replication, transcription, segregation,



encapsidation. Regulation of cellular gene expression;



cell cycle and apoptosis regulation. Several isoforms of



the virus replication/transcription factor E2 have also



been noted for a number of HPVs. E2 has an N-terminal



domain that mediates protein-protein interactions, a



flexible hinge region and a C-terminal DNA binding



domain. Truncated E2 proteins may be translated from



alternatively spliced E2 RNAs to generate El ″E2 and



E8″E2 protein isoforms present in HPV16 and 31-



infected cells. These E2 isoforms may act in a dominant-



negative manner to modulate the function of full length



E2. For example, a full length E2/E8″E2 dimer may bind



DNA but fail to recruit El to initiate virus replication.



Similarly, such a dimer may be unable to interact with



cellular transcription factors to alter virus genome



transcription.


E4
Remodels cytokeratin network; cell cycle arrest; virion



assembly


E5
Control of cell growth and differentiation; immune



modulation


E6
Inhibits apoptosis and differentiation; regulates cell shape,



polarity, mobility and signaling. Four mRNA isoforms



(FLE6, E6*I, E6*11, E6*X) have been observed in



HPV16 infected cervical epithelial cells and two in



HPV18 infection. A role for the E6*I isoform in



antagonizing FLE6 function has been suggested,



as has opposing roles for FLE6 and E6*I in regulation



of procaspase 8 in the extrinsic apoptotic pathway.



More recently, a stand-alone function of the E6*I isoform



has been determined in cellular protein degradation.


E7
Cell cycle control; controls centrosome duplication


Ll
Major capsid protein


L2
Minor capsid protein; recruits Ll; virus assembly


LCR
Viral long control region (location of early promoters)


Keratinocyte/



auxiliary



enhancer




P91 Promoter

Early (E) gene promoter for subtype HPV16


P10s Promoter
Early (E) gene promoter for subtype HPV18


P670 Promoter
Late (L) gene promoter for HPV16



P142 Promoter

Late (L) gene promoter for HPV31









Influenza A Target Sequences


Influenza A is the most common flu virus that infects humans. The influenza A virion is made up of 8 different single stranded RNA segments which encodes 11-14 proteins. These segments can vary in sequence, with most variation occurring in the hemagglutinin (H or HA) surface protein and neuraminidase (NA or N). The eight RNA segments (and the proteins they encode) are:

    • HA—encodes hemagglutinin (about 500 molecules of hemagglutinin are needed to make one virion).
    • NA—encodes neuraminidase (about 100 molecules of neuraminidase are needed to make one virion).
    • NP encodes nucleoprotein.
    • M encodes two matrix proteins (the M1 and the M2) by using different reading frames from the same RNA segment (about 3000 matrix protein molecules are needed to make one virion). M42 is produced by alternative splicing, and can partially replace an M2.
    • NS encodes two distinct non-structural proteins (NS1 and NEP) by using different reading frames from the same RNA segment.
    • PA encodes an RNA polymerase; an alternate form is sometimes made through a ribosomal skip, with +1 frameshift, reading through to the next stop codon.
    • PB1 encodes an RNA polymerase, plus two other transcripts read from alternate start sites, named PB1-N40 and PB1-F2 protein (induces apoptosis) by using different reading frames from the same RNA segment.
    • PB2 encodes an RNA polymerase.



M. tuberculosis Target Sequences


The methods and composition described herein can be used to target M. tuberculosis and treat a subject suffering from an infection with M. tuberculosis.


Other


In an embodiment, the target gene is associated with multiple drug resistance (MDR), e.g., in bacterial infection. Infectious pathogens can use a number of mechanisms in attaining multi-drug resistance. e.g., no longer relying on a glycoprotein cell wall, enzymatic deactivation of antibiotics, decreased cell wall permeability to antibiotics, altered target sites of antibiotic, efflux pumps to remove antibiotics, increased mutation rate as a stress response, or a combination thereof.


IX. Targets: Gene Editing/Correction


Candidate Cas9 molecules, candidate gRNA molecules, and/or candidate Cas9 molecule/gRNA molecule complexes, can be used to modulate genes (e.g., mutated genes) responsible for diseases. In an embodiment, the gene is modulated by editing or correcting a target gene, e.g., as described herein. In an embodiment, the human gene is modulated by delivery of one or more regulators/effectors (e.g., as described herein) inside cells to the target gene. For example, the genes described herein can be modulated, in vitro, ex vivo, or in vivo.









TABLE IX-1





Selected Diseases in which a gene can be therapeutically targeted.















Kinases (cancer)


Energy metabolism (cancer)


CFTR (cystic fibrosis)


Color blindness


Hemochromatosis


Hemophilia


Phenylketonuria


Polycystic kidney disease


Sickle-cell disease


Tay-Sachs disease


Siderius X-linked mental retardation syndrome


Lysosomal storage disorders, e.g., Alpha-galactosidase A deficiency


Anderson-Fabry disease


Angiokeratoma Corporis Diffusum


CADASIL syndrome


Carboxylase Deficiency, Multiple, Late-Onset


Cerebelloretinal Angiomatosis, familial


Cerebral arteriopathy with subcortical infarcts and leukoencephalopathy


Cerebral autosomal dominant arteriopathy with subcortical infarcts and


leukoencephalopathy


Cerebroside Lipidosis syndrome


Choreoathetosis self-mutilation hyperuricemia syndrome


Classic Galactosemia


Crohn's disease, fibrostenosing


Phenylalanine Hydroxylase Deficiency disease,


Fabry disease


Hereditary coproporphyria


Incontinentia pigmenti


Microcephal y


Polycystic kidney disease


Rett's


Alpha-1 antitrypsin deficiency


Wilson's Disease


Tyrosineima


Frameshift related diseases


Cystic fibrosis


Triplet repeat diseases (also referred herein as trinucleotide repeat diseases)









Trinucleotide repeat diseases (also known as triplet repeat disease, trinucleotide repeat expansion disorders, triplet repeat expansion disorders, or codon reiteration disorders) are a set of genetic disorders caused by trinucleotide repeat expansion, e.g., a type of mutation where trinucleotide repeats in certain genes exceed the normal and/or stable threshold. The mutation can be a subset of unstable microsatellite repeats that occur in multiple or all genomic sequences. The mutation can increase the repeat count (e.g., result in extra or expanded repeats) and result in a defective gene, e.g., producing an abnormal protein. Trinucleotide repeats can be classified as insertion mutations or as a separate class of mutations. Candidate Cas9 molecules, candidate gRNA molecules, and/or candidate Cas9 molecule/gRNA molecule complexes, can be used to modulate one or more genes (e.g., mutated genes) associated with a trinucleotide repeat disease, e.g., by reducing the number of (e.g., removing) the extra or expanded repeats, such that the normal or wild-type gene product (e.g., protein) can be produced.


Exemplary trinucleotide repeat diseases and target genes involved in trinucleotide repeat diseases are shown in Table IX-1A.


Table IX-1A. Exemplary Trinucleotide Repeat Diseases and Target Genes Involved in Trinucleotide Repeat Diseases


Exemplary target genes include those genes involved in various diseases or conditions, e.g., cancer (e.g., kinases), energy metabolism, cystic fibrosis (e.g., CFTR), color blindness, hemochromatosis, hemophilia, phenylketonuria, polycystic kidney disease, Sickle-cell disease, Tay-Sachs disease, Siderius X-linked mental retardation syndrome, Lysosomal storage disorders (e.g., Alpha-galactosidase A deficiency), Anderson-Fabry disease, Angiokeratoma Corporis Diffusum, CADASIL syndrome, Carboxylase Deficiency, Multiple, Late-Onset, Cerebelloretinal Angiomatosis, familial, Cerebral arteriopathy with subcortical infarcts and leukoencephalopathy, Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, Cerebroside Lipidosis syndrome, Choreoathetosis self-mutilation hyperuricemia syndrome, Classic Galactosemia, Crohn's disease, fibrostenosing, Phenylalanine Hydroxylase Deficiency disease. Fabry disease, Hereditary coproporphyria, Incontinentia pigmenti, Microcephaly, Polycystic kidney disease, Rett's, Alpha-1 antitrypsin deficiency, Wilson's Disease, Tyrosinemia, Frameshift related diseases, and Triplet repeat diseases.


Additional exemplary target genes include genes associated with diseases including, e.g., Crigler-Najjer syndrome, Glycogen storage disease type IV (GSD type IV), Familial hemophagocytic lymphohistiocytosis (FHL-Perforin deficiency), Omithine transcarbamylase deficiency (OTC deficiency) or other Urea Cycle Disorders. Primary Hyperoxaluria, Leber congenital amaurosis (LCA), Batten disease, Chronic Granulomatous Disease, Wiskott-Aldrich syndrome, Usher Syndrome, and hemoglobinoapthies.


Crigler-Najjer Syndrome.


Crigler-Najjer syndrome is a severe condition characterized by high levels of bilirubin in the blood (hyperbilirubinemia). Bilirubin is produced when red blood cells are broken down. This substance is removed from the body only after it undergoes a chemical reaction in the liver, which converts the toxic form of bilirubin (unconjugated bilirubin) to a nontoxic form (conjugated bilirubin). People with Crigler-Najjar syndrome have a buildup of unconjugated bilirubin in their blood (unconjugated hyperbilirubinemia). Crigler-Najjar syndrome is divided into two types. Type 1 (CN1) is very severe and Type 2 (CN2) is less severe.


Mutations in the UGT1A1 gene can cause Crigler-Najjar syndrome. This gene provides instructions for making the bilirubin uridine diphosphate glucuronosyl transferase (bilirubin-UGT) enzyme, which is found primarily in liver cells and is necessary for the removal of bilirubin from the body. The bilirubin-UGT enzyme is involved in glucuronidation, in which the enzyme transfers glucuronic acid to unconjugated bilirubin, converting it to conjugated bilirubin. Glucuronidation makes bilirubin dissolvable in water so that it can be removed from the body.


Mutations in the UGT1A1 gene that cause Crigler-Najjar syndrome result in reduced or absent function of the bilirubin-UGT enzyme. People with CN1 have no enzyme function, while people with CN2 can have less than 20 percent of normal function. The loss of bilirubin-UGT function decreases glucuronidation of unconjugated bilirubin. This toxic substance then builds up in the body, causing unconjugated hyperbilirubinemia and jaundice.


Glycogen Storage Disease Type IV.


Glycogen storage disease type IV (also known as GSD type IV. Glycogenosis type IV. Glycogen Branching Enzyme Deficiency (OBED), polyglucosan body disease, or Amylopectinosis) is an inherited disorder caused by the buildup of a complex sugar called glycogen in the body's cells. The accumulated glycogen is structurally abnormal and impairs the function of certain organs and tissues, especially the liver and muscles.


Mutations in the GBE1 gene cause GSD IV. The GBE1 gene provides instructions for making the glycogen branching enzyme. This enzyme is involved in the production of glycogen, which is a major source of stored energy in the body. GBE1 gene mutations that cause GSD IV lead to a shortage (deficiency) of the glycogen branching enzyme. As a result, glycogen is not formed properly. Abnormal glycogen molecules called polyglucosan bodies accumulate in cells, leading to damage and cell death. Polyglucosan bodies accumulate in cells throughout the body, but liver cells and muscle cells are most severely affected in GSD IV. Glycogen accumulation in the liver leads to hepatomegaly and interferes with liver functioning. The inability of muscle cells to break down glycogen for energy leads to muscle weakness and wasting.


Generally, the severity of the disorder is linked to the amount of functional glycogen branching enzyme that is produced. Individuals with the fatal perinatal neuromuscular type tend to produce less than 5 percent of usable enzyme, while those with the childhood neuromuscular type may have around 20 percent of enzyme function. The other types of GSD IV are usually associated with between 5 and 20 percent of working enzyme. These estimates, however, vary among the different types.


Familial Hemophagocytic Lymphohistiocytosis.


Familial hemophagocytic lymphohistiocytosis (FHL) is a disorder in which the immune system produces too many activated immune cells (lymphocytes), e.g., T cells, natural killer cells, B cells, and macrophages (histiocytes). Excessive amounts of cytokines are also produced. This overactivation of the immune system causes fever and damages the liver and spleen, resulting in enlargement of these organs.


Familial hemophagocytic lymphohistiocytosis also destroys blood-producing cells in the bone marrow, a process called hemophagocytosis. The brain may also be affected in familial hemophagocytic lymphohistiocytosis. In addition to neurological problems, familial hemophagocytic lymphohistiocytosis can cause abnormalities of the heart, kidneys, and other organs and tissues. Affected individuals also have an increased risk of developing cancers of blood-forming cells (leukemia and lymphoma).


Familial hemophagocytic lymphohistiocytosis may be caused by mutations in any of several genes. These genes provide instructions for making proteins that help destroy or deactivate lymphocytes that are no longer needed. By controlling the number of activated lymphocytes, these genes help regulate immune system function.


Approximately 40 to 60 percent of cases of familial hemophagocytic lymphohistiocytosis are caused by mutations in the PRF1 or UNC13D genes. Smaller numbers of cases are caused by mutations in other known genes such as STXl 1 or STXBP2. The gene mutations that cause familial hemophagocytic lymphohistiocytosis can impair the body's ability to regulate the immune system. These changes result in the exaggerated immune response characteristic of this condition.


Ornithine Transcarbamylase Deficiency.


Omithine transcarbamylase deficiency (OTC) is an inherited disorder that causes ammonia to accumulate in the blood.


Mutations in the OTC gene cause omithine transcarbamylase deficiency.


Omithine transcarbamylase deficiency belongs to a class of genetic diseases called urea cycle disorders. The urea cycle is a sequence of reactions that occurs in liver cells. It processes excess nitrogen, generated when protein is used by the body, to make a compound called urea that is excreted by the kidneys.


In omithine transcarbamylase deficiency, the enzyme that starts a specific reaction within the urea cycle is damaged or missing. The urea cycle cannot proceed normally, and nitrogen accumulates in the bloodstream in the form of ammonia.


Ammonia is especially damaging to the nervous system, so omithine transcarbamylase deficiency causes neurological problems as well as eventual damage to the liver.


Other urea cycle disorders and associate genes include, e.g., N-Acetylglutamate synthase deficiency (NAGS), Carbamoyl phosphate synthetase I deficiency (CPS1), “AS deficiency” or citrullinemia (ASS), “AL deficiency” or argininosuccinic aciduria (ASL), and “Arginase deficiency” or argininemia (ARG).


Primary Hyperoxaluria.


Primary hyperoxaluria, e.g., primary hyperoxaluria type 1 (PHI), is a rare, autosomal recessive inherited genetic condition in which an error in the glyoxylate metabolism pathway in the liver leads to an overproduction of oxalate, which crystallizes in soft tissues including the kidney, bone marrow, and eyes. The disease manifests as progressive deterioration of the kidneys, and treatment is a complicated double transplant of kidney (the damaged organ) and liver (the diseased organ).


Primary hyperoxaluria is caused by the deficiency of an enzyme that normally prevents the buildup of oxalate. There are two types of primary hyperoxaluria, distinguished by the enzyme that is deficient. People with type 1 primary hyperoxaluria have a shortage of a liver enzyme called alanine-glyoxylate aminotransferase (AGXT). Type 2 primary hyperoxaluria is characterized by a shortage of an enzyme called glyoxylate reductase/hydroxypyruvate reductase (GRHPR).


Mutations in the AGXT and GRHPR genes cause primary hyperoxaluria. The breakdown and processing of certain sugars and amino acids produces aglyoxylate. Normally, glyoxylate is converted to the amino acid glycine or to glycolate through the action of two enzymes, alanine-glyoxylate aminotransferase and glyoxylate reductase/hydroxypyruvate reductase, respectively. Mutations in the AGXT or GRHPR gene cause a shortage of these enzymes, which prevents the conversion of glyoxylate to glycine or glycolate. As levels of glyoxylate build up, it is converted to oxalate. Oxalate combines with calcium to form calcium oxalate deposits, which can damage the kidneys and other organs.


In an embodiment, the genetic defect in AGXT is corrected, e.g., by homologous recombination, using the Cas9 molecule and gRNA molecule described herein. For example, the functional enzyme encoded by the corrected AGXT gene can be redirected to its proper subcellular organelle. Though >50 mutations have been identified in the gene, the most common (40% in Caucasians) is a missense G170R mutation. This mutation causes the AGT enzyme to be localized to the mitochondria rather than to the peroxisome, where it must reside to perform its function. Other common mutations include, e.g., I244T (Canary Islands), F1521, G41R, G630A (Italy), and G588A (Italy).


In an embodiment, one or more genes encoding enzymes upstream in the glyoxylate metabolism pathway are targeted, using the Cas9 molecule and gRNA molecule described herein. Exemplary targets include, e.g., glycolate oxidase (gene HAO1, OMIM ID 605023). Glycolate oxidase converts glycolate into glyoxylate, the substrate for AGT. Glycolate oxidase is only expressed in the liver and, because of its peroxisomal localization, makes it a suitable target in this metabolic pathway. In an embodiment, a double-strand break in the HAO1 gene is introduced and upon repair by NHEJ a frame-shift results in a truncated protein. In an embodiment, a transcriptional repressor (e.g., a transcriptional repressor described herein) is delivered as a payload to the HAO1 gene to reduce the expression of HAO1.


Leber Congenital Amaurosis.


Leber congenital amaurosis (LCA) is an eye disorder that primarily affects the retina. People with this disorder typically have severe visual impairment beginning in infancy. The visual impairment tends to be stable, although it may worsen very slowly over time. At least 13 types of Leber congenital amaurosis have been described. The types are distinguished by their genetic cause, patterns of vision loss, and related eye abnormalities.


Leber congenital amaurosis can result from mutations in at least 14 genes, all of which are necessary for normal vision. These genes play a variety of roles in the development and function of the retina. For example, some of the genes associated with this disorder are necessary for the normal development of photoreceptors. Other genes are involved in phototransduction. Still other genes play a role in the function of cilia, which are necessary for the perception of several types of sensory input, including vision.


Mutations in any of the genes associated with Leber congenital amaurosis (e.g., AIPL1, CEP290, CRB1, CRX, GUCY2D, IMPDH1, LCA5, LRAT, RD3, RDH12, RPE65, RPGRIP1, SPATA7, TULP1) can disrupt the development and function of the retina, resulting in early vision loss. Mutations in the CEP290, CRB1. GUCY2D, and RPE65 genes are the most common causes of the disorder, while mutations in the other genes generally account for a smaller percentage of cases.


Batten Disease.


Batten disease or juvenile Batten disease is an inherited disorder that primarily affects the nervous system. After a few years of normal development, children with this condition develop progressive vision loss, intellectual and motor disability, and seizures.


Juvenile Batten disease is one of a group of disorders known as neuronal ceroid lipofuscinoses (NCLs). These disorders all affect the nervous system and typically cause progressive problems with vision, movement, and thinking ability. Some people refer to the entire group of NCLs as Batten disease, while others limit that designation to the juvenile form of the disorder. The different types of NCLs are distinguished by the age at which signs and symptoms first appear.


Most cases of juvenile Batten disease are caused by mutations in the CLN3 gene. These mutations can disrupt the function of cellular structures called lysosomes. Lysosome malfunction leads to a buildup of lipopigments within these cell structures. These accumulations occur in cells throughout the body, but neurons in the brain seem to be particularly vulnerable to the damage caused by lipopigments. The progressive death of cells, especially in the brain, leads to vision loss, seizures, and intellectual decline in people with juvenile Batten disease.


A small percentage of cases of juvenile Batten disease are caused by mutations in other genes (e.g., ATP13A2, CLN5, PPT1, TPP1). Many of these genes are involved in lysosomal function, and when mutated, can cause this or other forms of NCL.


Chronic Granulomatous Disease.


Chronic granulomatous disease is a disorder that causes the immune system to malfunction, resulting in a form of immunodeficiency. Individuals with chronic granulomatous disease have recurrent bacterial and fungal infections. People with this condition often have areas of inflammation (granulomas) in various tissues that can be damaging to those tissues. The features of chronic granulomatous disease usually first appear in childhood, although some individuals do not show symptoms until later in life.


Mutations in the CYBA, CYBB, NCF1, NCF2, or NCF4 gene can cause chronic granulomatous disease. There are five types of this condition that are distinguished by the gene that is involved. The proteins produced from the affected genes are subunits of NADPH oxidase, which plays an important role in the immune system. Specifically, NADPH oxidase is primarily active in phagocytes. Within phagocytes, NADPH oxidase is involved in the production of superoxide, which plays a role in killing foreign invaders and preventing them from reproducing in the body and causing illness. NADPH oxidase also regulates the activity of neutrophils, which play a role in adjusting the inflammatory response to optimize healing and reduce injury to the body.


Mutations in the CYBA, CYBB, NCF1, NCF2, and NCF4 genes result in the production of proteins with little or no function or the production of no protein at all. Without any one of its subunit proteins, NADPH oxidase cannot assemble or function properly. As a result, phagocytes are unable to kill foreign invaders and neutrophil activity is not regulated. A lack of NADPH oxidase leaves affected individuals vulnerable to many types of infection and excessive inflammation.


Wiskot-Aldrich Syndrome.


Wiskott-Aldrich syndrome is characterized by abnormal immune system function (immune deficiency) and a reduced ability to form blood clots. This condition primarily affects males. Individuals with Wiskott-Aldrich syndrome have microthrombocytopenia, which is a decrease in the number and size of blood cells involved in clotting (platelets), which can lead to easy bruising or episodes of prolonged bleeding following minor trauma. Wiskott-Aldrich syndrome causes many types of white blood cells to be abnormal or nonfunctional, leading to an increased risk of several immune and inflammatory disorders. Many people with this condition develop eczema, an inflammatory skin disorder characterized by abnormal patches of red, irritated skin. Affected individuals also have an increased susceptibility to infection. People with Wiskott-Aldrich syndrome are at greater risk of developing autoimmune disorders. The chance of developing some types of cancer, such as cancer of the immune system cells (lymphoma), is also greater in people with Wiskott-Aldrich syndrome.


Mutations in the WAS gene cause Wiskott-Aldrich syndrome. The WAS gene provides instructions for making WASP protein, which is found in all blood cells. WASP is involved in relaying signals from the surface of blood cells to the actin cytoskeleton. WASP signaling activates the cell when it is needed and triggers its movement and attachment to other cells and tissues (adhesion). In white blood cells, this signaling allows the actin cytoskeleton to establish the interaction between cells and the foreign invaders that they target (immune synapse).


WAS gene mutations that cause Wiskott-Aldrich syndrome lead to a lack of any functional WASP. Loss of WASP signaling disrupts the function of the actin cytoskeleton in developing blood cells. White blood cells that lack WASP have a decreased ability to respond to their environment and form immune synapses. As a result, white blood cells are less able to respond to foreign invaders, causing many of the immune problems related to Wiskott-Aldrich syndrome. Similarly, a lack of functional WASP in platelets impairs their development, leading to reduced size and early cell death.


Usher Syndrome.


Usher syndrome is a condition characterized by hearing loss or deafness and progressive vision loss. The loss of vision is caused by retinitis pigmentosa (RP), which affects the layer of light-sensitive tissue at the back of the eye (the retina). Vision loss occurs as the light-sensing cells of the retina gradually deteriorate.


Three major types of Usher syndrome, designated as types I (subtypes IA through IG), II (subtypes IIA, IIB, and IIC), and III, have been identified. These types are distinguished by their severity and the age when signs and symptoms appear.


Mutations in the CDH23, CLRN1, GPR98, MY07A, PCDH15, USH1C, USH1G, and USH2A genes can cause Usher syndrome. The genes related to Usher syndrome provide instructions for making proteins that play important roles in normal hearing, balance, and vision. They function in the development and maintenance of hair cells, which are sensory cells in the inner ear that help transmit sound and motion signals to the brain. In the retina, these genes are also involved in determining the structure and function of light-sensing cells called rods and cones. In some cases, the exact role of these genes in hearing and vision is unknown. Most of the mutations responsible for Usher syndrome lead to a loss of hair cells in the inner ear and a gradual loss of rods and cones in the retina. Degeneration of these sensory cells causes hearing loss, balance problems, and vision loss characteristic of this condition.


Usher syndrome type I can result from mutations in the CDH23, MY07A, PCDH15, USH1C, or USH1G gene. Usher syndrome type II can be caused by mutations in, e.g., USH2A or GPR98 (also called VLGR1) gene. Usher syndrome type III can be caused by mutations in e.g., CLRN1.


Hemoglobinopathies.


Hemoglobinopathies are a group of genetic defects that result in abnormal structure of one of the globin chains of the hemoglobin molecule. Exemplary hemoglobinopathies include, e.g., sickle cell disease, alpha thalassemia, and beta thalassemia.


In an embodiment, a genetic defect in alpha globulin or beta globulin is corrected, e.g., by homologous recombination, using the Cas9 molecule and gRNA molecule described herein.


In an embodiment, a hemoglobinopathies-associated gene is targeted, using the Cas9 molecule and gRNA molecule described herein. Exemplary targets include, e.g., genes associated with control of the gamma-globin genes. In an embodiment, the target is BCL11A.


Fetal hemoglobin (also hemoglobin For HbF or a2y2) is a tetramer of two adult alpha-globin polypeptides and two fetal beta-like gamma-globin polypeptides. HbF is the main oxygen transport protein in the human fetus during the last seven months of development in the uterus and in the newborn until roughly 6 months old. Functionally, fetal hemoglobin differs most from adult hemoglobin in that it is able to bind oxygen with greater affinity than the adult form, giving the developing fetus better access to oxygen from the mother's bloodstream.


In newborns, fetal hemoglobin is nearly completely replaced by adult hemoglobin by approximately 6 months postnatally. In adults, fetal hemoglobin production can be reactivated pharmacologically, which is useful in the treatment of diseases such as hemoglobinopathies. For example, in certain patients with hemoglobinopathies, higher levels of gamma-globin expression can partially compensate for defective or impaired beta-globin gene production, which can ameliorate the clinical severity in these diseases. Increased HbF levels or F-cell (HbF containing erythrocyte) numbers can ameliorate the disease severity of hemoglobinopathies, e.g., beta-thalassemia major and sickle cellanemia.


Increased HbF levels or F-cell can be associated reduced BCL11A expression in cells. The BCL11A gene encodes a multi-zinc finger transcription factor. In an embodiment, the expression of BCL11A is modulated, e.g., down-regulated. In an embodiment, the BCL11A gene is edited. In an embodiment, the cell is a hemopoietic stem cell or progenitor cell.


Sickle Cell Diseases


Sickle cell disease is a group of disorders that affects hemoglobin. People with this disorder have atypical hemoglobin molecules (hemoglobin S), which can distort red blood cells into a sickle, or crescent, shape. Characteristic features of this disorder include a low number of red blood cells (anemia), repeated infections, and periodic episodes of pain.


Mutations in the HBB gene cause sickle cell disease. The HBB gene provides instructions for making beta-globin. Various versions of beta-globin result from different mutations in the HBB gene. One particular HBB gene mutation produces an abnormal version of beta-globin known as hemoglobin S (HbS). Other mutations in the HBB gene lead to additional abnormal versions of beta-globin such as hemoglobin C (HbC) and hemoglobin E (HbE). HBB gene mutations can also result in an unusually low level of beta-globin, i.e., beta thalassemia.


In people with sickle cell disease, at least one of the beta-globin subunits in hemoglobin is replaced with hemoglobin S. In sickle cell anemia, which is a common form of sickle cell disease, hemoglobin S replaces both beta-globin subunits in hemoglobin. In other types of sickle cell disease, just one beta-globin subunit in hemoglobin is replaced with hemoglobin S. The other beta-globin subunit is replaced with a different abnormal variant, such as hemoglobin C. For example, people with sickle-hemoglobin C (HbSC) disease have hemoglobin molecules with hemoglobin S and hemoglobin C instead of beta-globin. If mutations that produce hemoglobin S and beta thalassemia occur together, individuals have hemoglobin S-beta thalassemia (HbSBetaThal) disease.


Aloha Thalassemia


Alpha thalassemia is a blood disorder that reduces the production of hemoglobin. In people with the characteristic features of alpha thalassemia, a reduction in the amount of hemoglobin prevents enough oxygen from reaching the body's tissues. Affected individuals also have a shortage of red blood cells (anemia), which can cause pale skin, weakness, fatigue, and more serious complications.


Two types of alpha thalassemia can cause health problems. The more severe type is hemoglobin Bart hydrops fetalis syndrome or Hb Bart syndrome. The milder form is HbH disease. Hb Bart syndrome is characterized, e.g., by hydrops fetalis, a condition in which excess fluid builds up in the body before birth. HbH disease can cause, e.g., mild to moderate anemia, hepatosplenomegaly, and yellowing of the eyes and skin Uaundice).


Alpha thalassemia typically results from deletions involving the HBA1 and HBA2 genes. Both of these genes provide instructions for making alpha-globin, which is a subunit of hemoglobin. The different types of alpha thalassemia result from the loss of some or all of these alleles.


Hb Bart syndrome can result from the loss of all four alpha-globin alleles. HbH disease can be caused by a loss of three of the four alpha-globin alleles. In these two conditions, a shortage of alpha-globin prevents cells from making normal hemoglobin. Instead, cells produce abnormal forms of hemoglobin, i.e., hemoglobin Bart (Hb Bart) or hemoglobin H (HbH), which cannot effectively carry oxygen to the body's tissues. The substitution of Hb Ban or HbH for normal hemoglobin can cause anemia and the other serious health problems associated with alpha thalassemia.


Two additional variants of alpha thalassemia are related to a reduced amount of alpha-globin. A loss of two of the four alpha-globin alleles can result in alpha thalassemia trait. People with alpha thalassemia trait may have unusually small, pale red blood cells and mild anemia. A loss of one alpha-globin allele can be found in alpha thalassemia silent carriers.


Beta Thalassemia


Beta thalassemia is a blood disorder that reduces the production of hemoglobin. In people with beta thalassemia, low levels of hemoglobin lead to a lack of oxygen in many parts of the body. Affected individuals also have a shortage of red blood cells (anemia), which can cause pale skin, weakness, fatigue, and more serious complications. People with beta thalassemia are at an increased risk of developing abnormal blood clots.


Beta thalassemia is classified into two types depending on the severity of symptoms; thalassemia major (also known as Cooley's anemia) and thalassemia intermedia. Of the two types, thalassemia major is more severe.


Mutations in the HBB gene cause beta thalassemia. The HBB gene provides instructions for making beta-globin. Some mutations in the HBB gene prevent the production of any beta-globin. The absence of beta-globin is referred to as beta-zero (Bo) thalassemia. Other HBB gene mutations allow some beta-globin to be produced but in reduced amounts, i.e., beta-plus (B+) thalassemia. People with both types have been diagnosed with thalassemia major and thalassemia intermedia.


In an embodiment, a Cas9 molecule/gRNA molecule complex targeting a first gene is used to treat a disorder characterized by second gene, e.g., a mutation in a second gene. By way of example, targeting of the first gene, e.g., by editing or payload delivery, can compensate for, or inhibit further damage from, the affect of a second gene, e.g., a mutant second gene. In an embodiment the allele(s) of the first gene carried by the subject is not causative of the disorder.









TABLE IX-3





Selected Disorders and Targets for Compensatory Targeting

















Indication









Prevention of










Non-Hodgkin's
organ transplant













Age-Related Macular
Atypical Hemolytic
lymphoma, Chronic
Rheumatoid
rejection, renal



Degeneration
Uremic Syndrome
lymphocytic leukemia
Arthritis
cell carcinoma


















Target
Factor H
C5
Factor H
C5
CD20
CD2I
mTORCl


Up-
up-
down-
up-
down-
down-
down-
down-


regulate/
regulate
regulate
regulate
regulate
regulate
regulate
regulate


Down-


regulate


Level of
animal

Factor H
Eculizumab/
Rituxan
Rituxan
everolimus


evidence:
models

concentrate
Soliris cSAb
(Genentech)
(Genentech)


Market



(Alexion)
CD20
CD20


proxy or



successful in
antibody
antibody


animal



decreasing


model



mortality












Comment
Muti-genetic origin. Factor H
aHUS due to fH deficiency.






deficiency is a risk factor.
CS antibody has been shown



Controlling the complement
to vastly improve prognosis.



cascade, through fH
Can approach disease directly



upregulation or CS
through increasing fH levels



downregulation, may have a
or controlling complement



beneficial effect.
through CS downregulation.












Indication















Devices:









stent,



pacemaker,



hernia mesh-
Graft
orthopedics-



Barrett's



local delivery
healing/wound
articular



esophagus,



to prevent
healing/
cartilage



Stomach



restenosis/
prevention of
repair,
Parkinson's
Allergic

ulcer,



fibrosis
fibrosis
arthritis
Disease
rhinitis
Epilepsy
gastritis





Target
mTORC2,
VEGF
IL-11
SNCA,
HI
H1
H2



others


LRRK2,
Receptors
receptors
receptor






EIF4GI
nasal
CNS
pylorus,







mucosa

esophagus


Upregulate/
down-
up-
up-
up-
down-
up-
down-


Downregulate
regulate
regulate
regulate
regulate
regulate
regulate
regulate






or fix






mutations


Level of
everolimus
VEGFlocal
animal

HI-anti-
animal
H2-specific


evidence:

administration
model of

histamines,
models
antihistamines,


Market

aids in
cartilage

e.g. Zyrtec

e.g.


proxy or

tracheal
repair



omeprazole,


animal

transplant




etc.


model

animal




models


Comment
Embodiments
Useful, e.g.,
In an



In an



include, e.g.,
in the
embodiment,



embodiment,



local delivery
promoting
the subject



the subject is



to tissue via
wound
sufferes from



treated for



device or
healing
arthritis or is



late-stage



injection to
(burns, etc);
in need of



barrett's.



prevent
Embodiments
healing after



fibrosis,
include, e.g.,
injury. In



restenosis
local delivery
embodiments,




of growth
chondrocytes




factors
are targeted




Cpost-injury to
post-injury to





promote





healing.









In an embodiment, Cas9 molecules, gRNA molecules, and/or Cas9 molecule/gRNA molecule complexes can be used to activate genes that regulate growth factors, such as up regulation of Epo to drive RBC production.


In an embodiment, Cas9 molecules, gRNA molecules, and/or Cas9 molecule/gRNA molecule complexes can be used to target, e.g., result in repression of, knockout of, or alteration of promoter for key transcription factors, such as BCL11A and KLF1 for up-regulating of fetal hemoglobin, e.g., for cure for sickle cell anemia and thalassemia.


Candidate Cas9 molecules, candidate gRNA molecules, and/or candidate Cas9 molecule/gRNA molecule complexes, as described herein, can be used to edit/correct a target gene or to deliver a regulator/effector inside cells, e.g., as described herein, at various subcellular locations. In an embodiment, the location is in the nucleus. In an embodiment, the location is in a sub-nuclear domain, e.g., the chromosome territories, nucleolus, nuclear speckles, Cajal bodies, Gems (gemini of Cajal bodies), or promyelocytic leukemia (PML) nuclear bodies. In an embodiment, the location is in the mitochondrion.


Candidate Cas9 molecules, candidate gRNA molecules, and/or candidate Cas9 molecule/gRNA molecule complexes, as described herein, can be used to edit/correct a target gene or to deliver a regulator/effector inside cells, as described herein, at various time points


For example, the editing/correction or delivery can occur at different phases of cell cycle, e.g., GO phase, Interphase (e.g., G1 phase, S phase, 02 phase), or M phase. As another example, the editing/correction or delivery can occur at different stages of disease progression, e.g., at latent stage or active stage of a disorder (e.g., viral infection), or at any stage or subclassification of a disorder (e.g., cancer).


Methods of the invention allow for the treatment of a disorder characterized by unwanted cell proliferation, e.g., cancer. In an embodiment, cancer cells are manipulated to make them more susceptible to treatment or to endogenous immune surveillance. In an embodiment a cancer cell is modulated to make it more susceptible to a therapeutic. In an embodiment, a cancer cell is manipulated so as to increase the expression of a gene that increases the ability of the immune system to recognize or kill the cancer cell. E.g., a Cas9 molecule/gRNA molecule complex can be used to deliver a payload, or edit a target nucleic acid so as to increase the expression of an antigen, e.g., in the case where the cancer cell has down regulated expression of the antigen. In an embodiment, a payload, e.g., a payload comprising a transcription factor or other activator of expression is delivered to the cancer cell. In an embodiment, an increase in expression is effected by cleavage of the target nucleic acid, e.g., cleavage and correction or alteration of the target nucleic acid by a template nucleic acid. In an embodiment, a payload that overrides epigenetic silencing, e.g., a modulator of methylation, is delivered.


In an embodiment, the treatment further comprises administering a second anti-cancer therapy, e.g., immunotherapy, e.g., an antibody that binds the upregulated antigen.


In an embodiment, methods described herein, e.g., targeting of a genomic signature, e.g., a somatic translocation, can be used to target the Cas9 molecule/gRNA molecule to a cancer cell.


In another aspect, the invention features a method of immunizing a subject against an antigen. The method comprises using a method described herein to promote the expression of the antigen from a cell, e.g., a blood cell, such that the antigen promotes an immune response. In an embodiment, the cell is manipulated ex vivo and then returned or introduced into the subject.


X. Modified Nucleosides, Nucleotides, and Nucleic Acids


Modified nucleosides and modified nucleotides can be present in nucleic acids, e.g., particularly gRNA, but also other forms of RNA, e.g., mRNA, RNAi, or siRNA. As described herein “nucleoside” is defined as a compound containing a five-carbon sugar molecule (a pentose or ribose) or derivative thereof, and an organic base, purine or pyrimidine, or a derivative thereof. As described herein, “nucleotide” is defined as a nucleoside further comprising a phosphate group.


Modified nucleosides and nucleotides can include one or more of:


(i) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage;


(ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar;


(iii) wholesale replacement of the phosphate moiety with“dephospho” linkers;


(iv) modification or replacement of a naturally occurring nucleobase;


(v) replacement or modification of the ribose-phosphate backbone;


(vi) modification of the 3′ end or 5′ end of the oligonucleotide, e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety; and


(vii) modification of the sugar.


The modifications listed above can be combined to provide modified nucleosides and nucleotides that can have two, three, four, or more modifications. For example, a modified nucleoside or nucleotide can have a modified sugar and a modified nucleobase. In an embodiment, every nucleotide of a gRNA or template nucleic acid is modified, e.g., all nucleotides have a modified phosphate group, e.g., all are phosphorothioate groups. In an embodiment, all, or substantially all, of the phosphate groups of a unimolecular or modular gRNA molecule or template nucleic acid are replaced with phosphorothioate groups.


In an embodiment, modified nucleotides, e.g., nucleotides having modifications as described herein, can be incorporated into a nucleic acid, e.g., a “modified nucleic acid.” In an embodiment, the modified nucleic acids comprise one, two, three or more modified nucleotides. In an embodiment, at least 5% (e.g., at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%) of the positions in a modified nucleic acid are a modified nucleotides.


Unmodified nucleic acids can be prone to degradation by, e.g., cellular nucleases. For example, nucleases can hydrolyze nucleic acid phosphodiester bonds. Accordingly, in one aspect the modified nucleic acids described herein can contain one or more modified nucleosides or nucleotides, e.g., to introduce stability toward nucleases.


In an embodiment, the modified nucleosides, modified nucleotides, and modified nucleic acids described herein can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo. The term “innate immune response” includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, generally of viral or bacterial origin, which involves the induction of cytokine expression and release, particularly the interferons, and cell death. In an embodiment, the modified nucleosides, modified nucleotides, and modified nucleic acids described herein can disrupt binding of a major groove interacting partner with the nucleic acid. In an embodiment, the modified nucleosides, modified nucleotides, and modified nucleic acids described herein can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo, and also disrupt binding of a major groove interacting partner with the nucleic acid.


In an embodiment, a governing gRNA comprises modifications, e.g., modified nucleotides, modifications to the backbone, and other modifications described herein.


In an embodiment, a template nucleic acid comprises modifications, e.g., modified nucleotides, modifications to the backbone, and other modifications described herein. In an embodiment, the modification improves the stability of the template nucleic acid, e.g., by increasing its resistance to endonucleases and/or exonucleases.


In an embodiment, a template nucleic acid that comprises modifications is double stranded, e.g., is double stranded DNA. In such embodiment, all the modifications are confined to one strand. In an embodiment, modifications are present on both strands. Modifications may be present in the 5′ homology arm, the 3′ homology arm, or the replacement sequence, or any combination thereof. In an embodiment, modifications are present in one or both homology arms but not the replacement sequence.


In an embodiment, a template nucleic acid that comprises modifications is single stranded, e.g., is single stranded DNA.


Definitions of Chemical Groups


As used herein, “alkyl” is meant to refer to a saturated hydrocarbon group which is straight-chained or branched. Example alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, t-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like. An alkyl group can contain from 1 to about 20, from 2 to about 20, from 1 to about 12, from 1 to about 8, from 1 to about 6, from 1 to about 4, or from 1 to about 3 carbon atoms.


As used herein, “aryl” refers to monocyclic or polycyclic (e.g., having 2, 3 or 4 fused rings) aromatic hydrocarbons such as, for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and the like. In an embodiment, aryl groups have from 6 to about 20 carbon atoms.


As used herein, “alkenyl” refers to an aliphatic group containing at least one double bond.


As used herein, “alkynyl” refers to a straight or branched hydrocarbon chain containing 2-12 carbon atoms and characterized in having one or more triple bonds. Examples of alkynyl groups include, but are not limited to, ethynyl, propargyl, and 3-hexynyl.


As used herein, “arylalkyl” or “aralkyl” refers to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. Aralkyl includes groups in which more than one hydrogen atom has been replaced by an aryl group. Examples of “arylalkyl” or “aralkyl” include benzyl, 2-phenylethyl, 3-phenylpropyl, 9-fluorenyl, benzhydryl, and trityl groups.


As used herein, “cycloalkyl” refers to a cyclic, bicyclic, tricyclic, or polycyclic non-aromatic hydrocarbon groups having 3 to 12 carbons. Examples of cycloalkyl moieties include, but are not limited to, cyclopropyl, cyclopentyl, and cyclohexyl.


As used herein, “heterocyclyl” refers to a monovalent radical of a heterocyclic ring system. Representative heterocyclyls include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, and morpholinyl.


As used herein, “heteroaryl” refers to a monovalent radical of a heteroaromatic ring system. Examples of heteroaryl moieties include, but are not limited to, imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrrolyl, furanyl, indolyl, thiophenyl pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, indolizinyl, purinyl, naphthyridinyl, quinolyl, and pteridinyl.


Phosphate Backbone Modifications


The Phosphate Group


In an embodiment, the phosphate group of a modified nucleotide can be modified by replacing one or more of the oxygens with a different substituent. Further, the modified nucleotide, e.g., modified nucleotide present in a modified nucleic acid, can include the wholesale replacement of an unmodified phosphate moiety with a modified phosphate as described herein. In an embodiment, the modification of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with unsymmetrical charge distribution.


Examples of modified phosphate groups include, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. In an embodiment, one of the non-bridging phosphate oxygen atoms in the phosphate backbone moiety can be replaced by any of the following groups: sulfur (S), selenium (Se), BR3 (wherein R can be, e.g., hydrogen, alkyl, or aryl), C (e.g., an alkyl group, an aryl group, and the like), H, NR2 (wherein R can be, e.g., hydrogen, alkyl, or aryl), or OR (wherein R can be, e.g., alkyl or aryl). The phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral; that is to say that a phosphorous atom in a phosphate group modified in this way is a stereogenic center. The stereogenic phosphorous atom can possess either the “R” configuration (herein Rp) or the “S” configuration (herein Sp).


Phosphorodithioates have both non-bridging oxygens replaced by sulfur. The phosphorus center in the phosphorodithioates is achiral which precludes the formation of oligoribonucleotide diastereomers. In an embodiment, modifications to one or both non-bridging oxygens can also include the replacement of the non-bridging oxygens with a group independently selected from S, Se, B, C, H, N, and OR (R can be, e.g., alkyl or aryl).


The phosphate linker can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at either linking oxygen or at both of the linking oxygens.


Replacement of the Phosphate Group


The phosphate group can be replaced by non-phosphorus containing connectors. In an embodiment, the charge phosphate group can be replaced by a neutral moiety.


Examples of moieties which can replace the phosphate group can include, without limitation, e.g., methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.


Replacement of the Ribophosphate Backbone


Scaffolds that can mimic nucleic acids can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates. In an embodiment, the nucleobases can be tethered by a surrogate backbone. Examples can include, without limitation, the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates.


Sugar Modifications


The modified nucleosides and modified nucleotides can include one or more modifications to the sugar group. For example, the 2′ hydroxyl group (OH) can be modified or replaced with a number of different “oxy” or “deoxy” substituents. In an embodiment, modifications to the 2′ hydroxyl group can enhance the stability of the nucleic acid since the hydroxyl can no longer be deprotonated to form a 2′-alkoxide ion. The 2′-alkoxide can catalyze degradation by intramolecular nucleophilic attack on the linker phosphorus atom.


Examples of “oxy”-2′ hydroxyl group modifications can include alkoxy or aryloxy (OR, wherein “R” can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar); polyethyleneglycols (PEG), O(CH2CH2O)nCH2CH2OR wherein R can be, e.g., H or optionally substituted alkyl, and n can be an integer from O to 20 (e.g., from O to 4, from O to 8, from O to 10, from O to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20). In an embodiment, the “oxy”-2′ hydroxyl group modification can include “locked” nucleic acids (LNA) in which the 2′ hydroxyl can be connected, e.g., by a C1-6 alkylene or C1-6 heteroalkylene bridge, to the 4′ carbon of the same ribose sugar, where exemplary bridges can include methylene, propylene, ether, or amino bridges; 0-amino (wherein amino can be, e.g., NH2: alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy, O(CH2)n-amino, (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino). In an embodiment, the “oxy”-2′ hydroxyl group modification can include the methoxyethyl group (MOE), (OCH 2CH2O CH3. e.g., a PEG derivative).


“Deoxy” modifications can include hydrogen (i.e. deoxyribose sugars, e.g., at the overhang portions of partially ds RNA); halo (e.g., bromo, chloro, fluoro, or iodo); amino (wherein amino can be, e.g., NH2: alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH2CH2NH)nCH2CHr amino (wherein amino can be, e.g., as described herein), —NHC(O)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino as described herein.


The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified nucleic acid can include nucleotides containing e.g., arabinose, as the sugar. The nucleotide “monomer” can have an alpha linkage at the 1′ position on the sugar, e.g., alpha-nucleosides. The modified nucleic acids can also include “abasic” sugars, which lack a nucleobase at C-1′. These abasic sugars can also be further modified at one or more of the constituent sugar atoms. The modified nucleic acids can also include one or more sugars that are in the L form, e.g. L-nucleosides.


Generally, RNA includes the sugar group ribose, which is a 5-membered ring having an oxygen. Exemplary modified nucleosides and modified nucleotides can include, without limitation, replacement of the oxygen in ribose (e.g., with sulfur (S), selenium (Se), or alkylene, such as, e.g., methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for example, anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone). In an embodiment, the modified nucleotides can include multicyclic forms (e.g., tricyclo; and “unlocked” forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), threose nucleic acid (TNA, where ribose is replaced with a-L-threofuranosyl-(3′ 2′)).


Modifications on the Nucleobase


The modified nucleosides and modified nucleotides described herein, which can be incorporated into a modified nucleic acid, can include a modified nucleobase. Examples of nucleobases include, but are not limited to, adenine (A), guanine (G), cytosine (C), and uracil (U). These nucleobases can be modified or wholly replaced to provide modified nucleosides and modified nucleotides that can be incorporated into modified nucleic acids. The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine or pyrimidine analog. In an embodiment, the nucleobase can include, for example, naturally-occurring and synthetic derivatives of abase.


Uracil


In an embodiment, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having a modified uracil include without limitation pseudouridine (\jf), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5U), 5-methoxycarbonylmethyl-uridine (mcm 5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm 5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm 5U), 5-methylaminomethyl-2-thio-uridine (mnm 5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm 5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (Tc m5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (Tm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m5U, i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m1\jf), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1S4\jf), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3\jf), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3\jf), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), a-thio-uridine, 2′-0-methyl-uridine (s2Um), 5,2′-0-dimethyl-uridine (m5Um), 2′-0-methyl-pseudouridine (\jfm), 2-thio-2′-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2′-0-methyl-uridine (mcm 5Um), 5-carbamoylmethyl-2′-0-methyl-uridine (ncm 5Um), 5-carboxymethylaminomethyl-2′-0-methyl-uridine (cmnm 5Um), 3,2′-0-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2′-0-methyl-uridine (inm 5Um), 1-thio-uridine, deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-0H-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, 5-[3-(1-E-propenylamino)uridine, pyrazolo[3,4-d]pyrimidines, xanthine, and hypoxanthine.


Cytosine


In an embodiment, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include without limitation 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (act), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, i-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), a-thio-cytidine, 2′-0-methyl-cytidine (Cm), 5,2′-0-dimethyl-cytidine (m5Cm), N4-acetyl-2′-0-methyl-cytidine (ac4Cm), N4,2′-0-dimethyl-cytidine (m4Cm), 5-formyl-2′-0-methyl-cytidine (f 5Cm), N4,N4,2′-0-trimethyl-cytidine (m\Cm), 1-thio-cytidine, 2′-F-ara-cytidine, 2′-F-cytidine, and 2′-0H-ara-cytidine.


Adenine


In an embodiment, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include without limitation 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m 6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A). N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-glycinylcarbamoyl-adenosine (g6A), N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m\A), N6-hydroxynorvalylcarbamoyl-adenosine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, a-thio-adenosine, 2′-0-methyl-adenosine (Am), N6,2′-0-dimethyl-adenosine (m6Am), N6-Methyl-2′-deoxyadenosine, N6,N6,2′-0-trimethyl-adenosine (m62Am), 1,2′-0-dimethyl-adenosine (m1Am), 2′-0-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2′-F-ara-adenosine, 2′-F-adenosine, 2′-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.


Guanine


In an embodiment, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include without limitation inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW), hydroxywybutosine (OHyW), undermodified hydroxywybutosine (OHyW*), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), archaeosine (G+), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m′G), N2-methyl-guanosine (m2G). N2,N2-dimethyl-guanosine (m2O). N2,7-dimethyl-guanosine (m2,7G), N2, N2,7-dimethyl-guanosine (m2,2,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-meth thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, a-thio-guanosine, 2′-0-methyl-guanosine (Gm), N2-methyl-2′-0-methyl-guanosine (m2Gm), N2,N2-dimethyl-2′-0-methyl-guanosine (m2 2Gm), 1-methyl-2′-0-methyl-guanosine (m′Gm), N2,7-dimethyl-2′-0-methyl-guanosine (m2,7Gm), 2′-0-methyl-inosine (Im), 1,2′-0-dimethyl-inosine (m′Im), 06-phenyl-2′-deoxyinosine, 2′-0-ribosylguanosine (phosphate) (Gr(p)), 1-thio-guanosine, 06-methyl-guanosine, 06-Methyl-2′-deoxyguanosine, 2′-F-ara-guanosine, and 2′-F-guanosine.


Modified gRNAs


In an embodiment, the modified nucleic acids can be modified gRNAs. In an embodiment, gRNAs can be modified at the 3′ end. In this embodiment, the gRNAs can be modified at the 3′ terminal U ribose. For example, the two terminal hydroxyl groups of the U ribose can be oxidized to aldehyde groups and a concomitant opening of the ribose ring to afford a modified nucleoside as sown below:




embedded image


wherein “U” can be an unmodified or modified uridine.


In another embodiment, the 3′ terminal U can be modified with a 2′3′ cyclic phosphate as shown below:




embedded image


wherein “U” can be an unmodified or modified uridine.


In an embodiment, the gRNA molecules may contain 3′ nucleotides which can be stabilized against degradation, e.g., by incorporating one or more of the modified nucleotides described herein. In this embodiment, e.g., uridines can be replaced with modified uridines, e.g., 5-(2-amino)propyl uridine, and 5-bromo uridine, or with any of the modified uridines described herein; adenosines and guanosines can be replaced with modified adenosines and guanosines, e.g., with modifications at the 8-position, e.g., 8-bromo guanosine, or with any of the modified adenosines or guanosines described herein. In an embodiment, deaza nucleotides, e.g., 7-deaza-adenosine, can be incorporated into the gRNA. In an embodiment, 0- and N-alkylated nucleotides, e.g., N6-methyl andenosine, can be incorporated into the gRNA. In an embodiment, sugar-modified ribonucleotides can be incorporated, e.g., wherein the 2′ OH-group is replaced by a group selected from H, —OR, —R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), halo, —SH, —SR (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), amino (wherein amino can be, e.g., NH2: alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or cyano (—CN). In an embodiment, the phosphate backbone can be modified as described herein, e.g., with a phosphothioate group. In an embodiment, the nucleotides in the overhang region of the gRNA can each independently be a modified or unmodified nucleotide including, but not limited to 2′-sugar modified, such as, 2-F 2′-0-methyl, thymidine (T), 2′-O-methoxyethyl-5-methyluridine (Teo), 2′-0-methoxyethyladenosine (Aeo), 2′-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combinations thereof.


In an embodiment, a one or more or all of the nucleotides in single stranded overhang of an RNA molecule, e.g., a gRNA molecule, are deoxynucleotides.


XI. Linkers


In an embodiment, the payload can be linked to the Cas9 molecules or the gRNA, e.g., by a covalent linker. This linker may be cleavable or non-cleavable. In an embodiment, a cleavable linker may be used to release the payload after transport to the desired target.


Linkers can comprise a direct bond or an atom such as, e.g., an oxygen (O) or sulfur (S), a unit such as —NR— wherein R is hydrogen or alkyl, —C(O)—, —C(O)O—, —C(O)NH—, SO, S02, —S02NH— or a chain of atoms, such as substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, heteroarylalkyl. In an embodiment, one or more methylenes in the chain of atoms can be replaced with one or more of 0, S, S(O), S02. —S02NH—, —NR—, —C(O)—, —C(O)O—, —C(O)NH—, a cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocyclic.


Non-Cleavable Linkages


In an embodiment, the payload is attached to the Cas9 molecule or gRNA through a linker that is itself is stable under physiological conditions, such as an alkylene chain, and does not result in release of the payload from the Cas9 molecule and/or gRNA for at least 2, 3, 4, 5, 10, 15, 24 or 48 hours or for at least 1, 2, 3, 4, 5, or 10 days when administered to a subject. In an embodiment, the payload and the Cas9 molecule and/or gRNA comprise residues of a functional groups through which reaction and linkage of the payload to the Cas9 molecule or gRNA was achieved. In an embodiment, the functional groups, which may be the same or different, terminal or internal, of the payload or Cas9 molecule and/or gRNA comprise an amino, acid, imidazole, hydroxyl, thio, acyl halide, —HC═CH—, -c=c- group, or derivative thereof. In an embodiment, the linker comprises a hydrocarbylene group wherein one or more methylene groups is optionally replaced by a group Y (provided that none of the Y groups are adjacent to each other), wherein each Y, independently for each occurrence, is selected from, substituted or unsubstituted aryl, heteroaryl, cycloalkyl, heterocycloalkyl, or —O—, —C(═X)— (wherein X is NR1, 0 or S), —NR1-, —NR1C(O)—, —C(O)NR1-, —S(O)n-, —NR1S(O)n-, —S(O)nNR1-, —NR1C(O)—NR1-; and R1, independently for each occurrence, represents H or a lower alkyl and wherein n is 0, 1, or 2.


In an embodiment, the linker comprises an alkylene moiety or a heteroalkylene moiety (e.g., an alkylene glycol moiety such as ethylene glycol). In an embodiment, a linker comprises a poly-L-glutamic acid, polylactic acid, poly(ethyleneimine), an oligosaccharide, an amino acid (e.g., glycine), an amino acid chain, or any other suitable linkage. The linker groups can be biologically inactive, such as a PEG, polyglycolic acid, or polylactic acid chain. In an embodiment, the linker group represents a derivatized or non-derivatized amino acid (e.g., glycine).


Cleavable Linkages


A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In one embodiment, the cleavable linking group is cleaved at least 10 times or more, or at least 100 times faster in the target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).


Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.


A cleavable linkage group, such as a disulfide bond (—S—S—) can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH. A linker can include a cleavable linking group that is cleavable by a particular enzyme.


In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. The candidate cleavable linking group can also be tested for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It may be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals.


In an embodiment, the cleavable linkers include redox cleavable linkers, such as a disulfide group (—S—S—) and phosphate cleavable linkers, such as, e.g., —O—P(O)(OR)-0-, —O—P(S)(OR)-0-, —O—P(S)(SR)-0-, —S—P(O)(OR)-0-, -0-P(O)(OR)—S—, —S—P(O)(OR)—S—, -0-P(S)(OR)—S—, —S—P(S)OR)-0-, —O—P(O)(R)—, —O—P(S)R)-0-, —S—P(O)(R)-0-, —S—P(S)(R)-0-, —S—P(O)(R)—S—, —OP(S)(R)—S—, wherein R is hydrogen or alkyl.


Acid Cleavable Linking Groups


Acid cleavable linking groups are linking groups that are cleaved under acidic conditions. In an embodiment, acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such as enzymes that can act as a general acid. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula-C(═N—)N—, —C(O)O—, or —OC(O)—.


Ester-Based Linking Groups


Ester-based cleavable linking groups are cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O—, or —OC(O)—.


XII. Targeting of Genomic Signatures


Cas9 molecules, gRNA molecules, and in particular, Cas9 molecule/gRNA molecule complexes, can be used to target a cell by virtue of sequence specific interaction with a target nucleic acid comprising a selected genomic signature. This provides for targeted destruction of cells having a selected genomic signature. Method and compositions disclosed herein can be used to treat disorders characterized by a selected genomic signature, e.g., a genomic signature present in the germline or a genomic signature that arise as a result of a sporadic or somatic change in the genome, e.g., a germline or acquired mutation in a cancer cell, a viral infection, or other germline or acquired changes to the genome.


While not wishing to be bound by theory, it is believed that complementarity between the targeting domain of a gRNA molecule and the target sequence of a target nucleic acid mediates target sequence-specific interaction of the Cas9 molecule/gRNA molecule complex with the target sequence. This allows targeting of specific sequences or genomic signatures, e.g., rearrangements, e.g., translocations, insertions, deletions, and inversions, and other mutations. A Cas9 molecule/gRNA molecule complex can be used to target specific sequence, e.g., mutations, that are germline, mitochondrial, or somatic. Depending on the Cas9 molecule/gRNA molecule complex used, specific editing, the delivery of a payload, or both, can be effected. In an embodiment, both cleavage and delivery of a payload is effected.


In an embodiment, the Cas9 molecule/gRNA molecule complex that promotes cell death upon recognition of its target genomic sequence. In an embodiment, an eaCas9 molecule/gRNA molecule complex cleaves the target nucleic acid. In an embodiment, it does not deliver a payload. While not wishing to be bound by theory is it believed that endogenous cellular elements, e.g., elements of the DNA damage apoptosis signaling cascade promote apoptosis in these embodiments.


In an embodiment, an eaCas9 molecule/gRNA molecule complex cleaves the target nucleic acid and delivers a payload. The payload can comprises a compound that inhibits growth or cell division, or promotes apoptosis, e.g., an element of the DNA damage apoptosis signaling cascade. In an embodiment, a second Cas9 molecule/gRNA molecule complex is used to deliver a payload comprising a second compound that inhibits growth or cell division, or promotes apoptosis, e.g., an element of the DNA damage apoptosis signaling cascade. The Cas9 molecule/gRNA molecule complex that delivers the second payload can comprise an eiCas9 molecule or an eaCas9 molecule. An additional, e.g., third or fourth, Cas9 molecule/gRNA molecule complex, can be used to deliver additional payload, e.g., an additional compound that inhibits growth or cell division, or promotes apoptosis, e.g., an additional element of the DNA damage apoptosis signaling cascade promote.


In an embodiment, the Cas9 molecule/gRNA molecule complex delivers a payload comprising a compound that inhibits growth or cell division, or promotes apoptosis, e.g., an element of the DNA damage apoptosis signaling cascade, but does not cleave the target nucleic acid. While not wishing to be bound by theory is it believed that endogenous cellular elements, e.g., elements of the DNA damage apoptosis signaling cascade promote apoptosis in these embodiments.


Exemplary compounds that inhibit growth or cell division, or promote apoptosis, e.g., an element of the DNA damage apoptosis signaling cascade, are described herein, e.g., in Table









TABLE XII-1







ATM kinases (double-strand breaks)


ATR kinases (single-strand breaks)


RF-C related protein (RAD17)


The 9-1-1 Complex: RAD1, RAD9, and HUS1


Checkpoint proteins CHK1, CHK2


P53


ZIP Kinase (ZIPK)


Fast Death-Domain Associated Protein XX (DAXX)


Promyelocytic leukemia protein (PML)


Apoptosis-inducing factor (AIF)


Caspase-activated DNAse (CAD) (in the absence of its inhibitor ICAD)









In an embodiment, a Cas9 molecule/gRNA molecule complex targets a sequence that includes or is near the breakpoint of a rearrangement, e.g., a translocation, inversion, insertion, or deletion. In an embodiment, the rearrangement confers unwanted properties, e.g., unwanted proliferation, on the cell. In an embodiment, the cell harboring the rearrangement is a cancer cell. In an embodiment, the rearrangement comprises a kinase gene and results in unwanted, increased, or constitutive expression of the kinase activity. In an embodiment, the rearrangement disrupts the expression of a tumor suppressor.


In an embodiment, the Cas9 molecule/gRNA molecule complex:


specifically targets, and e.g., cleaves, the genome of a cell comprising a rearrangement, e.g., by targeting a mutation, e.g., a breakpoint or junction of a rearrangement, or


targets, e.g., for cleavage or payload delivery, a nucleotide sequence within 200, 100, 150, 100, 50, 25, 10, or 5 nucleotides of a mutation, e.g., a rearrangement breakpoint.


The invention includes a method of manipulating a cell comprising a genomic signature, comprising:


administering a Cas9 molecule/gRNA molecule complex that targets said genomic signature, thereby manipulating said cell.


In an embodiment, manipulating comprises inhibiting the growth or division of, or killing, said cell.


In an embodiment, said cell is a cancer cell or cell having a viral infection.


In an embodiment, the method comprises treating a subject, e.g., a human subject, for a disorder characterized by a cell having said genomic signature, e.g., a cancer or a viral infection.


In an embodiment, a Cas9 molecule/gRNA molecule complex disrupts a rearrangement, e.g., by introduction of a stop codon from a template nucleic acid, e.g., a stop codon is inserted into a fusion protein, e.g., a fusion protein comprising kinase activity.


The invention includes a method of treating a cancer having a translocation of a kinase gene to a non-kinase gene, which places the kinase domain under the control of the non-kinase gene control region comprising:


administering a Cas9 molecule/gRNA molecule complex that targets the translocation. In an embodiment, the control region, e.g., the promoter, or the coding sequence, of the kinase translocation, is edited to reduce expression.


XIII. Combination Therapy


The Cas9 molecules, gRNA molecules, and in particular, Cas9 molecule/gRNA molecule complexes, can be used in combination with a second therapeutic agent, e.g., a cancer drug. In an embodiment, the second therapeutic agent (e.g., a cancer drug) and the Cas9 molecule, gRNA molecule, and in particular, Cas9 molecule/gRNA molecule complex target different (e.g., non-overlapping) pathways. In an embodiment, the second therapeutic agent (e.g., a cancer drug) and the Cas9 molecule, gRNA molecule, and in particular, Cas9 molecule/gRNA molecule complex target a same or overlapping pathway.

    • Exemplary combination therapies include, e.g.:
    • mTOR inhibitors (e.g., Temsirolimus (Torisel®) or Everolimus (Afinitor®)) together with a AKT-specific Cas9/gRNA molecule;
    • Tyrosine kinase inhibitors such as Imatinib mesylate (Gleevec®); Dasatinib (Sprycel®); Bosutinib (Bosulif®); Trastuzumab (Herceptin®); Pertuzumab (Perjetar™); Lapatinib (Tykerb®); Gefitinib (Lressa®); Erlotinib (Tarceva®) together with a HDAC-specific Cas9/gRNA molecule; and
    • Any chemotherapeutic agent together with one or more Cas9/gRNAs against multidrug resistance genes such as MDR1 gene.


      XIV. Treatment of Genetic Disorder, e.g., Duchenne Muscular Dystrophy (DMD)


In another aspect, the invention features, a method of altering a cell, e.g., reducing or abolishing the effect of a genetic signature, e.g., a stop codon, e.g., a premature stop codon. The method comprises contacting said cell with:


a Cas9 molecule/gRNA molecule complex that cleaves at or upstream from the genetic signature, e.g., a premature stop codon,


thereby altering the cell.


While not wishing to be bound by theory it is believed that, in an embodiment, cleavage and subsequent exonuclease activity, and non-homologous end joining results in an altered sequence in which the genetic signature, e.g., a premature stop codon is eliminated, e.g., by being placed in a different frame. In an embodiment, the same series of events restores the proper reading frame to the sequence that follows the signature, e.g., premature stop codon.


When the method is carried out to correct a frameshift mutation in order to remove a premature stop codon, repair can be carried out at various sites in the DNA. One may direct cleavage at the mutation, thereby correcting the frameshift entirely and returning the protein to its wild-type (or nearly wild-type) sequence. One may also direct cleavage at or near the premature stop codon, so that all (or nearly all) amino acids of the protein C-terminal of the codon where repair was effected are wild-type. In the latter case, the resulting protein may have one or more frame shifted amino acids between the mutation and the repair site; however the protein may still be functional because it is full-length and has wild-type sequence across most of its length.


A genetic signature is a particular DNA sequence at a particular portion of the genome, that causes a phenotype (such as a genetic disease or a symptom thereof). For instance, the genetic signature may be a premature stop codon that prevents expression of a protein. In this scenario, the premature stop codon can arise from a mutation that directly creates a stop codon, or from a mutation that causes a frameshift leading to a premature stop codon being formed downstream. A genetic signature may also be a point mutation that alters the identity of an important amino acid in a protein, disrupting the protein's function.


In an embodiment, the Cas9 molecule/gRNA molecule complex mediates a double stranded break in said target nucleic acid.


In an embodiment, the genetic signature, e.g., a premature stop codon, results from a point mutation, an insertion, a deletion, or a rearrangement. In an embodiment, a mutation causes a frameshift, resulting in a genetic signature. e.g., a premature stop codon downstream of the mutation.


In an embodiment, the premature stop codon is within the target nucleic acid. In an embodiment, the target nucleic acid is upstream of the premature stop codon. The mutation may be upstream of the target nucleic acid, within the target nucleic acid, or downstream of the target nucleic acid.


In an embodiment the double stranded break is within 500, 200, 100, 50, 30, 20, 10, 5, or 2 nucleotides of the mutation. In an embodiment, the double stranded break is within 500, 200, 100, 50, 30, 20, 10, 5, or 2 nucleotides of the genetic signature, e.g., a premature stop codon.


In an embodiment, the Cas9 molecule/gRNA molecule complex mediates exonuclease digestion of the target nucleic acid. In an embodiment, the Cas9 molecule/gRNA molecule complex removes 1, 2, 3, 4, or 5 nucleotides at the double stranded break.


In an embodiment, the double stranded break is resolved by non-homologous end joining.


In an embodiment the mutation and/or genetic signature, e.g., premature stop codon is in the dystrophin gene, e.g., in exon 51, or in the intron preceding or following exon 51. The premature stop codon may also be caused by a mutation in the dystrophin gene at one or more of codons 54, 645, 773, 3335, and 3340. In an embodiment, the premature stop codon in the dystrophin gene results from a deletion of codons 2305 through 2366.


In an embodiment, contacting the cell with a Cas9 molecule/gRNA molecule complex comprises contacting the cell with a nucleic acid encoding a Cas9 molecule. In an embodiment, contacting the cell with a Cas9 molecule/gRNA molecule complex comprises transfecting the cell with a nucleic acid, e.g., a plasmid, or using a viral vector such as adeno-associated virus (AAV).


In an embodiment, the method results in increased levels of the protein in which the genetic signature, e.g., a premature stop codon, was previously located. For instance, protein levels (e.g., dystrophin levels) may be increased by at least 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30% in a cell or in a tissue. In an embodiment, the method results in increased levels of the mRNA in which the premature stop codon was previously located, for instance by preventing the mRNA from undergoing nonsense-mediated mRNA decay.


In an embodiment, one or more of the target nucleic acid, the genetic signature, e.g., premature stop codon, and the mutation are located in the dystrophin gene (which is mutated in DMD). One or more of the target nucleic acid, the genetic signature, e.g., premature stop codon, and the mutation may also be located in the COL7A1 gene (mutated in type VII-associated dystrophic epidermolysis bullosa), the FKTN gene (mutated in Fukuyama congenital muscular dystrophy), the dysferlin gene (mutated in limb-girdle muscular dystrophy type 2B), the CFTR gene (mutated in cystic fibrosis), HEXA (mutated in Tay-Sachs disease), the IDS gene (mutated in Hunter syndrome), the FVIII gene (mutated in hemophilia), the IDUA gene (mutated in Hurler syndrome), the PPT1 gene (mutated in infantile neuronal ceroid lipofuscinosis), a tumor suppressor such as the ATM gene (mutated in cancers like gliomas and B-Cell Chronic Lymphocytic Leukemia), RP2 (mutated in X-linked retinitis pigmentosa), the CTNS gene (mutated in nephropathic cystinosis), and the AVPR2 gene (mutated in Congenital nephrogenic diabetes insipidus).


In an embodiment, the method is performed in cultured cells. In an embodiment, the method further comprises administering the cell to a patient. The cell may be, for example, an induced pluripotent stem cell, a bone marrow derived progenitor, a skeletal muscle progenitor, a CD133+ cell, a mesoangioblast, or a MyoD-transduced dermal fibroblast.


In an embodiment, the method comprises contacting the cell with a template nucleic acid under conditions that allow for homology-directed repair between the target nucleic acid and the template nucleic acid to correct the mutation or the premature stop codon.


In another aspect, the invention features a method of treating a human subject having a disorder associated with a genetic signature, e.g., premature stop codon, e.g., DMD, comprising providing to the human subject:


1) a Cas9 molecule/gRNA molecule complex that cleaves at or upstream from the premature stop codon or


2) a cell that has been contacted with such complex,


thereby treating the subject.


In an embodiment, the Cas9 molecule/gRNA molecule complex mediates a double stranded break in said target nucleic acid.


In an embodiment, genetic signature, e.g., premature stop codon results from a point mutation, an insertion, a deletion, or a rearrangement. In an embodiment, a mutation causes a frameshift, resulting in a premature stop codon downstream of the mutation.


In an embodiment the double stranded break is within 500, 200, 100, 50, 30, 20, 10, 5, or 2 nucleotides of the mutation. In an embodiment the double stranded break is within 500, 200, 100, 50, 30, 20, 10, 5, or 2 nucleotides of the premature stop codon.


In an embodiment, the genetic signature, e.g., premature stop codon is within the target nucleic acid of the Cas9 molecule/gRNA molecule complex. In an embodiment, the target nucleic acid is upstream of the genetic signature, e.g., premature stop codon. The mutation may be upstream of the target nucleic acid, within the target nucleic acid, or downstream of the target nucleic acid.


In an embodiment, the Cas9 molecule/gRNA molecule complex mediates exonuclease digestion of the target nucleic acid. In an embodiment, the Cas9 molecule/gRNA molecule complex removes 1, 2, 3, 4, or 5 nucleotides at the double stranded break.


In an embodiment, the double stranded break is resolved by non-homologous end joining.


In an embodiment, the mutation and/or genetic signature, e.g., premature stop codon is in the dystrophin gene, e.g., in exon 51, or in the intron preceding or following exon 51. The premature stop codon may also be caused by a mutation in the dystrophin gene at one or more of codons 54, 645, 773, 3335, and 3340. In an embodiment, the premature stop codon in the dystrophin gene results from a deletion of codons 2305 through 2366.


In an embodiment, contacting the cell with a Cas9 molecule/gRNA molecule complex comprises contacting the cell with a nucleic acid encoding a Cas9 molecule. In an embodiment, contacting the cell with a Cas9 molecule/gRNA molecule complex comprises transfecting the cell with a nucleic acid, e.g., a plasmid, or using a viral vector such as adeno-associated virus (AAV).


In an embodiment, the method results in increased levels of the protein in which the genetic signature, e.g., premature stop codon was previously located. For instance, protein levels (e.g., dystrophin levels) may be increased by at least 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30% in a cell or in a tissue. In an embodiment, the method results in increased levels of the mRNA in which the premature stop codon was previously located, for instance by preventing the mRNA from undergoing nonsense-mediated mRNA decay.


In an embodiment, one or more of the target nucleic acid, the genetic signature, e.g., premature stop codon, and the mutation are located in the dystrophin gene (which is mutated in DMD). One or more of the target nucleic acid, the genetic signature, e.g., premature stop codon, and the mutation may also be located in the COL7A1 gene (mutated in type VII-associated dystrophic epidermolysis bullosa), the FKTN gene (mutated in Fukuyama congenital muscular dystrophy), the dysferlin gene (mutated in limb-girdle muscular dystrophy type 2B), the CFTR gene (mutated in cystic fibrosis), HEXA (mutated in Tay-Sachs disease), the IDS gene (mutated in Hunter syndrome), the FVIII gene (mutated in hemophilia), the IDUA gene (mutated in Hurler syndrome), the PPT1 gene (mutated in infantile neuronal ceroid lipofuscinosis), a tumor suppressor such as the ATM gene (mutated in cancers like gliomas and B-Cell Chronic Lymphocytic Leukemia), RP2 (mutated in X-linked retinitis pigmentosa), the CTNS gene (mutated in nephropathic cystinosis), and the AVPR2 gene (mutated in Congenital nephrogenic diabetes insipidus).


In an embodiment, the method is performed in cultured cells. In an embodiment, the method further comprises administering the cell to a patient. The cell may be, for example, an induced pluripotent stem cell, a bone marrow derived progenitor, a skeletal muscle progenitor, a CD133+ cell, a mesoangioblast, or a MyoD-transduced dermal fibroblast.


In an embodiment, the method comprises contacting the cell with a template nucleic acid under conditions that allow for homology-directed repair between the target nucleic acid and the template nucleic acid to correct the mutation or the premature stop codon.


In an embodiment, the subject has a disorder selected from Duchenne Muscular Dystrophy (DMD), collagen type VII-associated dystrophic epidermolysis bullosa, Fukuyama congenital muscular dystrophy, and limb-girdle muscular dystrophy type 2B, cystic fibrosis, lysosomal storage disorders (such as Tay-Sachs disease, Hunter syndrome, and nephropathic cystinosis), hemophilia, Hurler syndrome, infantile neuronal ceroid lipofuscinosis, X-linked retinitis pigmentosa (RP2), cancers (such as gliomas and B-Cell Chronic Lymphocytic Leukemia), and Congenital nephrogenic diabetes insipidus.


XV. Treatment of Disorders Characterized by Lack of Mature Specialized Cells. e.g., Impaired Hearing, with Loss of Hair Cells, Supporting Cells, or Spiral Ganglion Neurons; or for Diabetes, with Loss of Beta Islet Cells


In another aspect, the invention features, a method of altering a cell, e.g., to promote the development of other mature specialized cells, e.g, in regeneration therapy. For example, proliferation genes can be upregulated and/or checkpoint inhibitors can be inhibited, e.g., to drive down one or more differentiation pathways.


In an embodiment, the method includes induction of proliferation and specified lineage maturation.


In an embodiment, the method comprises, e.g., for restoration or improvement of hearing, contacting said cell with:


a Cas9 molecule/gRNA molecule complex that up-regulates a gene that promotes the development of hair cells, or down-regulates a gene that inhibits the development of hair cells thereby altering the cell.


In an embodiment, the Cas9 molecule/gRNA molecule delivers a payload that up-regulates a gene that promotes hair cell development.


In an embodiment, the Cas9 molecule/gRNA molecule delivers a payload that down-regulates a gene that inhibits hair growth.


In an embodiment, the Cas9 molecule/gRNA molecule complex edits the genome of a cell to up-regulate a gene that promotes hair growth. In an embodiment, a template nucleic acid is used to effect a Cas9 molecule/gRNA molecule complex alteration to the genome that up-regulates a gene that promotes hair growth.


In an embodiment, the Cas9 molecule/gRNA molecule complex edits the genome of a cell to down-regulate a gene that inhibits hair growth. In an embodiment, a template nucleic acid is used to effect a Cas9 molecule/gRNA molecule complex alteration to the genome that down-regulates a gene that promotes hair growth.


In an embodiment, said cell is an iPS cell, a native hair cell progenitor, or a mature hair cell.


In an embodiment, the Cas9 molecule/gRNA molecule and modifies expression of a gene, e.g., by modifying the structure of the gene (e.g., by editing the genome) or by delivery of a payload that modulates a gene. In an embodiment, the gene is a transcription factor or other regulatory gene.


In an embodiment, for hair cell or other mature cell regeneration, the method includes one or more or all of the following:


contacting the cell with a Cas9 molecule/gRNA molecule complex that results in up-regulation one or more of the following for cell proliferation: c-Myc, GATA3, Oct4, Sox2, Wnt, TCF3;


contacting the cell with a Cas9 molecule/gRNA molecule complex that results in downregulation one or more of the following for check point: BCL2, BMP, Hes1, Hes5, Notch, p27. Prox 1, TGF; and


contacting the cell with a Cas9 molecule/gRNA molecule complex that results in turning on a maturation pathway. For hair cells this would include one or more of the following: Atoh1 (Math1), Barh1L Gfil, Myo7a, p63, PAX2, PAX8, Pou4f3 and for neurons would include one or more of the following: NEFH, Nemodl, Nemogl, POU4FL


In an embodiment, the method comprises generation of inner ear hair cells, outer ear hair cells, spiral ganglion neurons, and ear supporting cells.


In an embodiment, one or more growth factors can be modulated, e.g., upregulated, e.g., TPO can be upregulated for production of platelets and GCSF can be upregulated for production of neutrophils.


In another aspect, the invention provides altered cell described herein, e.g., in this Section XV.


In another aspect, the invention features a method of treating impaired hearing. The method comprises administering to said subject, an altered cell described herein, e.g., in this section XV. In an embodiment, the cell is autologous. In an embodiment, the cell is allogeneic. In an embodiment, the cell is xenogeneic.


In another aspect, the invention features a method of treating subject, e.g., for impaired hearing. The method comprises administering to said subject:


a Cas9 molecule/gRNA molecule complex that up-regulates a gene that promotes the growth of hair, or down-regulates a gene that inhibits the growth of hair thereby altering the cell.


In an embodiment, the Cas9 molecule/gRNA molecule delivers a payload that up-regulates a gene that promotes hair growth.


In an embodiment, the Cas9 molecule/gRNA molecule delivers a payload that down-regulates a gene that inhibits hair growth.


In an embodiment, the Cas9 molecule/gRNA molecule complex edits the genome of a cell to up-regulate a gene that promotes hair growth. In an embodiment, a template nucleic acid is used to effect a Cas9 molecule/gRNA molecule complex alteration to the genome that up-regulates a gene that promotes hair growth.


In an embodiment, the Cas9 molecule/gRNA molecule complex edits the genome of a cell to down-regulate a gene that inhibits hair growth. In an embodiment, a template nucleic acid is used to effect a Cas9 molecule/gRNA molecule complex alteration to the genome that down-regulates a gene that promotes hair growth.


In an embodiment, the Cas9 molecule/gRNA molecule and modifies expression of a gene, e.g., by modifying the structure of the gene (e.g., by editing the genome) or by delivery of a payload that modulates a gene. In an embodiment, the gene is a transcription factor or other regulatory gene.


In an embodiment, the method includes one or more or all of the following:


administering a Cas9 molecule/gRNA molecule complex that results in up-regulation one or more of the following: c-Myc, GATA3, Oct4, Sox2, Wnt, or TCF3:


administering a Cas9 molecule/gRNA molecule complex that results in turning on a maturation pathway. For hair cells this would include one or more of the following: Atoh1 (Math1), Barhl1, Gfi1, Myo7a, p63, PAX2, PAX8, or Pou4f3 and for neurons would include one or more of the following: NEFH. Neurodl, Neurogl, or POU4FL


XVI. Governing gRNA Molecules and their Use to Limit the Activity of a Cas9 System


As discussed herein, methods and compositions that use, or include, a nucleic acid, e.g., DNA, that encodes a Cas9 molecule or a gRNA molecule, can, in addition, use or include a governing gRNA molecule. The governing gRNA molecule can complex with the Cas9 molecule to inactivate or silence a component of the system, e.g., the nucleic acid that encodes the Cas9 molecule or the nucleic acid that encodes the gRNA molecule. In either case, the governing gRNA, e.g., a Cas9-targeting gRNA molecule, or a gRNA targeting gRNA molecule, limits the effect of the Cas9/gRNA complex mediated gene targeting, and can place temporal limits on activity or reduce off-target activity. Governing gRNA molecules can act as to inhibit, e.g., entirely or substantially inhibit, the production of a component of the Cas9 system and thereby limit, or govern, its activity.


Typically a nucleic acid sequence encoding a governing gRNA molecule, e.g., a Cas9-targeting gRNA molecule, is under the control of a different control region, e.g., promoter, than is the component it negatively modulates, e.g., a nucleic acid encoding the Cas9 molecule. In an embodiment, different refers to simply not being under the control of one control region, e.g., promoter, that is functionally coupled to both controlled sequences. In an embodiment, different refers to different in kind or type. For example, the sequence encoding a governing gRNA molecule, e.g., a Cas9-targeting gRNA molecule, is under the control of a control region, e.g., a promoter, that has a lower level of expression, or is expressed later than the sequence which encodes the component it negatively modulates, e.g., a nucleic acid encoding the Cas9 molecule.


By way of example a sequence that encodes a governing gRNA molecule, e.g., a Cas9-targeting gRNA molecule, can be under the control of a control region (e.g., a promoter) described herein, e.g., human U6 small nuclear promoter, or human H1 promoter. In an embodiment, a sequence that encodes the component it negatively regulates, e.g. a nucleic acid encoding the Cas9 molecule, can be under the control of a control region (e.g., a promoter) described herein, e.g., human U6 small nuclear promoter, human H1 promoter, or a Pol11 promoter, e.g., a CMV promoter, a CAGGS promoter, or a CB promoter.


EXAMPLES

The following Examples are merely illustrative and are not intended to limit the scope or content of the invention in any way.


Example 1: In Silica Design of Governing gRNA Sequences Targeting S. pyogenes and S. aureus Cas9

Governing guide RNAs (gRNAs) targeting S. pyogenes and S. aureus Cas9s were identified using a DNA sequence searching algorithm. In addition to identifying potential gRNA sites adjacent to PAM sequences, the software also identifies all PAM adjacent sequences that differ by 1, 2, 3 or more nucleotides from the selected gRNA sites in the human genome. Genomic DNA sequence for each Cas9 gene was obtained from the UCSC Genome browser and sequences were screened for repeat elements using the publically available RepeatMasker program. RepeatMasker searches input DNA sequences for repeated elements and regions of low complexity. The output is a detailed annotation of the repeats present in a given query sequence.


Following identification, governing gRNAs were ranked into tiers based on their cleavage position within the Cas9 coding sequence, their orthogonality and presence of a 5′ G (based on identification of close matches in the human genome containing a PAM). Orthogonality refers to the number of sequences in the human genome that contain a minimum number of mismatches to the target sequence. A “high level of orthogonality” or “good orthogonality” may, for example, refer to 20-mer gRNAs that have no identical sequences in the human genome besides the intended target, nor any sequences that contain one or two mismatches in the target sequence. Targeting domains with good orthogonality are selected to minimize off-target DNA cleavage.


Tier 1 includes all gRNAs that target the first 500 nucleotides of coding sequence of Cas9, have good orthogonality, and begin with a 5′ G. Tier 2 includes all gRNAs that target the first 500 nucleotides of coding sequence of Cas9, have good orthogonality, but don't begin with a 5′ G. Tier 3 includes all gRNAs that target the first 500 nucleotides of coding sequence of Cas9, have poor orthogonality, and begin with a 5′ G. Tier 4 includes all gRNAs that target the first 500 nucleotides of coding sequence of Cas9, have poor orthogonality, but don't begin with a 5′ G. Tier 5 includes all gRNAs that target the remaining coding sequence. In the case of S. aureus, there is a 6th tier that includes all gRNAs whose targets have a non-optimal PAM of NNGRRV.


For all S. pyogenes targets, 17-mer, or 20-mer gRNAs were designed. For all S. aureus targets, 20-mer gRNAs were designed, gRNAs were identified for both single-gRNA nuclease cleavage and for a dual-gRNA paired “nickase” strategy. The designed governing gRNAs are listed in Tables E-1 to E-12.












TABLE E-1







Exemplary guide
Group A
Group B
Group A guides can


RNA pairs for S.


be paired with any



pyogenes (SP)



from Group B.


nickase






antiSPCas9-2,
antiSPCas9-3,




antiSPCas9-18
antiSPCas9-24




Group C
Group D
Group C guides can





be paired with any





from Group D.



antiSPCas9-7,
antiSPCas9-30,




antiSPCas9-70
antiSPCas9-72


















TABLE E-2








First 500 bp of coding sequence  




downstream of start codon, good




orthogonality, starts with G













S. pyogenes



Target
SEQ


1st Tier
DNA

Site
ID


gRNA Name
Strand
Targeting Domain
Length
NO





antiSPCas9-1

GCCCUCCUAUUCGAUAG
17
304





antiSPCas9-2

GCCUGAAACGAACCGCU
17
305





antiSPCas9-3
+
GCCACUAUCGAAUAGGA
17
306





antiSPCas9-4

GCACUAAUUCCGUUGGA
17
307





antiSPCas9-5

GAACGGCACCCCAUCUU
17
308





antiSPCas9-6

GGUGCCCUCCUAUUCGAUAG
20
309





antiSPCas9-7

GCCCAUAUGAUAAAGUUCCG
20
310





antiSPCas9-8
+
GCCCACGGAACUUUAUCAUA
20
311


















TABLE E-3








First 500 bp of coding sequence 




downstream of start codon, good




orthogonality, does not start




with G













S. pyogenes



Target
SEQ


2nd Tier
DNA

Site
ID


gRN AName
Strand
Targeting Domain
Length
NO





antiSPCas9-9
+
UCCGAGCGGUUCGUUUC
17
312





antiSPCas9-10

AACGAACCGCUCGGAGA
17
313





antiSPCas9-11
+
CGCCACUAUCGAAUAGG
17
314





antiSPCas9-12

AUCGGCACUAAUUCCGU
17
315





antiSPCas9-13
+
UUUCGCCACUAUCGAAU
17
316





antiSPCas9-14

AUUCGAUAGUGGCGAAA
17
317





antiSPCas9-15

UAGUGGCGAAACGGCAG
17
318





antiSPCas9-16
+
CCGUUUCGCCACUAUCGAAU
20
319





antiSPCas9-17

GACAUCGGCACUAAUUCCGU
20
320





antiSPCas9-18

CUCGCCUGAAACGAACCGCU
20
321





antiSPCas9-19
+
UUCUCCGAGCGGUUCGUUUC
20
322





antiSPCas9-20

UGAAACGAACCGCUCGGAGA
20
323





antiSPCas9-21
+
UUUCGCCACUAUCGAAUAGG
20
324





antiSPCas9-22

UCGGCACUAAUUCCGUUGGA
20
325





antiSPCas9-23

CGAUAGUGGCGAAACGGCAG
20
326





antiSPCas9-24
+
UUCGCCACUAUCGAAUAGGA
20
327





antiSPCas9-25
+
ACGUGUAUACCUUCUCCGAG
20
328





antiSPCas9-26

CGGCACUAAUUCCGUUGGAU
20
329





antiSPCas9-27

CCUAUUCGAUAGUGGCGAAA
20
330





antiSPCas9-28

CCCAUAUGAUAAAGUUCCGU
20
331


















TABLE E-4








First 500 bp of coding sequence 




downstream of start codon, poor




orthogonality, starts with G













S. pyogenes



Target
SEQ


3rd Tier
DNA

Site
ID


gRNA Name
Strand
Targeting Domain
Length
NO





antiSPCas9-29
+
GAAAGAAUCGUCAACUU
17
332





antiSPCas9-30
+
GAGCCAAGUAGAUUAACCUC
20
333





antiSPCas9-31

GAUAAAAAGUAUUCUAU
17
334





antiSPCas9-32
+
GAUUCUUUUUAAUCGAAUGA
20
335





antiSPCas9-33
+
GGAAGGACUCUUCCAAA
17
336





antiSPCas9-34

GGACCUGAGGUUAAUCUACU
20
337





antiSPCas9-35

GGGCACUUUCUCAUUGA
17
338





antiSPCas9-36
+
GGUUAUGACAGCCCAUCCAA
20
339





antiSPCas9-37
+
GUCCUCUUCGACAAGGA
17
340





antiSPCas9-38

GUCCUUCCUUGUCGAAG
17
341





antiSPCas9-39
+
GUUUCUUGUCCUCUUCGACA
20
342


















TABLE E-5








First 500 bp of coding sequence




downstream of start codon, poor,




orthogonality, does not start with G













S. pyogenes



Target
SEQ


4th Tier
Name
Trageting
Site
ID


gRNA Name
Strand
Domain
Lenght
No





antiSPCas9-40

AAACAUAGUAGAUGAGG
17
343





antiSPCas9-41
+
AAAGAAAGAAUCGUCAACUU
20
344





antiSPCas9-42

AAAGAAAUUUAAGGUGU
17
345





antiSPCas9-43
+
AACACCUUAAAUUUCUUUGA
20
346





antiSPCas9-44

AAGAAAUUUAAGGUGUU
17
347





antiSPCas9-45

AAGAGGACAAGAAACAUGAA
20
348





antiSPCas9-46

AAUUUUUAGCAAUGAGA
17
349





antiSPCas9-47
+
ACCUUAAAUUUCUUUGA
17
350





antiSPCas9-48

ACCUUCAAAGAAAUUUA
17
351





antiSPCas9-49
+
ACGGAACUUUAUCAUAU
17
352





antiSPCas9-50
+
ACUAUGUUUCCAAAGAU
17
353





antiSPCas9-51

AGAAAUUUAAGGUGUUG
17
354





antiSPCas9-52

AGAAAUUUUUAGCAAUGAGA
20
355





antiSPCas9-53

AGAGUCCUUCCUUGUCGAAG
20
356





antiSPCas9-54

AGGACAAGAAACAUGAA
17
357





antiSPCas9-55
+
AGGUACUUUGUAUUCAU
17
358





antiSPCas9-56

AGUACCUUCAAAGAAAUUUA
20
359





antiSPCas9-57
+
AGUCAACUAGCUUUUUUCUG
20
360





antiSPCas9-58

AGUUGACUCAACUGAUAAAG
20
361





antiSPCas9-59

AUAUGAUAAAGUUCCGU
17
362





antiSPCas9-60
+
AUCUACUAUGUUUCCAAAGA
20
363





antiSPCas9-61

AUGGAUAAAAAGUAUUCUAU
20
364





antiSPCas9-62

AUUAAAAAGAAUCUUAU
17
365





antiSPCas9-63

CAAAGAAAUUUAAGGUGUUG
20
366





antiSPCas9-64
+
CAACUAGCUUUUUUCUG
17
367





antiSPCas9-65

CAACUGAUAAAGCGGACCUG
20
368





antiSPCas9-66
+
CAAGGAAGGACUCUUCCAAA
20
369





antiSPCas9-67
+
CAAUGAGAAAGUGCCCA
17
370





antiSPCas9-68
+
CACGGAACUUUAUCAUA
17
371





antiSPCas9-69

CACUAAUUCCGUUGGAU
17
372





antiSPCas9-70

CAUAUGAUAAAGUUCCG
17
373





antiSPCas9-71

CAUGAACGGCACCCCAUCUU
20
374





antiSPCas9-72
+
CCAAGUAGAUUAACCUC
17
375





antiSPCas9-73
+
CCCACGGAACUUUAUCAUAU
20
376





antiSPCas9-74

CCGUGGGCACUUUCUCAUUG
20
377





antiSPCas9-75
+
CCUCAAUGAGAAAGUGCCCA
20
378





antiSPCas9-76

CCUGAGGUUAAUCUACU
17
379





antiSPCas9-77

CGAUUCUUUCUUUCACCGUU
20
380





antiSPCas9-78

CGUGGGCACUUUCUCAUUGA
20
381





antiSPCas9-79
+
CUACUAUGUUUCCAAAGAUG
20
382





antiSPCas9-80
+
CUAUGUUUCCAAAGAUG
17
383





antiSPCas9-81
+
CUGAGGUGAUAAAUCGU
17
384





antiSPCas9-82

CUGAUAAAGCGGACCUG
17
385





antiSPCas9-83
+
CUUGUAAGUAACAUAUU
17
386





antiSPCas9-84
+
CUUGUCCUCUUCGACAAGGA
20
387





antiSPCas9-85

CUUUGGAAACAUAGUAGAUG
20
388





antiSPCas9-86
+
UACUAUGUUUCCAAAGA
17
389





antiSPCas9-87
+
UAUGACAGCCCAUCCAA
17
390





antiSPCas9-88

UAUUCUAUUGGUUUAGACAU
20
391





antiSPCas9-89

UCAAAGAAAUUUAAGGUGUU
20
392





antiSPCas9-90

UCGAUUAAAAAGAAUCUUAU
20
393





antiSPCas9-91
+
UCUACUAUGUUUCCAAAGAU
20
394





antiSPCas9-92

UCUAUUGGUUUAGACAU
17
395





antiSPCas9-93
+
UCUUGUCCUCUUCGACA
17
396





antiSPCas9-94
+
UCUUUUUAAUCGAAUGA
17
397





antiSPCas9-95
+
UGAAGGUACUUUGUAUUCAU
20
398





antiSPCas9-96

UGACUCAACUGAUAAAG
17
399





antiSPCas9-97
+
UGAGGUGAUAAAUCGUU
17
400





antiSPCas9-98

UGGAAACAUAGUAGAUG
17
401





antiSPCas9-99

UGGAAACAUAGUAGAUGAGG
20
402





antiSPCas9-100

UGGGCACUUUCUCAUUG
17
403





antiSPCas9-101
+
UGUAUACCUUCUCCGAG
17
404





antiSPCas9-102

UUCAAAGAAAUUUAAGGUGU
20
405





antiSPCas9-103
+
UUCUGAGGUGAUAAAUCGUU
20
406





antiSPCas9-104

UUCUUUCUUUCACCGUU
17
407





antiSPCas9-105
+
UUUCUGAGGUGAUAAAUCGU
20
408





antiSPCas9-106
+
UUUCUUGUAAGUAACAUAUU
20
409


















TABLE E-6








Rest of gene















Target
SEQ


5th Tier
Name

Site
ID


gRNA Name
Strand
Trageting Domain
Lenght
No














antiSPCas9-107

GCGUCGCUAUACGGGCU
17
410





antiSPCas9-108

GGCGUCGCUAUACGGGC
17
411





antiSPCas9-109

UCGAAAAACGGGUACGC
17
412





antiSPCas9-110

CGCUCGGAUAAGAACCG
17
413





antiSPCas9-111

CGUGGCUAUACGGGCUG
17
414





antiSPCas9-112
+
GAUAAGCGUCGUGCGCA
17
415





antiSPCas9-113
+
CGGGUAAUUGUGCGAUC
17
416





antiSPCas9-114

GAUCGCGAAAAGCGAAC
17
417





antiSPCas9-115

GCGACGGCUUCGCCAAU
17
418





antiSPCas9-116
+
UAACCUUAUCGUCGAAC
17
419





antiSPCas9-117
+
AGCGGAUAACGGCGCCU
17
420





antiSPCas9-118

AAGAGGCGUCGCUAUAC
17
421





antiSPCas9-119
+
AAGCGUCGUGCGCAUGG
17
422





antiSPCas9-120

CGGAUGUUGGCUAGCGC
17
423





antiSPCas9-121

CGAUCUCGACAAUCUAC
17
424





antiSPCas9-122
+
UCGACAUCCGAGUUGUC
17
425





antiSPCas9-123
+
GAGUGCGGUCCCUACGA
17
426





antiSPCas9-124

CGCGGAAACUUAUCAAC
17
427





antiSPCas9-125

AUCUUAAUGCCGUCGUA
17
428





antiSPCas9-126

CUCAAUCGUUCAUCGAG
17
429





antiSPCas9-127
+
UCCGCCUGCUCACGUAU
17
430





antiSPCas9-128

UCACCUGUUCGACGAUA
17
431





antiSPCas9-129

GCUCAUCGCUCGUAAAA
17
432





antiSPCas9-130

GCUCGGAUAAGAACCGA
17
433





antiSPCas9-131

UAUCUUAAUGCCGUCGU
17
434





antiSPCas9-132

CGGCGGAGCGAGUCAAG
17
435





antiSPCas9-133

UCGCGGAAACUUAUCAA
17
436





antiSPCas9-134

CGAUUUAAUGCGUCACU
17
437





antiSPCas9-135

UCGCUCGUAAAAAGGAC
17
438





antiSPCas9-136

GACCCGAAAAAGUACGG
17
439





antiSPCas9-137
+
GCCGUCGGGAUUUAGAG
17
440





antiSPCas9-138

AAAGAGGCGUCGCUAUA
17
441





antiSPCas9-139

CGCAUACCUUACUAUGU
17
442





antiSPCas9-140
+
CGAACCGAGAGUUCCCU
17
443





antiSPCas9-141

GGAACUCUCGGUUCGCA
17
444





antiSPCas9-142

UGGCCGAAAACGGAUGU
17
445





antiSPCas9-143
+
CGCGUCUAGCACCUCCU
17
446





antiSPCas9-144
+
UCACCGUCGCGAAGUCC
17
447





antiSPCas9-145

UAUUAAACGUCAGCUCG
17
448





antiSPCas9-146

CGCUCGUAAAAAGGACU
17
449





antiSPCas9-147
+
UUCCGCCUGCUCACGUA
17
450





antiSPCas9-148
+
CUAGUGAGAGCGCUAUA
17
451





antiSPCas9-149
+
CGUCGUAAUCAGAUAAA
17
452





antiSPCas9-150

CUGAUCGCACAAUUACC
17
453





antiSPCas9-151

UCGCAUACCUUACUAUG
17
454





antiSPCas9-152

CGCCGGAGAGCUUCAAA
17
455





antiSPCas9-153

GCUGGGGACGAUUGUCG
17
456





antiSPCas9-154
+
CGCCUCAAGGAAGUCGA
17
457





antiSPCas9-155

CUUAUAGCGCUCUCACU
17
458





antiSPCas9-156

UUCUGUCGAGAUCUCCG
17
459





antiSPCas9-157

CAUAUUGCGAAUCUUGC
17
460





antiSPCas9-158

GGGCCGGGACUUCGCGA
17
461





antiSPCas9-159

UAAACCCAUACGUGAGC
17
462





antiSPCas9-160
+
UUUACGACUUCCUCGCU
17
463





antiSPCas9-161

GCGCAUACAACAAGCAC
17
464





antiSPCas9-162

GCAGGUUAUAUUGACGG
17
465





antiSPCas9-163
+
UUUUCGCGAUCAUCUUA
17
466





antiSPCas9-164

UCGUAUGGGGAUAAGGC
17
467





antiSPCas9-165
+
AUAAUCCCGUGAUGGAU
17
468





antiSPCas9-166

ACGAUACACUUCUACCA
17
469





antiSPCas9-167
+
CAGUUGCUGACGGACUA
17
470





antiSPCas9-168

GAACGAUAAGCUGAUUC
17
471





antiSPCas9-169

UCUAAAUCCGGACAACU
17
472





antiSPCas9-170

CAGCGGACUUUCGACAA
17
473





antiSPCas9-171

CAUGGGACGUCACAAAC
17
474





antiSPCas9-172
+
CAGGUUUUCUAGCCGUC
17
475





antiSPCas9-173

CCGCUUCAAUGAUCAAA
17
476





antiSPCas9-174
+
UAGCCAACAUCCGUUUU
17
477





antiSPCas9-175
+
GGAGGAUUGCAUCGCUA
17
478





antiSPCas9-176
+
CAUGCAAUUCGCCUAAG
17
479





antiSPCas9-177

AAGUGGCGUGGAUGCGA
17
480





antiSPCas9-178

CGUAUGGGAUAAGGGCC
17
481





antiSPCas9-179

GACAGGUGAAAUCGUAU
17
482





antiSPCas9-180
+
GAACCGAGAGUUCCCUC
17
483





antiSPCas9-181
+
AAAUCGAUCUUCUACCC
17
484





antiSPCas9-182
+
AAUGGGACGCUAAAUAC
17
485





antiSPCas9-183

UAAAGUGCUUACACGCU
17
486





antiSPCas9-184

UACGCAGGUUAUAUUGA
17
487





antiSPCas9-185

AUUCUGUCGAGAUCUCC
17
488





antiSPCas9-186

GAUUCUGUCGAGAUCUC
17
489





antiSPCas9-187

GCCUCUCUAAAUCCCGA
17
490





antiSPCas9-188
+
UGCAUCGCUAAGGUUUU
17
491





antiSPCas9-189
+
GCAGUUGCUGACGGACU
17
492





antiSPCas9-190

CGGGAUUAUAUGAAACU
17
493





antiSPCas9-191

ACCCAUACGUGAGCAGG
17
494





antiSPCas9-192
+
AGGGGUCCCACAUAGUA
17
495





antiSPCas9-193
+
AGCCACCGUACUUUUUC
17
496





antiSPCas9-194

AUUGGGGAUAACGAUUA
17
497





antiSPCas9-195

UGACAAUGUUCCAAGCG
17
498





antiSPCas9-196

GGUGUCGGACUUCAGAA
17
499





antiSPCas9-197
+
UCUCCGCUUAGAAAGGC
17
500





antiSPCas9-198
+
UGUAACCUUUCGCCUCA
17
501





antiSPCas9-199

ACAGGUUUCCGGACAAG
17
502





antiSPCas9-200

UGGGACCCGAAAAAGUA
17
503





antiSPCas9-201
+
UUCAUAUAAUCCCGUGA
17
504





antiSPCas9-202

CACAGGUUUCCGGACAA
17
505





antiSPCas9-203
+
AUAGUAAGGUAUGCGAA
17
506





antiSPCas9-204
+
AACUGUCACUUUGCGGU
17
507





antiSPCas9-205

UUACUAUGUGGGACCCC
17
508





antiSPCas9-206
+
AUGCGGCUGGAGCGCCG
17
509





antiSPCas9-207

UAAGACGGAAAUCACUC
17
510





antiSPCas9-208
+
CGCCACUUGCAUUUAUA
17
511





antiSPCas9-209

CCCCAUCGACUUCCUUG
17
512





antiSPCas9-2IO
+
GCCUCAAGGAAGUCGAU
17
513





antiSPCas9-211

UUGAUCAGUCGAAAAAC
17
514





antiSPCas9-212
+
UCGGGAUUUAGAGAGGC
17
515





antiSPCas9-213
+
CAAUGAGUCCCCUUGUC
17
516





antiSPCas9-214
+
UCAGGUUUUCUAGCCGU
17
517





antiSPCas9-215

GACCGGAGGGUUUUCAA
17
518





antiSPCas9-216
+
AAGCCACCGUACUUUUU
17
519





antiSPCas9-217
+
CUGUUCUCCGCUUAGAA
17
520





antiSPCas9-218
+
AUCAUUGAAGCGGAUAA
17
521





antiSPCas9-219

UUCGCCAGCCAUCAAAA
17
522





antiSPCas9-220

GCCGGAGAGCUUCAAAA
17
523





antiSPCas9-221

AGAACGAUAAGCUGAUU
17
524





antiSPCas9-222

GCACAGGUUUCCGGACA
17
525





antiSPCas9-223

UCGCCAGCCAUCAAAAA
17
526





antiSPCas9-224
+
AAGGUAUGCGAAAGGUU
17
527





antiSPCas9-225
+
GGCAAUACUUUUUCGUU
17
528





antiSPCas9-226

GAGGAAGUUGUCGAUAA
17
529





antiSPCas9-227
+
CUGACGUUUAAUAAAUC
17
530





antiSPCas9-228

AAACCCGCCUUUCUAAG
17
531





antiSPCas9-229

UAUAAAUGCAAGUGGCG
17
532





antiSPCas9-230
+
CCACUACUUUGACUGUC
17
533





antiSPCas9-231
+
GAGAGUUCCCUCGGGCC
17
534





antiSPCas9-232
+
UAUAGGUUUGUACUAAC
17
535





antiSPCas9-233

CAUUACGAGAAGUUGAA
17
536





antiSPCas9-234

GUAUGUUGAUCAGGAAC
17
537





antiSPCas9-235
+
CGUAUUUCGUAUUCAUU
17
538





antiSPCas9-236

UGAGGGUGAUCUAAAUC
17
539





antiSPCas9-237
+
CCUCAAGGAAGUCGAUG
17
540





antiSPCas9-238

GAAAUCGUAUGGGAUAA
17
541





antiSPCas9-239
+
UGGAGUAAUCGUUUCUU
17
542





antiSPCas9-240

UGCUAUACUUAGAAGGC
17
543





antiSPCas9-241

CCGGAGAGCUUCAAAAG
17
544





antiSPCas9-242

AGACAGGUGAAAUCGUA
17
545





antiSPCas9-243

GAGCUAGUUAAGGUCAU
17
546





antiSPCas9-244
+
GAGUUCCCUCGGGCCAG
17
547





antiSPCas9-245
+
CUUUCAACUUCUCGUAA
17
548





antiSPCas9-246
+
AGAGUUCCCUCGGGCCA
17
549





antiSPCas9-247

AGAUCGGGAAAUGAUUG
17
550





antiSPCas9-248

UGAGGCGAAAGGUUACA
17
551





antiSPCas9-249

UGGCCCGAGGGAACUCU
17
552





antiSPCas9-250
+
GUUUCUCGUUCUGCAAU
17
553





antiSPCas9-251
+
ACGCCACUUGCAUUUAU
17
554





antiSPCas9-252
+
CACUACUAGGACAGAAU
17
555





antiSPCas9-253

AUCUACUGCGAAAGCAG
17
556





antiSPCas9-254
+
GUCGGGAUUUAGAGAGG
17
557





antiSPCas9-255

CCUAGCUGAUGCCAAUC
17
558





antiSPCas9-256
+
UUCUCCGCUUAGAAAGG
17
559





antiSPCas9-257

ACUGAGGUGCAGACCGG
17
560





antiSPCas9-258

UGCAUGCUAUACUUAGA
17
561





antiSPCas9-259

CUGAGGUGCAGACCGGA
17
562





antiSPCas9-260
+
AUGCCCUUUUUGAUGGC
17
563





antiSPCas9-261

AUUCACCAAUCCAUCAC
17
564





antiSPCas9-262

UGGGACCCCUGGCCCGA
17
565





antiSPCas9-263
+
UUUCAACUUCUCGUAAU
17
566





antiSPCas9-264
+
GUAUUUCGUAUUCAUUC
17
567





antiSPCas9-265

AGUGGAUGAGCUAGUUA
17
568





antiSPCas9-266
+
GAGUAUGCCCUUUUUGA
17
569





antiSPCas9-267

AUCAAACGACUCAGAAG
17
570





antiSPCas9-268
+
CAUUCUCUUCGUUAUCC
17
571





antiSPCas9-269

AUACACUUCUACCAAGG
17
572





antiSPCas9-270

GACUUCCUUGAGGCGAA
17
573





antiSPCas9-271

ACCCCAAUCCUUUUUGA
17
574





antiSPCas9-272
+
UGAAUCGUCCUUCAAAA
17
575





antiSPCas9-273

UACCCUCUUUGAAGAUC
17
576





antiSPCas9-274

UCUGAACUUGACAAGGC
17
577





antiSPCas9-275
+
CGAUUGUCUUUGAGGAA
17
578





antiSPCas9-276

GGACAUGUAUGUUGAUC
17
579





antiSPCas9-277

CGCAUACAACAAGCACA
17
580





antiSPCas9-278
+
UCGAUUACAAUGUUUUC
17
581





antiSPCas9-279
+
CGUCCUUCAAAAAGGAU
17
582





antiSPCas9-280
+
CUUACUAAGCUGCAAUU
17
583





antiSPCas9-281

AAGAAACGAUUACUCCA
17
584





antiSPCas9-282
+
CAACUUUUGCCACUACU
17
585





antiSPCas9-283

CUAUUCUGUCCUAGUAG
17
586





antiSPCas9-284
+
CUGCAUAAAGUUCCUAU
17
587





antiSPCas9-285

GUGGGACCCCUGGCCCG
17
588





antiSPCas9-286

GUAUGCGGACUUAUUUU
17
589





antiSPCas9-287
+
CCUAAGUGGAUUUGAUG
17
590





antiSPCas9-288

GAUUUUCUAAAGAGCGA
17
591





antiSPCas9-289

GGGCAGCCAGAUCUUAA
17
592





antiSPCas9-290

CAAAUUGCAGCUUAGUA
17
593





antiSPCas9-291

UGAAAUCGUAUGGGAUA
17
594





antiSPCas9-292

UUUACUCUUACCAACCU
17
595





antiSPCas9-293
+
GAUGCUCCUUUAAGAUC
17
596





antiSPCas9-294
+
CUCUCAGUAUGUCAGAU
17
597





antiSPCas9-295
+
AAAUACUUGAAUGCGGC
17
598





antiSPCas9-296

ACUUAACUAAAGCUGAG
17
599





antiSPCas9-297

CGAACAGGAGAUAGGCA
17
600





antiSPCas9-298

GAUUCACCAAUCCAUCA
17
601





antiSPCas9-299

UUUGAUCAGUCGAAAAA
17
602





antiSPCas9-300

CUUAAAGGAGCAUCCUG
17
603





antiSPCas9-301
+
CCUUUUGAUCAUUGAAG
17
604





antiSPCas9-302

UGAGCUAGUUAAGGUCA
17
605





antiSPCas9-303

AAACAUUGUAAUCGAGA
17
606





antiSPCas9-304
+
CCAGAUUGGCAUCAGCU
17
607





antiSPCas9-305
+
AAUUGGGUAUUUUCCAC
17
608





antiSPCas9-306
+
UCAAACAGACUAUACUU
17
609





antiSPCas9-307

CCACAUCAAAUCCACUU
17
610





antiSPCas9-308

GGAUAUACAAAAGGCAC
17
611





antiSPCas9-309

AAUCUACUGGCACAAAU
17
612





antiSPCas9-310
+
UCAGUAUGUCAGAUAGG
17
613





antiSPCas9-311

AGUUAAGUAUGUCACUG
17
614





antiSPCas9-312
+
AAGUUCGACUUAAAAUU
17
615





antiSPCas9-313
+
UUCUGUUCGUUAUCUUC
17
616





antiSPCas9-314

GAGGGUAUUAAAGAACU
17
617





antiSPCas9-315

GUUAAGUAUGUCACUGA
17
618





antiSPCas9-316
+
GCUUAACUGUCACUUUG
17
619





antiSPCas9-317

AGAUUUGUCACAGCUUG
17
620





antiSPCas9-318

AGACUUGACACUUCUCA
17
621





antiSPCas9-319

CCAGACAGUCAAAGUAG
17
622





antiSPCas9-320
+
CCCCUUUUGAAGCUCUC
17
623





antiSPCas9-321
+
UUAUCAGUUUCGCAUUU
17
624





antiSPCas9-322

AAAUCAAACGACUCAGA
17
625





antiSPCas9-323

AAUCGAUUCUUCCAAAA
17
626





antiSPCas9-324
+
UUCCCGAUCUUCAAAGA
17
627





antiSPCas9-325

GUCAGUCAAAGAAUUAU
17
628





antiSPCas9-326

GAUUUGUCACAGCUUGG
17
629





antiSPCas9-327

GAAACCAAUGGGGAGAC
17
630





antiSPCas9-328
+
AGGUUAAAGAGUCAUCA
17
631





antiSPCas9-329

AGAGGGUAUUAAAGAAC
17
632





antiSPCas9-330
+
ACAGAAUAGGCAACUGU
17
633





antiSPCas9-331
+
UUUCACGAUUGUCUUUG
17
634





antiSPCas9-332
+
GUCCUUCAAAAAGGAUU
17
635





antiSPCas9-333
+
AUGCUUUGUGAUUUGGC
17
636





antiSPCas9-334
+
UGAGAAGUGUCAAGUCU
17
637





antiSPCas9-335

UAAAGAUAAGGACUUCC
17
638





antiSPCas9-336
+
UUUCUCGUUCUGCAAUU
17
639





antiSPCas9-337
+
CCAUUGGUUUCAAUUAA
17
640





antiSPCas9-338
+
AUCCAUCUUCUCUAAUA
17
641





antiSPCas9-339

UAAUACUGAGAUUACCA
17
642





antiSPCas9-340
+
UUCACCUGUCUCCCCAU
17
643





antiSPCas9-341

AUAGAUUUGUCACAGCU
17
644





antiSPCas9-342

CUUGUCUGAACUUGACA
17
645





antiSPCas9-343

CUUAACUAAAGCUGAGA
17
646





antiSPCas9-344

CAGUCAAAGAAUUAUUG
17
647





antiSPCas9-345
+
AAAUUCACGUAUUUAGA
17
648





antiSPCas9-346

UAGAUUUGUCACAGCUU
17
649





antiSPCas9-347
+
UAAUACUUUGUCCAGAU
17
650





antiSPCas9-348
+
ACUCACUUUCUAGCUUC
17
651





antiSPCas9-349

CCUUUAAUUGAAACCAA
17
652





antiSPCas9-350

UACUCCAUGGAAUUUUG
17
653





antiSPCas9-351
+
GUCAAAAUACUUGAAUG
17
654





antiSPCas9-352
+
ACUUAAAAUUUGGUGUC
17
655





antiSPCas9-353

CUCUAUUACCUACAAAA
17
656





antiSPCas9-354

CUCUUUAACCUUCAAAG
17
657





antiSPCas9-355
+
CUUUACUAUGUUGACUU
17
658





antiSPCas9-356
+
CAACAUGCUUUGUGAUU
17
659





antiSPCas9-357

AAUUGGAGAUCAGUAUG
17
660





antiSPCas9-358
+
CAUUUUGUAGGUAAUAG
17
661





antiSPCas9-359
+
GACUGACUUCAGUUUCU
17
662





antiSPCas9-360

ACUAAAGCUGAGAGGGG
17
663





antiSPCas9-361

AAAAGCGAACAGGAGAU
17
664





antiSPCas9-362

AACCCUAUAAAUGCAAG
17
665





antiSPCas9-363

ACCCAUAUUAGAGAAGA
17
666





antiSPCas9-364
+
GUAUUUCUUAAUGAGUG
17
667





antiSPCas9-365
+
UAUGUUGACUUGGGGCA
17
668





antiSPCas9-366

CAAAAGGCACAGGUUUC
17
669





antiSPCas9-367
+
UUUCCCGAUCUUCAAAG
17
670





antiSPCas9-368

UUACCCUCUUUGAAGAU
17
671





antiSPCas9-369

UAUUAGAGAAGAUGGAU
17
672





antiSPCas9-370

UUAACUAAAGCUGAGAG
17
673





antiSPCas9-371

AGUUAAGCAAUUGAAAG
17
674





antiSPCas9-372

AAAGUCAAAAUUGGUGU
17
675





antiSPCas9-373
+
UUCAAACAACUGAUUAU
17
676





antiSPCas9-374

GUGGCAAAAGUUGAGAA
17
677





antiSPCas9-375
+
UAAAGUAAACUGUGCUU
17
678





antiSPCas9-376
+
CAUUGUCACUUUUCCCU
17
679





antiSPCas9-377
+
UUCCUUUGAAAACCCUC
17
680





antiSPCas9-378
+
AACUCACUUUCUAGCUU
17
681





antiSPCas9-379

ACUGCCUGAGAAAUAUA
17
682





antiSPCas9-380
+
CAUGCUUUGUGAUUUGG
17
683





antiSPCas9-381

AGUGGCAAAAGUUGAGA
17
684





antiSPCas9-382

CUGUUUGAGUUAGAAAA
17
685





antiSPCas9-383

UAGAAAAUGGCCGAAAA
17
686





antiSPCas9-384
+
UUUACUAUGUUGACUUG
17
687





antiSPCas9-385

AUUACCUACAAAAUGGA
17
688





antiSPCas9-386

GAAAGUGAGUUUGUGUA
17
689





antiSPCas9-387
+
UCCAUCUUCUCUAAUAU
17
690





antiSPCas9-388

CUUUAAUUGAAACCAAU
17
691





antiSPCas9-389

UCAGUCAAAGAAUUAUU
17
692





antiSPCas9-390

AAUCAAACGACUCAGAA
17
693





antiSPCas9-391
+
UGUCCCUUCCAUUUUGU
17
694





antiSPCas9-392

UUUAAUUGAAACCAAUG
17
695





antiSPCas9-393
+
UAAAUUCUUGUCAAAGU
17
696





antiSPCas9-394
+
UUGUAUAUCCUCUUUGA
17
697





antiSPCas9-395
+
CUUUAAUUAUCUUUAGG
17
698





antiSPCas9-396

AAAUGAAGAACUAUUGG
17
699





antiSPCas9-397
+
UCUUUACUAUGUUGACU
17
700





antiSPCas9-398

CCGGAGAGAAGAAAAAU
17
701





antiSPCas9-399

AAUCAUAGAGCAAAUUU
17
702





antiSPCas9-400

CCCGGAGAGAAGAAAAA
17
703





antiSPCas9-401

UUACCUACAAAAUGGAA
17
704





antiSPCas9-402
+
UCUCAGGCAGUUGCUGA
17
705





antiSPCas9-403
+
UCCUUCAAAAAGGAUUG
17
706





antiSPCas9-404

UUCAAUUCUAUAAAGUU
17
707





antiSPCas9-405
+
UGGUAAGAGUAAACAAA
17
708





antiSPCas9-406

UCAAUUCUAUAAAGUUA
17
709





antiSPCas9-407

GAAAUCACUCUGGCAAA
17
710





antiSPCas9-408
+
ACUUCCUCAAAAUUCCA
17
711





antiSPCas9-409
+
UUAUCACUAUUCCUUUU
17
712





antiSPCas9-410

CACUUUAAAGUCAAAAU
17
713





antiSPCas9-411

AUAUUAGAGAAGAUGGA
17
714





antiSPCas9-412
+
CCCAUUUUUCUUCUCUC
17
715





antiSPCas9-413

AAAAAAACAGUCGAGAG
17
716





antiSPCas9-414

UUAUGAAACAGUUAAAG
17
717





antiSPCas9-415

AAGAAAAAUGGGUUGUU
17
718





antiSPCas9-416
+
CCAUUUUUCUUCUCUCC
17
719





antiSPCas9-417

CAUAGUAAAGAAAACUG
17
720





antiSPCas9-418

AUAAGAGACAAGCAAAG
17
721





antiSPCas9-419

GAUGAAGAGAAUAGAAG
17
722





antiSPCas9-420
+
GGCUGGAGCGCCGAGGU
17
723





antiSPCas9-421
+
AUUUCCUUAUAUUUCUC
17
724





antiSPCas9-422
+
CAGAAUAGGCAACUGUA
17
725





antiSPCas9-423

UAUGAAUUUCUUUAAGA
17
726





antiSPCas9-424

AGAAAAUGAAGAACUAU
17
727





antiSPCas9-425

UUACAAGGAAGUAAAAA
17
728





antiSPCas9-426

AGAGAAGAUGGAUGGGA
17
729





antiSPCas9-427

AAAACUGAGGUGCAGAC
17
730





antiSPCas9-428
+
UAUCUUUAAUUAUCUUU
17
731





antiSPCas9-429
+
AGAAUAAAAGAAGUAUU
17
732





antiSPCas9-430

AUGAAGAGAAUAGAAGA
17
733





antiSPCas9-431

AAAGAUAAUUAAAGAUA
17
734





antiSPCas9-432

UAUACUUAGAAGGCAGG
17
735





antiSPCas9-433

CAAAGAGGAUAUACAAA
17
736





antiSPCas9-434

CAGUCGAAAAACGGGUACGC
20
737





antiSPCas9-435

CACGCUCGGAUAAGAACCGA
20
738





antiSPCas9-436
+
UAAGAUAAGCGUCGUGCGCA
20
739





antiSPCas9-437

CGCUCACCUGUUCGACGAUA
20
740





antiSPCas9-438
+
GAUAAGCGUCGUGCGCAUGG
20
741





antiSPCas9-439

CGAUUCUGUCGAGAUCUCCG
20
742





antiSPCas9-440

AGAGGCGUCGCUAUACGGGC
20
743





antiSPCas9-441

UUAAAGAGGCGUCGCUAUAC
20
744





antiSPCas9-442

AGGCGUCGCUAUACGGGCUG
20
745





antiSPCas9-443
+
CGCUUUUCGCGAUCAUCUUA
20
746





antiSPCas9-444

AGAGCGACGGCUUCGCCAAU
20
747





antiSPCas9-445
+
UGAAGCGGAUAACGGCGCCU
20
748





antiSPCas9-446

ACACGCUCGGAUAAGAACCG
20
749





antiSPCas9-447

GAGGCGUCGCUAUACGGGCU
20
750





antiSPCas9-448

GAUGAUCGCGAAAAGCGAAC
20
751





antiSPCas9-449

UAAGGGCCGGGACUUCGCGA
20
752





antiSPCas9-450
+
CGUAAUGGGACGCUAAAUAC
20
753





antiSPCas9-451
+
CUCCGGGUAAUUGUGCGAUC
20
754





antiSPCas9-452

UGUCGCGGAAACUUAUCAAC
20
755





antiSPCas9-453
+
CGCUAGCCAACAUCCGUUUU
20
756





antiSPCas9-454
+
AAUGAGUGCGGUCCCUACGA
20
757





antiSPCas9-455

UACGCAGGUUAUAUUGACGG
20
758





antiSPCas9-456

UGACGAUCUCGACAAUCUAC
20
759





antiSPCas9-457

GUUAAAGAGGCGUCGCUAUA
20
760





antiSPCas9-458
+
CGACGUCGUAAUCAGAUAAA
20
761





antiSPCas9-459
+
UCAUAACCUUAUCGUCGAAC
20
762





antiSPCas9-460

GCUUAUCUUAAUGCCGUCGU
20
763





antiSPCas9-461

AAACGGAUGUUGGCUAGCGC
20
764





antiSPCas9-462
+
UUGUCGACAUCCGAGUUGUC
20
765





antiSPCas9-463

CAGCUCAAUCGUUCAUCGAG
20
766





antiSPCas9-464

GAUCGAUUUAAUGCGUCACU
20
767





antiSPCas9-465

CUUAUCUUAAUGCCGUCGUA
20
768





antiSPCas9-466

UGACGGCGGAGCGAGUCAAG
20
769





antiSPCas9-467
+
CUAGCCGUCGGGAUUUAGAG
20
770





antiSPCas9-468

UCAUCGCUCGUAAAAAGGAC
20
771





antiSPCas9-469

CGAGAACGAUAAGCUGAUUC
20
772





antiSPCas9-470
+
AUGCGAACCGAGAGUUCCCU
20
773





antiSPCas9-471

UAAGCUCAUCGCUCGUAAAA
20
774





antiSPCas9-472

UUGUCGCGGAAACUUAUCAA
20
775





antiSPCas9-473

UCGAUUCUGUCGAGAUCUCC
20
776





antiSPCas9-474
+
UGUCGCGUCUAGCACCUCCU
20
777





antiSPCas9-475

UGGGACCCGAAAAAGUACGG
20
778





antiSPCas9-476
+
GGCCUAGUGAGAGCGCUAUA
20
779





antiSPCas9-477

GGAUAAACCCAUACGUGAGC
20
780





antiSPCas9-478
+
CCGUCGGGAUUUAGAGAGGC
20
781





antiSPCas9-479
+
UUUUCCGCCUGCUCACGUAU
20
782





antiSPCas9-480

CAUCGCUCGUAAAAAGGACU
20
783





antiSPCas9-481

AACCUUAUAGCGCUCUCACU
20
784





antiSPCas9-482

AUCGACUUCCUUGAGGCGAA
20
785





antiSPCas9-483
+
CGAUCAGGUUUUCUAGCCGU
20
786





antiSPCas9-484
+
CGUCGUAUUUCGUAUUCAUU
20
787





antiSPCas9-485
+
UCGAAGCCACCGUACUUUUU
20
788





antiSPCas9-486
+
UGCGAACCGAGAGUUCCCUC
20
789





antiSPCas9-487

UAGCGCCGGAGAGCUUCAAA
20
790





antiSPCas9-488

AACCUGAUCGCACAAUUACC
20
791





antiSPCas9-489

GGGUACGCAGGUUAUAUUGA
20
792





antiSPCas9-490

GAGGGAACUCUCGGUUCGCA
20
793





antiSPCas9-491

ACGAGAACGAUAAGCUGAUU
20
794





antiSPCas9-492

CCCGCCUCUCUAAAUCCCGA
20
795





antiSPCas9-493

CGGGCUGGGGACGAUUGUCG
20
796





antiSPCas9-494

CGUAAACCCGCCUUUCUAAG
20
797





antiSPCas9-495
+
CAUAUAAUCCCGUGAUGGAU
20
798





antiSPCas9-496

UUCGAUUCUGUCGAGAUCUC
20
799





antiSPCas9-497
+
CGAAGCCACCGUACUUUUUC
20
800





antiSPCas9-498

CCGAAGAAACGAUUACUCCA
20
801





antiSPCas9-499
+
GUCGUAUUUCGUAUUCAUUC
20
802





antiSPCas9-500

AGCGCCGGAGAGCUUCAAAA
20
803





antiSPCas9-501
+
UUCUCACCGUCGCGAAGUCC
20
804





antiSPCas9-502

UGAUCUAAAUCCGGACAACU
20
805





antiSPCas9-503
+
GAUCAGGUUUUCUAGCCGUC
20
806





antiSPCas9-504

GGUUCGCCAGCCAUCAAAAA
20
807





antiSPCas9-505
+
UUCGCCUCAAGGAAGUCGAU
20
808





antiSPCas9-506
+
UCGCCUAAGUGGAUUUGAUG
20
809





antiSPCas9-507

UGGUUCGCCAGCCAUCAAAA
20
810





antiSPCas9-508
+
UCGCCUCAAGGAAGUCGAUG
20
811





antiSPCas9-509
+
ACCGAGAGUUCCCUCGGGCC
20
812





antiSPCas9-510
+
AUAGGAGGAUUGCAUCGCUA
20
813





antiSPCas9-511
+
GCCGUCGGGAUUUAGAGAGG
20
814





antiSPCas9-512

CAAUAAAGUGCUUACACGCU
20
815





antiSPCas9-513
+
AUUUUCCGCCUGCUCACGUA
20
816





antiSPCas9-514

GAACCCCAUCGACUUCCUUG
20
817





antiSPCas9-515

GCGCCGGAGAGCUUCAAAAG
20
818





antiSPCas9-516

ACGAUACACUUCUACCAAGG
20
819





antiSPCas9-517

AAAUGGCCGAAAACGGAUGU
20
820





antiSPCas9-518
+
UAGCAUGCAAUUCGCCUAAG
20
821





antiSPCas9-519

UAAGCGCAUACAACAAGCAC
20
822





antiSPCas9-520
+
UUUCGCCUCAAGGAAGUCGA
20
823





antiSPCas9-521
+
UGAAUGCGGCUGGAGCGCCG
20
824





antiSPCas9-522
+
GUGCAAUGAGUCCCCUUGUC
20
825





antiSPCas9-523

UCAAGUGGCGUGGAUGCGA
20
826





antiSPCas9-524

AAGCAGCGGACUUUCGACAA
20
827





antiSPCas9-525

UAAACCCAUACGUGAGCAGG
20
828





antiSPCas9-526

CCCUAUAAAUGCAAGUGGCG
20
829





antiSPCas9-527

UUUCGCAUACCUUACUAUGU
20
830





antiSPCas9-528
+
GCGUUAUCAGUUUCGCAUUU
20
831





antiSPCas9-529

GGUCAUGGGACGUCACAAAC
20
832





antiSPCas9-530
+
CGAGAGUUCCCUCGGGCCAG
20
833





antiSPCas9-531
+
UUCUUUACGACUUCCUCGCU
20
834





antiSPCas9-532

CAAACGAUACACUUCUACCA
20
835





antiSPCas9-533

ACCUUACUAUGUGGGACCCC
20
836





antiSPCas9-534
+
GAUUGCAUCGCUAAGGUUUU
20
837





antiSPCas9-535

CUUUCGCAUACCUUACUAUG
20
838





antiSPCas9-536
+
UUGAUCAUUGAAGCGGAUAA
20
839





antiSPCas9-537

CUCGAUUUUCUAAAGAGCGA
20
840





antiSPCas9-538
+
UCGAAGUUCGACUUAAAAUU
20
841





antiSPCas9-539
+
AGGCAGUUGCUGACGGACUA
20
842





antiSPCas9-540

UAUCCGCUUCAAUGAUCAAA
20
843





antiSPCas9-541

GACUGGGACCCGAAAAAGUA
20
844





antiSPCas9-542
+
CCGAGAGUUCCCUCGGGCCA
20
845





antiSPCas9-543

GCAUGCUAUACUUAGAAGGC
20
846





antiSPCas9-544

AAUCGUAUGGGAUAAGGGCC
20
847





antiSPCas9-545
+
GUCCUUACUAAGCUGCAAUU
20
848





antiSPCas9-546
+
UGUUCUCCGCUUAGAAAGGC
20
849





antiSPCas9-547

CGACCUUUAAUUGAAACCAA
20
850





antiSPCas9-548

AAAUCGUAUGGGAUAAGGGC
20
851





antiSPCas9-549

GAACAUAUUGCGAAUCUUGC
20
852





antiSPCas9-550
+
GAGCUGACGUUUAAUAAAUC
20
853





antiSPCas9-551
+
CUUAACUGUCACUUUGCGGU
20
854





antiSPCas9-552
+
AUUAAAUCGAUCUUCUACCC
20
855





antiSPCas9-553

GGCACAGGUUUCCGGACAAG
20
856





antiSPCas9-554
+
CACAUAGUAAGGUAUGCGAA
20
857





antiSPCas9-555
+
AGUAAGGUAUGCGAAAGGUU
20
858





antiSPCas9-556
+
CCAUGGAGUAAUCGUUUCUU
20
859





antiSPCas9-557
+
CGGGUAUUUCUUAAUGAGUG
20
860





antiSPCas9-558

UAUGUGGGACCCCUGGCCCG
20
861





antiSPCas9-559
+
CCUUGUAACCUUUCGCCUCA
20
862





antiSPCas9-560
+
UCCACGCCACUUGCAUUUAU
20
863





antiSPCas9-561

UCCCAUUACGAGAAGUUGAA
20
864





antiSPCas9-562

AUUGGUGUCGGACUUCAGAA
20
865





antiSPCas9-563
+
AGUUUCAUAUAAUCCCGUGA
20
866





antiSPCas9-564

GGUGAAAUCGUAUGGGAUAA
20
867





antiSPCas9-565

AUUAUUGGGGAUAACGAUUA
20
868





antiSPCas9-566

UCACGGGAUUAUAUGAAACU
20
869





antiSPCas9-567

AAGUGACAAUGUUCCAAGCG
20
870





antiSPCas9-568

GCGAAAAGCGAACAGGAGAU
20
871





antiSPCas9-569

GGAGACAGGUGAAAUCGUAU
20
872





antiSPCas9-570
+
ACCUUUCAACUUCUCGUAAU
20
873





antiSPCas9-571

GCAGACCGGAGGGUUUUCAA
20
874





antiSPCas9-572
+
UGCCACUACUAGGACAGAAU
20
875





antiSPCas9-573
+
CCACGCCACUUGCAUUUAUA
20
876





antiSPCas9-574

AUUUAUUAAACGUCAGCUCG
20
877





antiSPCas9-575
+
AACCUUUCAACUUCUCGUAA
20
878





antiSPCas9-576

CGAAAAUCAAACGACUCAGA
20
879





antiSPCas9-577

UCUUUGAUCAGUCGAAAAAC
20
880





antiSPCas9-578
+
CGGUAAAUUCUUGUCAAAGU
20
881





antiSPCas9-579

AUGUGGGACCCCUGGCCCGA
20
882





antiSPCas9-580

CGGAUAGAUUUGUCACAGCU
20
883





antiSPCas9-581
+
UCGACUUAAAAUUUGGUGUC
20
884





antiSPCas9-582

CAUCCUAGCUGAUGCCAAUC
20
885





antiSPCas9-583
+
CAUCCACUACUUUGACUGUC
20
886





antiSPCas9-584

AGGCACAGGUUUCCGGACAA
20
887





antiSPCas9-585
+
GCCAGGGGUCCCACAUAGUA
20
888





antiSPCas9-586
+
UCGUAAAGUAAACUGUGCUU
20
889





antiSPCas9-587
+
CAGGCAGUUGCUGACGGACU
20
890





antiSPCas9-588

CUGAUUCACCAAUCCAUCAC
20
891





antiSPCas9-589

UGCCUAUUCUGUCCUAGUAG
20
892





antiSPCas9-590

GAUGAGcuAGUUAAGGUCAU
20
893





antiSPCas9-591
+
AGUAUGCCCUUUUUGAUGGC
20
894





antiSPCas9-592

GGGAGACAGGUGAAAUCGUA
20
895





antiSPCas9-593
+
GAUUGAAUCGUCCUUCAAAA
20
896





antiSPCas9-594
+
CUGUUCUCCGCUUAGAAAGG
20
897





antiSPCas9-595

AAGAUCUACUGCGAAAGCAG
20
898





antiSPCas9-596

UUGUCUGAACUUGACAAGGC
20
899





antiSPCas9-597
+
UGCGGCUGGAGCGCCGAGGU
20
900





antiSPCas9-598

CAUGUAUGUUGAUCAGGAAC
20
901





antiSPCas9-599

GACAAUCUACUGGCACAAAU
20
902





antiSPCas9-600

CCUUGAGGCGAAAGGUUACA
20
903





antiSPCas9-601
+
GCUUAAUACUUUGUCCAGAU
20
904





antiSPCas9-602

AGGUGAAAUCGUAUGGGAUA
20
905





antiSPCas9-603

AAGGCACAGGUUUCCGGACA
20
906





antiSPCas9-604
+
AUCGUCCUUCAAAAAGGAUU
20
907





antiSPCas9-605
+
CAGCUGCAUAAAGUUCCUAU
20
908





antiSPCas9-606

GAUCUUAAAGGAGCAUCCUG
20
909





antiSPCas9-607
+
GUACCUUUUGAUCAUUGAAG
20
910





antiSPCas9-608

UGAAGAUCGGGAAAUGAUUG
20
911





antiSPCas9-609

UGCCAAAUUGCAGCUUAGUA
20
912





antiSPCas9-610

GGAUGAGCUAGUUAAGGUCA
20
913





antiSPCas9-611
+
CGAUUCCUUUGAAAACCCUC
20
914





antiSPCas9-612
+
CUUCUGUUCUCCGCUUAGAA
20
915





antiSPCas9-613

UUUGAGGAAGUUGUCGAUAA
20
916





antiSPCas9-614
+
UCAAAAUACUUGAAUGCGGC
20
917





antiSPCas9-615

ACUGGGCAGCCAGAUCUUAA
20
918





antiSPCas9-616

GAUAGAUUUGUCACAGCUUG
20
919





antiSPCas9-617
+
CCUUGAGAAGUGUCAAGUCU
20
920





antiSPCas9-618
+
AUCUCGAUUACAAUGUUUUC
20
921





antiSPCas9-619
+
UCACGAUUGUCUUUGAGGAA
20
922





antiSPCas9-620
+
CUGGAGUAUGCCCUUUUUGA
20
923





antiSPCas9-621
+
AAAGUUUCUCGUUCUGCAAU
20
924





antiSPCas9-622

UUGUUUACUCUUACCAACCU
20
925





antiSPCas9-623

UUGUUUACUCUUACCAACCU
20
926





antiSPCas9-624
+
AAGCGCAUACAACAAGCACA
20
927





antiSPCas9-625
+
AAUCGUCCUUCAAAAAGGAU
20
928





antiSPCas9-626
+
UGCAAUUGGGUAUUUUCCAC
20
929





antiSPCas9-627
+
UGCUUCUGUUCGUUAUCUUC
20
930





antiSPCas9-628

GGAUAGAUUUGUCACAGCUU
20
931





antiSPCas9-629

UGUACCCCAAUCCUUUUUGA 
20
932





antiSPCas9-630

UGGCUUGUCUGAACUUGACA
20
933





antiSPCas9-631

AAGGGACAUGUAUGUUGAUC
20
934





antiSPCas9-632
+
GAUUUCACCUGUCUCCCCAU
20
935





antiSPCas9-633
+
UUAGGCAAUACUUUUUCGUU
20
936





antiSPCas9-634
+
UAACUCUCAGUAUGUCAGAU
20
937





antiSPCas9-635
+
GUUCCCCUUUUGAAGCUCUC
20
938





antiSPCas9-636

UCAGUAUGCGGACUUAUUUU
20
939





antiSPCas9-637
+
UCUCAACUUUUGCCACUACU
20
940





antiSPCas9-638
+
GAUUAUAGGUUUGUACUAAC
20
941





antiSPCas9-639

GACCUUUAAUUGAAACCAAU
20
942





antiSPCas9-640

AGGAAUCGAUUCUUCCAAAA
20
943





antiSPCas9-641

CUUUAAGACGGAAAUCACUC
20
944





antiSPCas9-642
+
AAGUUUCUCGUUCUGCAAUU
20
945





antiSPCas9-643
+
GUGCAACAUGCUUUGUGAUU
20
946





antiSPCas9-644

AUUGAAACCAAUGGGGAGAC
20
947





antiSPCas9-645

ACUCCAGACAGUCAAAGUAG
20
948





antiSPCas9-646

AAUUGCAUGCUAUACUUAGA
20
949





antiSPCas9-647
+
AACUCAAACAGACUAUACUU
20
950





antiSPCas9-648

GAUUACUCCAUGGAAUUUUG
20
951





antiSPCas9-649

GGAAAACAUUGUAAUCGAGA
20
952





antiSPCas9-650

AGUAGUGGAUGAGCUAGUUA
20
953





antiSPCas9-651
+
CAGGAUGCUCCUUUAAGAUC
20
954





antiSPCas9-652

CAUUGAGGGUGAUCUAAAUC
20
955





antiSPCas9-653

CCAAGACUUGACACUUCUCA
20
956





antiSPCas9-654
+
UCCCCAUUGGUUUCAAUUAA
20
957





antiSPCas9-655

AUAACUUAACUAAAGCUGAG
20
958





antiSPCas9-656
+
CCCAUCCAUCUUCUCUAAUA
20
959





antiSPCas9-657

GAGAACCCUAUAAAUGCAAG
20
960





antiSPCas9-658

AAAACUGAGGUGCAGACCGG
20
961





antiSPCas9-659

GGCAAAAAAACAGUCGAGAG
20
962





antiSPCas9-660
+
AGGAAAUUCACGUAUUUAGA
20
963





antiSPCas9-661

AAGCGAACAGGAGAUAGGCA
20
964





antiSPCas9-662

UCUUACCCUCUUUGAAGAUC
20
965





antiSPCas9-663

CCCUGGCCCGAGGGAACUCU
20
966





antiSPCas9-664

CGAAAUCAUAGAGCAAAUUU
20
967





antiSPCas9-665

UUCUUUGAUCAGUCGAAAAA
20
968





antiSPCas9-666

UGACUCUUUAACCUUCAAAG
20
969





antiSPCas9-667
+
AGGACAGAAUAGGCAACUGU
20
970





antiSPCas9-668

AGUUAAUACUGAGAUUACCA
20
971





antiSPCas9-669

ACCUUUAAUUGAAACCAAUG
20
972





antiSPCas9-670

AUUCCACAUCAAAUCCACUU
20
973





antiSPCas9-671
+
AGCUUAUCACUAUUCCUUUU
20
974





antiSPCas9-672
+
UGUCCAGAUUGGCAUCAGCU
20
975





antiSPCas9-673

GAAAGUUAAGUAUGUCACUG
20
976





antiSPCas9-674
+
AACAUGCUUUGUGAUUUGGC
20
977





antiSPCas9-675
+
CAUUUCCCGAUCUUCAAAGA
20
978





antiSPCas9-676

CCAUAUUAGAGAAGAUGGAU
20
979





antiSPCas9-677

UACCUCUAUUACCUACAAAA
20
980





antiSPCas9-678

AUAGAUUUGUCACAGCUUGG
20
981





antiSPCas9-679
+
GGUUGGUAAGAGUAAACAAA
20
982





antiSPCas9-680
+
UCAUUUCCCGAUCUUCAAAG
20
983





antiSPCas9-681

ACGGAAAUCACUCUGGCAAA
20
984





antiSPCas9-682
+
UGAAGGUUAAAGAGUCAUCA
20
985





antiSPCas9-683
+
CUCUUCAAACAACUGAUUAU
20
986





antiSPCas9-684
+
GUUCAUUCUCUUCGUUAUCC
20
987





antiSPCas9-685

AUACAAAAGGCACAGGUUUC
20
988





antiSPCas9-686

GUAGUGGCAAAAGUUGAGAA
20
989





antiSPCas9-687

AGUCAGUCAAAGAAUUAUUG
20
990





antiSPCas9-688

GCAACUGCCUGAGAAAUAUA
20
991





antiSPCas9-689

CUAGAAAGUGAGUUUGUGUA
20
992





antiSPCas9-690
+
GAACAUUGUCACUUUUCCCU
20
993





antiSPCas9-691

CCCAUAUUAGAGAAGAUGGA
20
994





antiSPCas9-692
+
CAACAUGCUUUGUGAUUUGG
20
995





antiSPCas9-693
+
ACAUGUCCCUUCCAUUUUGU
20
996





antiSPCas9-694

CUCUUACCCUCUUUGAAGAU
20
997





antiSPCas9-695

ACAAAUUGGAGAUCAGUAUG
20
998





antiSPCas9-696

AAACUGAGGUGCAGACCUUA
20
999





antiSPCas9-697

UACCCGGAGAGAAGAAAAAU
20
1000





antiSPCas9-698

UUAACUAAAGCUGAGAGGGG
20
1001





antiSPCas9-699

GAAGUCAGUCAAAGAAUUAU
20
1002





antiSPCas9-700

AAAGUUAAGUAUGUCACUGA
20
1003





antiSPCas9-701

UCUAUUACCUACAAAAUGGA
20
1004





antiSPCas9-702

AAUUAAAGAUAAGGACUUCC
20
1005





antiSPCas9-703

AAUUAAAGAUAAGGACUUCC
20
1006





antiSPCas9-704

AGUAGUGGCAAAAGUUGAGA
20
1007





antiSPCas9-705

AGAGGAUAUACAAAAGGCAC
20
1008





antiSPCas9-706
+
UCUUUUCACGAUUGUCUUUG
20
1009





antiSPCas9-707

CAAACCCAUAUUAGAGAAGA
20
1010





antiSPCas9-708
+
UGUGUCAAAAAUACUUGAAU
20
1011





antiSPCas9-709

GACAGUUAAGCAAUUGAAAG
20
1012





antiSPCas9-710

GAAGAGGGUAUUAAAGAACU
20
1013





antiSPCas9-711
+
UUCCAUUUUGUAGGUAAUAG
20
1014





antiSPCas9-712

GCGGAUGAAGAGAAUAGAAG
20
1015





antiSPCas9-713

CUAUUACCUACAAAAUGGAA
20
1016





antiSPCas9-714
+
AUUGCUUAACUGUCACUUUG
20
1017





antiSPCas9-715

AGUCUGUUUGAGUUAGAAAA
20
1018





antiSPCas9-716
+
ACAAACUCACUUUCUAGCUU
20
1019





antiSPCas9-717

AAAAUCAAACGACUCAGAAG
20
1020





antiSPCas9-718

AGAAGAGGGUAUUAAAGAAC
20
1021





antiSPCas9-719
+
CAAACUCACUUUCUAGCUUC
20
1022





antiSPCas9-720

UUACCCGGAGAGAAGAAAAA
20
1023





antiSPCas9-721
+
AUUUCUCAGGCAGUUGCUGA
20
1024


antiSPCas9-722

AGUUAGAAAAUGGCCGAAAA
20
1025





antiSPCas9-723

UAACUUAACUAAAGCUGAGA
20
1026





antiSPCas9-724
+
GGACAGAAUAGGCAACUGUA
20
1027





antiSPCas9-725

AAGUCAGUCAAAGAAUUAUU
20
1028





antiSPCas9-726

AGGUUAUGAAACAGUUAAAG
20
1029





antiSPCas9-727
+
UUUGACUGACUUCAGUUUCU
20
1030





antiSPCas9-728
+
CAACCCAUUUUUCUUCUCUC
20
1031





antiSPCas9-729
+
UCGUCCUUCAAAAAGGAUUG
20
1032





antiSPCas9-730
+
UACUAUGUUGACUUGGGGCA
20
1033





antiSPCas9-731

UGCUAUACUUAGAAGGCAGG
20
1034





antiSPCas9-732
+
ACAACUUCCUCAAAAUUCCA
20
1035





antiSPCas9-733

UUUAAAGUCAAAAUUGGUGU
20
1036





antiSPCas9-734

CGGAUGAAGAGAAUAGAAGA
20
1037





antiSPCas9-735
+
CUUUUGUAUAUCCUCUUUGA
20
1038





antiSPCas9-736
+
AACCCAUUUUUCUUCUCUCC
20
1039





antiSPCas9-737
+
CCAUCCAUCUUCUCUAAUAU
20
1040





antiSPCas9-738

AUUAGAGAAGAUGGAUGGGA
20
1041





antiSPCas9-739

ACUGAUUCACCAAUCCAUCA
20
1042





antiSPCas9-740

CCUAAAGAUAAUUAAAGAUA
20
1043





antiSPCas9-741

GGGAUAAGAGACAAGCAAAG
20
1044





antiSPCas9-742
+
UUCUUUACUAUGUUGACUUG
20
1045





antiSPCas9-743

GAAAAUCAAACGACUCAGAA
20
1046





antiSPCas9-744
+
UUUCUUUACUAUGUUGACUU
20
1047





antiSPCas9-745
+
CCUUAUCUUUAAUUAUCUUU
20
1048





antiSPCas9-746

AAUCACUUUAAAGUCAAAAU
20
1049





antiSPCas9-747

AGAAAAUGAAGAACUAUUGG
20
1050





antiSPCas9-748
+
GUUAGAAUAAAAGAAGUAUU
20
1051





antiSPCas9-749

CAACAUAGUAAAGAAAACUG
20
1052





antiSPCas9-750

AGGUUACAAGGAAGUAAAAA
20
1053





antiSPCas9-751
+
UAUCUUUAAUUAUCUUUAGG
20
1054





antiSPCas9-752

GAGAAGAAAAAUGGGUUGUU
20
1055





antiSPCas9-753

CAUUAUGAAUUUCUUUAAGA
20
1056





antiSPCas9-754
+
UUUUCUUUACUAUGUUGACU
20
1057





antiSPCas9-755

AUUUUCAAUUCUAUAAAGUU
20
1058





antiSPCas9-756
+
AAUAUUUCCUUAUAUUUCUC
20
1059





antiSPCas9-757

UUUUCAAUUCUAUAAAGUUA
20
1060





antiSPCas9-758

UAAAGAAAAUGAAGAACUAU
20
1061





antiSPCas9-759

AAGAAAACUGAGGUGCAGAC
20
1062





antiSPCas9-760

CUUCAAAGAGGAUAUACAAA
20
1063



















TABLE E-7







Exemplary guide
Group A
Group B
Group A guides can


RNA pairs for S.


be paired with any



aureus (SA)



from Group B.


nickase






antiSACas9-8,
antiSACas9-5




antiSACas9-9
antiSACas9-2




Group C
Group D
Group C guides can





be paired with any





from Group D.



antiSACas9-7
antiSACas9-11


















TABLE E-8








First SOObp of coding sequence




downstream of start codon, good




orthogonality, starts with G












5. aureus


Target
SEQ


1st Tier
DNA

Site
ID


dRNA Name
Strand
Targeting Domain
Length
NO





antiSACas9-1

GGACAUCGGGAUUACAAGOG
20
1064





antiSACas9-2
+
GUAAUCCOGAUGUCCAGCCC
20
1065





antiSACas9-5

GGAAUUAAUCCUUAUGAAGC
20
1068





antiSACas9-6
+
GCCUUUCACCCUGGCUUCAU
20
1069


















TABLE E-9








First SOObp of coding sequence




downstream of start codon, good




orthogonaiity, does




not start with G












5. aureus


Target
SEQ


2nd Tier
DNA

Site
ID


gRNA Name
Strand
Targeting Domain
Length
NO














antiSACas9-7

CUGCACCUGGCUAAGCGCCG
20
1070





antiSACas9-8

ACCGACCAUUCUGAGCUGAG
20
1071





antiSACas9-9

UCCUGACCCACCAUUCUGAG
20
1072





antiSACas9-10
+
AACACCAGUUUCUUCACCCU
20
1073





antiSACas9-11
+
AGGAUUAAUUCCACUCAGCU
20
1074





antiSACas9-12

UACAUUCUGGGGCUGGACAU
20
1075





antiSACas9-13

AUGAAGCCAGGGUGAAAGGC
20
1076





antiSACas9-14

CUGAAACGACGGAGAAGGCA
20
1079





antiSACas9-15

CGGACAAGGCACAGAAUCCA
20
1080


















TABLE E-10








First SOObp of coding sequence




downstream of start codon, poor 




orthogonality, starts with G












5. aureus


Target
SEQ


3rd Tier
DNA

Site
ID


gRNA Name
Strand
Targeting Domain
Length
NO














antiSACas9-

GAGUCAGAAGCUGUCA
20
1081


16

GAGG







antiSACas9-

GAAGAAAGAUGGCGAG
20
1082


17

GUGA







antiSACas9-

GGGAUUACAAGCGUGG
20
1083


18

GGUA


















TABLE E-11








Rest of gene












5. aureus


Target
SEQ


5th Tier
DNA

Site
ID


gRNA Name
Strand
Targeting Domain
Length
NO














antiSACas9-19

GAUACGCUACUCGCGGCCUG
20
1084





antiSACas9-20
+
AUGAUCGACCUCGUAGUUGA
20
1085





antiSACas9-21

AUGAAUGAUAAGCGCCCCCC
20
1086





antiSACas9-22

ACGCAGAUCUGUACAACGCC
20
1087





antiSACas9-23

CGCCGUUGUCCAGAUAGACA
20
1088





antiSACas9-24
+
CGCCGUUGUCCAGAUAGACA
20
1089





antiSACas9-25

UCAUUGAGAACGCCGAACUG
20
1090





antiSACas9-26
+
UAAUAUGAUCGACCUCGUAG
20
1091





antiSACas9-27
+
GCGUUCUCUUUCCCGGUAGU
20
1092





antiSACas9-28

GGCGAACUGUAUAGGGUCAU
20
1093





antiSACas9-29

GCCCGAAAUCGAGACAGAAC
20
1094





antiSACas9-30

AUCUGCUGAACCGCAUUGAA
20
1095





antiSACas9-31
+
CCGUUCAGAUUGUUCACAAU
20
1096





antiSACas9-32
+
UCGGAGCUCUGGUAGAUAGU
20
1097





antiSACas9-33
+
GACCACCUUGUUGCGACUGU
20
1098





antiSACas9-34
+
UUUGUAUGCCACAGCUCAUC
20
1099





antiSACas9-35
+
GGGCUUUUUAUCCACCCGGU
20
1100





antiSACas9-36
+
GUUGAGUACUUUUUGAUACU
20
1101





antiSACas9-37

AAGAUCAAUGGCGAACUGUA
20
1102





antiSACas9-38

GAAAGUCAAGUCCAUCAACG
20
1103





antiSACas9-39
+
GGUCCCUCAUAGUAGGUUCU
20
1104





antiSACas9-40

CCUAUUUCCGGGUGAACAAU
20
1105





antiSACas9-41
+
CGCAGCAGAUUCAUCAGGCC
20
1106





antiSACas9-42
+
CAGGUUUCCCAGAAUGUCGG
20
1107





antiSACas9-43

CGAACAGAUUAGUAAUCUGA
20
1108





antiSACas9-44
+
CCAGAUUGUUCACCCGGAAA
20
1109





antiSACas9-45
+
AAAUCGUCCACCAGUGUGGU
20
1110





antiSACas9-46

CUUCGGAUGGAAAGACAUCA
20
1111





antiSACas9-47

AAAUGCCGACUUCAUCUUUA
20
1112





antiSACas9-48

UCAACAGAUUCUCCGUCCAG
20
1113





antiSACas9-49
+
GCGUUGAUCACUUUGAUGCU
20
1114





antiSACas9-50

AAGGACUACAAGUACUCUCA
20
1115





antiSACas9-51
+
CUCCGCUUGACCACGGGUGA
20
1116





antiSACas9-52

GGUGACAAGCACUGGAAAAC
20
1117





antiSACas9-53

ACCUGACCAAGUAUAGCAAA
20
1118





antiSACas9-54
+
GGAUGAAGCUCCGCUUGACC
20
1119





antiSACas9-55

CCGCAUCAGCAAGACCAAAA
20
1120





antiSACas9-56

UCCAGAAGGAUUUUAUUAAC
20
1121





antiSACas9-57
+
UUGAUAUGCUUGAUCUGGUG
20
1122





antiSACas9-58

UGUAUAAAUUUGUGACUGUC
20
1123





antiSACas9-59

CUCACCAGAUCAAGCAUAUC
20
1124





antiSACas9-60

UGCAGAAGGCUUACCACCAG
20
1125





antiSACas9-61
+
UUGAUGUCCUCUUCGUUGAC
20
1126





antiSACas9-62
+
CUAAUCUGUUCGAUCUCUUC
20
1127





antiSACas9-63
+
GACCAGCACCUUGUUGUUAA
20
1128





antiSACas9-64

GAAGAGGACAUCAAGGGCUA
20
1129





antiSACas9-65
+
UAAUAAAAUCCUUCUGGACG
20
1130





antiSACas9-66

UGGUCCCAAAAAAGGUGGAC
20
1131





antiSACas9-67
+
CUCUUCAUAGUACUUAUACA
20
1132





antiSACas9-68

AAUCUGCUGCGAUCCUAUUU
20
1133





antiSACas9-69
+
GAACUAGACAGGUACUGGAA
20
1134





antiSACas9-70
+
AAAGGUUUCGUAAGAGAUCU
20
1135





antiSACas9-71
+
CCCUUUCCUUUGGCCAGAUU
20
1136





antiSACas9-72
+
UCGUCUUUUCUUGUACUAUA
20
1137





antiSACas9-73
+
AGGAGUCCUAUUGCCCUUUU
20
1138





antiSACas9-74

UAUGAUUGACAUCACUUACC
20
1139





antiSACas9-75
+
UUCACCUCAUACAGGUUUCC
20
1140





antiSACas9-76
+
CUCCAGGGGGAUGGCCUCCA
20
1141





antiSACas9-77

UGAAAGCUAUCAAUCUGAUU
20
1142





antiSACas9-78

UCAAGUACUAUGGGAACAAG
20
1143





antiSACas9-79

UGAACAACCUGGUCAUCACC
20
1144





antiSACas9-80
+
UUGUUCAGCAGGUCCUCCAG
20
1145





antiSACas9-81

ACCGAGAGUAUCUGGAAAAC
20
1146





antiSACas9-82

GUUUAAAAAGGAGCGCAACA
20
1147





antiSACas9-83
+
CUUCAGUUUCUGAUAUGUCU
20
1148





antiSACas9-84

AAACAAUUGCCUCUAAGACU
20
1149





antiSACas9-85

GAAAAAGAUUAGCAACCAGG
20
1150





antiSACas9-86

CAGGGAUGAAAACGAGAAAC
20
1151





antiSACas9-87
+
UUCAGGUUAGUCAGCUCUUC
20
1152





antiSACas9-88
+
UUGUCGAAGGACACGCUUCU
20
1153





antiSACas9-89

AAACCUUUAAAAAGCACAUU
20
1154





antiSACas9-90

ACCAGGAGAAGGGAGCCCCU
20
1155





antiSACas9-91

GUACAAGAAAAGACGAUAAG
20
1156





antiSACas9-92

GAUGUUCGAAGAGAAGCAGG
20
1157





antiSACas9-93

AAAAGGAGAACUACUAUGAA
20
1158





antiSACas9-94

AAUUUGUGACUGUCAAGAAU
20
1159


















TABLE E-12








Suboptimal PAM-NNGRRV












5. aureus


Target
SEQ


6th Tier
DNA

Site
ID


gRNA Name
Strand
Targeting Domain
Length
NO














antiSACas9-95
+
GAAGCAGGCCGAAUCUAUGC
20
1160





antiSACas9-96
+
AGGAAUGGUACGAGAUGCUG
20
1161





antiSACas9-97
+
UCCGGGUGAACAAUCUGGAU
20
1162





antiSACas9-98
+
AGACUCGGAGAACCUACUAU
20
1163





antiSACas9-99
+
GGACGCACAGAAGAUGAUCA
20
1164





antiSACas9-100

UCACAUCCAGAUUGUUCACC
20
1165





antiSACas9-101
+
GGAGAAGGGAGCCCCUUCGG
20
1166





antiSACas9-102
+
CCGGCAACGAGCUGUCUACA
20
1167





antiSACas9-103
+
AAGUACUCAACCGACAUUCU
20
1168





antiSACas9-104
+
AAUGACACCCUGUAUAGUAC
20
1169





antiSACas9-105
+
ACUGUUCAAGGAGGCCAACG
20
1170





antiSACas9-106
+
CCGACUUCAUCUUUAAGGAG
20
1171





antiSACas9-107

GAAUCUGAACUAGACAGGUA
20
1172





antiSACas9-108
+
CGGGUGGAUAAAAAGCCCAA
20
1173





antiSACas9-109
+
ACCAGAGCUCCGAGGACAUC
20
1174





antiSACas9-110

GGUGGUACAUCAGCAGCUUC
20
1175





antiSACas9-111
+
CCGGAACACACAACCUGUCC
20
1176





antiSACas9-112

GUGCUUUUUAAAGGUUUCGU
20
1177





antiSACas9-113

UUCACAUCCAGAUUGUUCAC
20
1178





antiSACas9-114

UUCCAGAGCUUUGCUAUUGC
20
1179





antiSACas9-115
+
ACAUCUUUUCUGAGGCGCAA
20
1180





antiSACas9-116
+
AAAGCUGAUCAACAAAAGUC
20
1181





antiSACas9-117
+
GAAUCUGGAUGUCAUCAAAA
20
1182





antiSACas9-118
+
UAUAAGUACUAUGAAGAGAC
20
1183





antiSACas9-119
+
CAUCACUUACCGAGAGUAUC
20
1184





antiSACas9-120
+
UAUCAUUAUCGAGCUGGCUA
20
1185





antiSACas9-121
+
CAAGGUGCUGGUCAAGCAGG
20
1186





antiSACas9-122
+
AAAGUACUCAACCGACAUUC
20
1187





antiSACas9-123

AUUGUCGAAGGACACGCUUC
20
1188





antiSACas9-124
+
GAGUGCAUAACGUCAAUGAG
20
1189





antiSACas9-125
+
AGUAUGUCGCAGAGCUGCAG
20
1190





antiSACas9-126

GAAAUCGUCCACCAGUGUGG
20
1191





antiSACas9-127
+
ACCAUGAUCCUCAGACAUAU
20
1192





antiSACas9-128

CUUGACGCUUCUCAGCUCUU
20
1193





antiSACas9-129
+
GUAUAAGUACUAUGAAGAGA
20
1194





antiSACas9-130
+
GGGGUAUGGGAUUAUUGACU
20
1195





antiSACas9-131
+
CCCACUGUAUAAGUACUAUG
20
1196





antiSACas9-132
+
UCGAAAACGUGUUUAAGCAG
20
1197





antiSACas9-133
+
AGACCAAAAAGGAGUACCUG
20
1198





antiSACas9-134

UUAAACACGUUUUCGAUGAU
20
1199





antiSACas9-135

GCUUGACCACGGGUGACAGA
20
1200





antiSACas9-136

UUAAACUUCCAUUUGCGCCU
20
1201





antiSACas9-137
+
AUGAGGGACGGAGAAGCAAG
20
1202





antiSACas9-138

CUGACUCAGGUCCACCUUUU
20
1203





antiSACas9-139
+
GAAAGACAUCAAGGAAUGGU
20
1204





antiSACas9-140
+
UAAGGACAUCACAGCACGGA
20
1205





antiSACas9-141
+
AAAAGGUGGACCUGAGUCAG
20
1206





antiSACas9-142
+
AAUAUGAUUGACAUCACUUA
20
1207





antiSACas9-143
+
GAGAAGGGAGCCCCUUCGGA
20
1208





antiSACas9-144
+
GAUUAUCCGAACUACCGGGA
20
1209





antiSACas9-145
+
UCAAAGAAGCCAAGCAGCUG
20
1210





antiSACas9-146

GGUGAGAGUACUUGUAGUCC
20
1211





antiSACas9-147
+
ACCUGAACAGCGAGCUGACC
20
1212





antiSACas9-148
+
GACCGACCAUUCUGAGCUGA
20
1213





antiSACas9-149

GCUUGAUCUGGUGAGGAGUG
20
1214





antiSACas9-150

UGACCAGCACCUUGUUGUUA
20
1215





antiSACas9-151
+
CAAGCUGCACGAUAUGCAGG
20
1216





antiSACas9-152

UAAAGGUUUCGUAAGAGAUC
20
1217





antiSACas9-153
+
GCACCUAUUUUCCAGAAGAG
20
1218





antiSACas9-154
+
AAACGAGAAACUGGAAUACU
20
1219





antiSACas9-155
+
UGAAGAGAUUAUCCGAACUA
20
1220





antiSACas9-156
+
GGCUGAAGAAAGAUGGCGAG
20
1221





antiSACas9-157
+
GUCCAGAAGGAUUUUAUUAA
20
1222





antiSACas9-158
+
GAACAGCGAGCUGACCCAGG
20
1223





antiSACas9-159
+
AGAACCUACUAUGAGGGACC
20
1224





antiSACas9-160

AGCCAGGUGCAGCAGAGCUG
20
1225





antiSACas9-161
+
CUACUAUGAGGGACCAGGAG
20
1226





antiSACas9-162
+
GAAAACCAGAGUUCACCAAU
20
1227





antiSACas9-163
+
ACAACAAGGUGCUGGUCAAG
20
1228





antiSACas9-164
+
GCAGACCAAUGAACGCAUUG
20
1229





antiSACas9-165
+
GGAAAAAGCUGGACAAAGCC
20
1230





antiSACas9-166
+
UUCAGAUUCCAAGAUCUCUU
20
1231





antiSACas9-167
+
AGCUGCAGCUGGAACGGCUG
20
1232





antiSACas9-168
+
GAUUAUGGAGCAGUACGGCG
20
1233





antiSACas9-169

AGAAUCAGAUUGAUAGCUUU
20
1234





antiSACas9-170

UCCUCCAGGGGGAUGGCCUC
20
1235





antiSACas9-171
+
GGGAUUAUUGACUAUGAAAC
20
1236





antiSACas9-172
+
UCUCACGCAAUAGCAAAGCU
20
1237





antiSACas9-173
+
ACGUGGAAAACAAUGAGGGA
20
1238





antiSACas9-174

UUGACGCUUCUCAGCUCUUC
20
1239





antiSACas9-175
+
GAUAUCAUUAUCGAGCUGGC
20
1240





antiSACas9-176

GUUAAUAAAAUCCUUCUGGA
20
1241





antiSACas9-177
+
CAACCUGGUCAUCACCAGGG
20
1242





antiSACas9-178
+
GCACAUUCUGAAUCUGGCCA
20
1243





antiSACas9-179
+
GGUGGACCUGAGUCAGCAGA
20
1244





antiSACas9-180
+
AUAUUAAGGACAUCACAGCA
20
1245





antiSACas9-181
+
CUACAUUCUGGGGCUGGACA
20
1246





antiSACas9-182
+
GCAAGAGGGGAGCCAGGCGC
20
1247





antiSACas9-183
+
CCAGGGAUGAAAACGAGAAA
20
1248





antiSACas9-184

UAGCCAGGUGCAGCAGAGCU
20
1249





antiSACas9-185
+
GGGCUACCGGGUGACAAGCA
20
1250





antiSACas9-186
+
GGUCAUCACCAGGGAUGAAA
20
1251





antiSACas9-187
+
GAGCCAGGCGCCUGAAACGA
20
1252





antiSACas9-188
+
GCUACGAAGAGGCUAAAAAG
20
1253





antiSACas9-189
+
GGACAAAGCCAAGAAAGUGA
20
1254





antiSACas9-190

UUGGGCUUUUUAUCCACCCG
20
1255





antiSACas9-191
+
GACUGUUCAAGGAGGCCAAC
20
1256





antiSACas9-192
+
AAAAGUACUCAACCGACAUU
20
1257





antiSACas9-193
+
UAGUAAUCUGAAGGGGUACA
20
1258





antiSACas9-194
+
GGCCGAAUCUAUGCCCGAAA
20
1259





antiSACas9-195
+
UCAAGCUGCACGAUAUGCAG
20
1260





antiSACas9-196
+
CUGAACAACCUGGUCAUCAC
20
1261





antiSACas9-197
+
GGCACAGAAUCCAGAGGGUG
20
1262





antiSACas9-198
+
ACGCAAUAGCAAAGCUCUGG
20
1263





antiSACas9-199
+
AGAGAACGCAAAGUACCUGA
20
1264





antiSACas9-200
+
UCAUCACCAGGGAUGAAAAC
20
1265





antiSACas9-201
+
AAGGAGUACCUGCUGGAAGA
20
1266





antiSACas9-202
+
AGCAGAAGAAAAAGCCUACA
20
1267





antiSACas9-203
+
ACAUCACUUACCGAGAGUAU
20
1268





antiSACas9-204
+
CACAGCACGGAAAGAAAUCA
20
1269





antiSACas9-205
+
AGAAGAUGAUCAAUGAGAUG
20
1270





antiSACas9-206
+
AAGCUGCACGAUAUGCAGGA
20
1271





antiSACas9-207

AUUGUUCAGCAGGUCCUCCA
20
1272





antiSACas9-208
+
GAUAUUAAGGACAUCACAGC
20
1273





antiSACas9-209
+
CUAUGAGAAGUUCCAGAUCA
20
1274





antiSACas9-210
+
GAAGAGAUUAUCCGAACUAC
20
1275





antiSACas9-211
+
UACACUGAAACAGAUUGCUA
20
1276





antiSACas9-212
+
AGUACAAGAAAAGACGAUAA
20
1277





antiSACas9-213
+
CGGGAUUACAAGCGUGGGGU
20
1278





antiSACas9-214
+
GGCUACCGGGUGACAAGCAC
20
1279





antiSACas9-215
+
AUAUCGACCUGCUGGAGACU
20
1280





antiSACas9-216
+
UUAAUCCUUAUGAAGCCAGG
20
1281





antiSACas9-217
+
CGAGACAGAACAGGAGUACA
20
1282





antiSACas9-218
+
GAUCAAGAAGAUCAAGUACU
20
1283





antiSACas9-219
+
GCCGACUUCAUCUUUAAGGA
20
1284





antiSACas9-220
+
AAGAGAUUAUCCGAACUACC
20
1285





antiSACas9-221
+
AUGAUCAAUGAGAUGCAGAA
20
1286





antiSACas9-222
+
AGGCCAACGUGGAAAACAAU
20
1287





antiSACas9-223
+
AUCAAGAAGAUCAAGUACUA
20
1288





antiSACas9-224
+
CACAUUCUGAAUCUGGCCAA
20
1289





antiSACas9-225
+
ACGACAAAGAUAAUGACAAG
20
1290





antiSACas9-226

AAUCUGAACUAGACAGGUAC
20
1291





antiSACas9-227
+
GCCUGAGUCAGAAGCUGUCA
20
1292





antiSACas9-228
+
CGAUACUUAUAUCGACCUGC
20
1293





antiSACas9-229
+
UAGUACAAGAAAAGACGAUA
20
1294





antiSACas9-230
+
CCUUCGGAUGGAAAGACAUC
20
1295





antiSACas9-231
+
GAUGGAGAACCAGAUGUUCG
20
1296





antiSACas9-232

CGGCGCUUAGCCAGGUGCAG
20
1297





antiSACas9-233
+
AGUAAUCUGAAGGGGUACAC
20
1298





antiSACas9-234
+
CGACCUGAUUAAGAUCAAUG
20
1299





antiSACas9-235
+
AGCUAUCAAUCUGAUUCUGG
20
1300





antiSACas9-236
+
AAAGGAACUACAUUCUGGGG
20
1301





antiSACas9-237
+
GGCCAACGUGGAAAACAAUG
20
1302





antiSACas9-238
+
UCUUUAAGGAGUGGAAAAAG
20
1303





antiSACas9-239
+
AGGAGUACCUGCUGGAAGAG
20
1304





antiSACas9-240
+
UGACUAUCUACCAGAGCUCA
20
1305





antiSACas9-241

GUGGUACAUCAGCAGCUUCU
20
1306





antiSACas9-242
+
AGGAGAUCCUGGUCAACGAA
20
1307





antiSACas9-243

UGUUGUUAAAGGAAUUGUCG
20
1308





antiSACas9-244
+
ACAUUGCACCUAUUUUCCAG
20
1309





antiSACas9-245
+
GAGAACUCUAAAAAGGGCAA
20
1310





antiSACas9-246
+
AGAGCUGACUAACCUGAACA
20
1311





antiSACas9-247
+
CAUACAGAUUCGAUGUCUAU
20
1312





antiSACas9-248
+
UUCUGAAUCUGGCCAAAGGA
20
1313





antiSACas9-249

UGACUCAGGUCCACCUUUUU
20
1314





antiSACas9-250
+
AACUGUAUAGGGACAUGGGG
20
1315





antiSACas9-251

GAAUCAGAUUGAUAGCUUUC
20
1316





antiSACas9-252
+
UGGCUAGGGAGAAGAACAGC
20
1317





antiSACas9-253
+
UCCAGGAAGAGCUGACUAAC
20
1318





antiSACas9-254

CUUUGACGUAGUCGCUUGUC
20
1319





antiSACas9-255
+
AUAAGUACUAUGAAGAGACU
20
1320





antiSACas9-256

UUCCGGUUAAUAAAAUCCUU
20
1321





antiSACas9-257
+
UAGAUUCGGCCUGCUUCUCU
20
1322





antiSACas9-258

UAGAUUCGGCCUGCUUCUCU
20
1323





antiSACas9-259
+
UCAAGAAGAUCAAGUACUAU
20
1324





antiSACas9-260
+
AUUUUAUUAACCGGAAUCUG
20
1325





antiSACas9-261
+
UGGGAAACCUGUAUGAGGUG
20
1326





antiSACas9-262
+
UCAGAAACUGAAGCUGAUUA
20
1327





antiSACas9-263
+
AGGCUUACCACCAGCUGGAU
20
1328





antiSACas9-264
+
UGCUGCGAUCCUAUUUCCGG
20
1329





antiSACas9-265
+
AUAACGUCAAUGAGGUGGAA
20
1330





antiSACas9-266
+
GAACGCAUUGAAGAGAUUAU
20
1331





antiSACas9-267
+
UUCUGUCACCCGUGGUCAAG
20
1332





antiSACas9-268
+
GCGCAAAUGGAAGUUUAAAA
20
1333





antiSACas9-269

UAAACACGUUUUCGAUGAUC
20
1334





antiSACas9-270

GUACUUGAUCUUCUUGAUCA
20
1335





antiSACas9-271

AAGUCGGCAUUUGCGAUAAU
20
1336





antiSACas9-272
+
CGAGCUGACCCAGGAAGAGA
20
1337





antiSACas9-273
+
UCAAGCAUAUCAAGGAUUUC
20
1338





antiSACas9-274
+
AGGUGCUGGUCAAGCAGGAA
20
1339





antiSACas9-275
+
CUCUGGAGGCCAUCCCCCUG
20
1340





antiSACas9-276
+
ACUAUGAGGGACCAGGAGAA
20
1341





antiSACas9-277
+
GAGCUCCGAGGACAUCCAGG
20
1342





antiSACas9-278
+
CAAAAAGGAGUACCUGCUGG
20
1343





antiSACas9-279
+
AUACCCUGAUUGUGAACAAU
20
1344





antiSACas9-280
+
UCUGGAAGAGAAGUAUGUCG
20
1345





antiSACas9-281
+
AUAAGGGGAAUACCCUGAUU
20
1346





antiSACas9-282

CCAGCUCGAUAAUGAUAUCA
20
1347





antiSACas9-283
+
GGAUUAUUGACUAUGAAACA
20
1348





antiSACas9-284
+
GAGGGACGGAGAAGCAAGAG
20
1349





antiSACas9-285

CAGCUCUUCCUGGAUGUCCU
20
1350





antiSACas9-286
+
GAAAGAAAUCAUUGAGAACG
20
1351





antiSACas9-287
+
CAGCACGGAAAGAAAUCAUU
20
1352





antiSACas9-288
+
GACUCGGAGAACCUACUAUG
20
1353





antiSACas9-289

AAGAUGUGAACCCGCCGUUG
20
1354





antiSACas9-290
+
GGUGGAUAAAAAGCCCAACA
20
1355





antiSACas9-291
+
UCGGGGUGAACAAUGAUCUG
20
1356





antiSACas9-292
+
UGUACAACGCCCUGAAUGAC
20
1357





antiSACas9-293

ACUCCUGUUCUGUCUCGAUU
20
1358





antiSACas9-294
+
AUCUAUGCCCGAAAUCGAGA
20
1359





antiSACas9-295
+
GGAACAAGCUGAAUGCCCAU
20
1360





antiSACas9-296

ACCAGGUUGUUCAGGUCAUU
20
1361





antiSACas9-297
+
ACAAAGCCAAGAAAGUGAUG
20
1362





antiSACas9-298
+
CAACCUGCUGACCGACCAUU
20
1363





antiSACas9-299
+
AUCUGGAUGUCAUCAAAAAG
20
1364





antiSACas9-300
+
CCAUCCCCCUGGAGGACCUG
20
1365





antiSACas9-301

GGUCGGUCAGCAGGUUGUAA
20
1366





antiSACas9-302
+
UGAAAGUGUAUCACGAUAUU
20
1367





antiSACas9-303
+
CUAAGAUCCUGACUAUCUAC
20
1368





antiSACas9-304
+
UGGUCAAGCGGAGCUUCAUC
20
1369





antiSACas9-305
+
CGGCAACGAGCUGUCUACAA
20
1370





antiSACas9-306
+
AGCAGGAAGAGAACUCUAAA
20
1371





antiSACas9-307
+
GAAACGAAACCGGCAGACCA
20
1372





antiSACas9-308

CUUGAUGUCUUUCCAUCCGA
20
1373





antiSACas9-309
+
CCUGAUUGUGAACAAUCUGA
20
1374





antiSACas9-310
+
UUAUCGAGCUGGCUAGGGAG
20
1375





antiSACas9-311

CGUGAUACACUUUCAGAUUG
20
1376





antiSACas9-312
+
AAGAGAUCCCAACCACACUG
20
1377





antiSACas9-313

UUCGUAAGAGAUCUUGGAAU
20
1378





antiSACas9-314
+
CCUGCCCAAUGAUAUCAUUA
20
1379





antiSACas9-315
+
AUCGACCUGCUGGAGACUCG
20
1380





antiSACas9-316

UGUCAUUGAUCAGCUCUCUG
20
1381





antiSACas9-317
+
UGGAGCAGUACGGCGACGAG
20
1382





antiSACas9-318

AUUUGCGCCUCAGAAAAGAU
20
1383





antiSACas9-319

UGUUUUCCACGUUGGCCUCC
20
1384





antiSACas9-320

CGUUGUUGUAAAAGGAGGCG
20
1385





antiSACas9-321
+
AGCACCCUCAGAUUAUCAAA
20
1386





antiSACas9-322

UCUGCGACAUACUUCUCUUC
20
1387





antiSACas9-323
+
GGAAUGGUACGAGAUGCUGA
20
1388





antiSACas9-324
+
GUAUGUCGCAGAGCUGCAGC
20
1389





antiSACas9-325
+
UGGUCAACGAAGAGGACAUC
20
1390





antiSACas9-326
+
GGUGGAAGAGGACACCGGCA
20
1391





antiSACas9-327

UAAUCGUCUGUGAUGUCCAG
20
1392





antiSACas9-328
+
UUAUCCGAACUACCGGGAAA
20
1393





antiSACas9-329

AAUUGUUUUGAUAAUUCGAG
20
1394





antiSACas9-330
+
GACCAAAAAGGAGUACCUGC
20
1395





antiSACas9-331
+
CAUCAAAAAGGAGAACUACU
20
1396





antiSACas9-332
+
AGUGAAUAGCAAGUGCUACG
20
1397





antiSACas9-333
+
CGACAUUCUGGGAAACCUGU
20
1398





antiSACas9-334
+
CCGAGGAGUGCAUAACGUCA
20
1399





antiSACas9-335
+
AGGCCUGAGUCAGAAGCUGU
20
1400





antiSACas9-336
+
GCAAUAGCAAAGCUCUGGAA
20
1401





antiSACas9-337
+
ACCUAUUUUCCAGAAGAGCU
20
1402





antiSACas9-338

UCAGCUCUUCCUGGAUGUCC
20
1403





antiSACas9-339

GCAGGUCGAUAUAAGUAUCG
20
1404





antiSACas9-340

CUUGAUAUGCUUGAUCUGGU
20
1405





antiSACas9-341
+
AGUGCAUAACGUCAAUGAGG
20
1406





antiSACas9-342

AGCUUCAGUUUCUGAUAUGU
20
1407





antiSACas9-343

GGCAAUUGUUUUGAUAAUUC
20
1408





antiSACas9-344
+
CAACAAGGUGCUGGUCAAGC
20
1409





antiSACas9-345
+
AGUGAUGGAGAACCAGAUGU
20
1410





antiSACas9-346
+
GGGAAAGUGUCUGUAUUCUC
20
1411





antiSACas9-347
+
AGGGAAAGUGUCUGUAUUCU
20
1412





antiSACas9-348
+
AGAUCGAACAGAUUAGUAAU
20
1413





antiSACas9-349

GAUUGUUCAGCAGGUCCUCC
20
1414





antiSACas9-350
+
AAUCAAGCUGCACGAUAUGC
20
1415





antiSACas9-351
+
GUGGAAAACAAUGAGGGACG
20
1416





antiSACas9-352
+
GCCAGGCGCCUGAAACGACG
20
1417





antiSACas9-353
+
UCGAUACUUAUAUCGACCUG
20
1418





antiSACas9-354
+
AGGAUAAUGGCCCCGUGAUC
20
1419





antiSACas9-355
+
UCAUUAUCGAGCUGGCUAGG
20
1420





antiSACas9-356
+
AACCUACUAUGAGGGACCAG
20
1421





antiSACas9-357
+
ACAAUGAGGGACGGAGAAGC
20
1422





antiSACas9-358
+
UGAAGUGAAUAGCAAGUGCU
20
1423





antiSACas9-359
+
AUUCUCUGGAGGCCAUCCCC
20
1424





antiSACas9-360
+
CUGAAGAAAGAUGGGGAGGU
20
1425





antiSACas9-361
+
UGGCGAACUGUAUAGGGUCA
20
1426





antiSACas9-362
+
UACUAUGAGGGACCAGGAGA
20
1427





antiSACas9-363

CAAUUGUUUUGAUAAUUCGA
20
1428





antiSACas9-364
+
GCUGAGUGGAAUUAAUGGCC
20
1429





antiSACas9-365
+
CUGCUGCACCUGGCUAAGCG
20
1430





antiSACas9-366
+
CCAGAGCUCCGAGGACAUCC
20
1431





antiSACas9-367

AAUUGUCGAAGGACACGCUU
20
1432





antiSACas9-368
+
UAUAUCGACCUGCUGGAGAC
20
1433





antiSACas9-369
+
AGCUGAUCAACAAAAGUCCC
20
1434





antiSACas9-370
+
AGAAGAACAGCAAGGACGCA
20
1435





antiSACas9-371
+
UGGAGAACCAGAUGUUCGAA
20
1436





antiSACas9-372
+
AGAUUGCAAUCUUUAACCGG
20
1437





antiSACas9-373
+
UUCUCUGGAGGCCAUCCCCC
20
1438





antiSACas9-374
+
AAUGAGGGACGGAGAAGCAA
20
1439





antiSACas9-375

UCCUUGAUAUGCUUGAUCUG
20
1440





antiSACas9-376
+
GGGACAUUGCACCUAUUUUC
20
1441





antiSACas9-377
+
AAGUGAUCAACGCCAUCAUC
20
1442





antiSACas9-378
+
GCUGCACCUGGCUAAGCGCC
20
1443





antiSACas9-379
+
GGAACGGCUGAAGAAAGAUG
20
1444





antiSACas9-380

CCUUGAUGUCUUUCCAUCCG
20
1445





antiSACas9-381
+
GGAGGCCAACGUGGAAAACA
20
1446





antiSACas9-382
+
ACGGCGGGUUCACAUCUUUU
20
1447





antiSACas9-383
+
GAGGUCGAUCAUAUUAUCCC
20
1448





antiSACas9-384
+
UCGAACAGAUUCGUAAUCUG
20
1449





antiSACas9-385
+
CAAUGAUCUGCUGAACCGCA
20
1450





antiSACas9-386
+
CCUGACUAUCUACCAGAGCU
20
1451





antiSACas9-387
+
ACGAGAAACUGGAAUACUAU
20
1452





antiSACas9-388
+
CGCAGGCGUCAGACUCUUCA
20
1453





antiSACas9-389

AUUCCUUGAUGUCUUUCCAU
20
1454





antiSACas9-390
+
CCGGCAGACCAAUGAACGCA
20
1455





antiSACas9-391
+
AGAAUCUGGAUGUCAUCAAA
20
1456





antiSACas9-392
+
UGCUAAGGAGAUCCUGGUCA
20
1457





antiSACas9-393

AGUACUUGAUCUUCUUGAUC
20
1458





antiSACas9-394
+
AUAUCAUUAUCGAGCUGGCU
20
1459





antiSACas9-395
+
AAAUCAAGCUGCACGAUAUG
20
1460





antiSACas9-396
+
CAGAGCUGCAGCUGGAACGG
20
1461





antiSACas9-397
+
GAACCCACUGUAUAAGUACU
20
1462





antiSACas9-398

UGAUCACUUUGAUGCUCUGG
20
1463





antiSACas9-399
+
GCUGAACAAUCCAUUCAACU
20
1464





antiSACas9-400
+
CAUCUUUUCUGAGGCGCAAA
20
1465





antiSACas9-401
+
GCCGCAUCAGCAAGACCAAA
20
1466





antiSACas9-402
+
AUCAGAAACUGAAGCUGAUU
20
1467





antiSACas9-403
+
AUCCUCAGACAUAUCAGAAA
20
1468





antiSACas9-404
+
ACGCAGGCGUCAGACUGUUC
20
1469





antiSACas9-405

CCUUGUUGUUAAAGGAAUUG
20
1470





antiSACas9-406
+
CUCACGCAAUAGCAAAGCUC
20
1471





antiSACas9-407
+
CAAAUGCCGACUUCAUCUUU
20
1472





antiSACas9-408
+
UUAUGGAGCAGUACGGCGAC
20
1473





antiSACas9-409

GCUUUUUCCACUCCUUAAAG
20
1474





antiSACas9-410
+
UCAUCGAAAACGUGUUUAAG
20
1475





antiSACas9-411
+
CCAGGGUGAAAGGCCUGAGU
20
1476





antiSACas9-412

GGUUAAUAAAAUCCUUCUGG
20
1477





antiSACas9-413
+
ACAAGGUGGUCAAGCUGUCA
20
1478





antiSACas9-414
+
GAGAACCUACUAUGAGGGAC
20
1479





antiSACas9-415
+
CAAAGGGUACAAGCACCAUG
20
1480





antiSACas9-416
+
AACGUGGAAAACAAUGAGGG
20
1481





antiSACas9-417
+
UUCUGGGAAACCUGUAUGAG
20
1482





antiSACas9-418
+
UGAGGGACGGAGAAGCAAGA
20
1483





antiSACas9-419
+
CAAGACAAGCGACUACGUCA
20
1484





antiSACas9-420
+
GGAGCCAGGCGCCUGAAACG
20
1485





antiSACas9-421
+
ACAUCAACAGAUUCUCCGUC
20
1486





antiSACas9-422

UGAACUAGACAGGUACUGGA
20
1487





antiSACas9-423
+
CUACACUGAAACAGAUUGCU
20
1488





antiSACas9-424
+
GACGGAGAAGGCACAGAAUC
20
1489





antiSACas9-425

CUUUUUGAUACUCUGAGUCU
20
1490





antiSACas9-426
+
UGGACAAAGCCAAGAAAGUG
20
1491





antiSACas9-427
+
AUUCUGUCACCCGUGGUCAA
20
1492





antiSACas9-428
+
AUAGUACAAGAAAAGACGAU
20
1493





antiSACas9-429
+
CCUGAACAGCGAGCUGACCC
20
1494





antiSACas9-430

GAGGCAAUUGUUUUGAUAAU
20
1495





antiSACas9-431

AAUCAGGUCGUUGUUGUAAA
20
1496





antiSACas9-432

GCAAUUGUUUUGAUAAUUCG
20
1497





antiSACas9-433
+
GGCGCAAAUGGAAGUUUAAA
20
1498





antiSACas9-434
+
UCGAGACAGAACAGGAGUAC
20
1499





antiSACas9-435
+
CCAAGCAGCUGCUGAAAGUG
20
1500





antiSACas9-436
+
UGGACAUCGGGAUUACAAGC
20
1501





antiSACas9-437
+
GAAGGCACAGAAUCCAGAGG
20
1502





antiSACas9-438
+
UAAGGAGAUCCUGGUCAACG
20
1503





antiSACas9-439

UAAUCAGGUCGUUGUUGUAA
20
1504





antiSACas9-440

GGAUUGUUCAGCAGGUCCUC
20
1505





antiSACas9-441
+
GCAUAACGUCAAUGAGGUGG
20
1506





antiSACas9-442
+
GGAGACUCGGAGAACCUACU
20
1507





antiSACas9-443
+
CCUGAGUCAGAAGCUGUCAG
20
1508





antiSACas9-444
+
UGCCCGAAAUCGAGACAGAA
20
1509









Example 2: Efficacy of Governing gRNAs Targeting Cas9 Co-Transfected with a gRNA Targeting VEGF in 293T Cells

In this study, 293T cells (120,000 cells per well in a 24 well plate) were transfected with 750 ng of a plasmid expressing epitope-tagged (3× Flag-tagged) S. pyogenes Cas9 together with 125 ng of a construct expressing a gRNA targeting the VEGF gene (gRNA sequence GGTGAGUGAGUGUGUGCGUG (SEQ ID NO: 1510), see the 20mer of VEGFA Site 3 (Target Site 3) from Figure 1d of Fu et al, Improving CIRSPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol 32, 279-284 (2014)). Simultaneously, the cells were transfected with 125 ng of one of three gRNA expression constructs: a construct expressing a gRNA targeting the CCR5 gene (serving as a control), a construct expressing governing gRNA anti-SPCas9-175 (see Example 1) targeting S. pyogenes Cas9, or a construct expressing governing gRNA anti-SPCas9-1 (see Example 1) targeting S. pyogenes Cas9. Cells were harvested for analysis one day, two days, three days, six days and nine days after transfection.


To quantify mutation rates of the endogenous VEGF and plasmid-borne Cas9 genes, total genomic DNA was isolated at each time point, and regions encompassing the VEGF gRNA and Cas9 governing gRNA targeting sequences were amplified by PCR. Amplified PCR products were denatured and re-annealed, followed by treatment with T7E1 nuclease. Mutation rates (indel frequency) were measured using a capillary electrophoresis instrument as described in Reyon, D. et al, FLASH assembly of TALENs for high-throughput genome editing. Nat Biotechnol 30, 460-465 (2012). Mutation frequencies (% indels±standard deviation) of VEGF and Cas9 for the three treatment groups are shown in Table E-13. These results confirm that both Cas9-targeted governing gRNAs induce mutations in the Cas9 gene, and that the endogenous VEGF locus is mutated to a similar extent in the presence or absence of a co-transfected Cas9-targeted governing gRNA.


To assess levels of Cas9 protein during the time course of the experiment, total protein lysates were prepared from each treatment group at each time point. Protein samples (15 ug) were separated by SDS-PAGE, blotted to PVDF membrane, and probed with an antibody specific for the 3×Flag epitope tag. An antibody specific for cytoskeletal actin was used as a loading control. As shown in FIG. 7, co-transfection with each of the Cas9-targeted governing gRNAs leads to reduced levels of Cas9 protein, especially at six and nine days following transfection.









TABLE E-13







VEGF and Cas9 mutation rates













Co-







transfected
target





Group
gRNA
locus
time point
% indel
std. dev.















1
CCR5
VEGF
day 1
14.6
0.39



(control)

day 2
30.6
5.16





day 3
23.4
1.38





day 6
25.7
n.d.





day 9
15.9
1.37




Cas9
day 1
n.a.
n.a.





day 2
n.a.
n.a.





day 3
n.a.
n.a.





day 6
n.a.
n.a.





day 9
n.a.
n.a.


2
Cas9
VEGF
day 1
9.7
1.33



governing

day 2
21.6
9.76



gRNA (anti-

day 3
23.5
0.77



SPCas9-175)

day 6
25.5
2.37





day 9
15.6
n.d.




Cas9
day 1
6.6
0.83





day 2
7.4
n.d.





day 3
11.7
1.19





day 6
29.1
4.19





day 9
24.0



3
Cas9
VEGF
day 1
13.2
2.84



governing

day 2
18.2
3.35



gRNA (anti-

day 3
26.9
2.13



SPCas9-1)

day 6
16.8
0.49





day 9
22.4
0.48




Cas9
day 1
0
0





day 2
10.1
1.15





day 3
11.7
n.d.





day 6
16.5
0.99





day 9
21.4
1.44









Example 3: Activity Comparison of S. aureus gRNAs of Various Lengths

In this study, HEK-293T cells stably expressing GFP were co-transfected with constructs expressing gRNAs with targeting domains of various lengths (from 15-20 nucleotides) together with a construct expressing S. aureus Cas9. The gRNAs targeted several different genes: VEGF (total of 22 gRNAs), CCR5 (total of 15 gRNAs) and GFP (total of 10 gRNAs). The targeting domains of all the tested gRNAs initiated with a G nucleotide, and all of the gRNA target sites were associated with NNGRRT PAM sequences.


To quantify activity of the VEGF and CCR5 targeting gRNAs, total genomic DNA was isolated from cells two days following transfection and regions encompassing the VEGF and CCR5 gRNA target sites were amplified by PCR. Amplified PCR products were denatured and re-annealed, followed by treatment with T7E1 nuclease. Mutation rates (indel frequency) were measured using a capillary electrophoresis instrument as described in Reyon, D. et al., FLASH assembly of TALENs for high-throughput genome editing. Nat Biotechnol 30.460-465 (2012). To quantify activity of the GFP targeting gRNAs, cells were harvested three days following transfection and the percentage of OPP-negative cells (indicating mutation of the GFP gene) were measured by flow cytometry. The mean activity of all gRNAs of each targeting domain length was calculated and compared to the mean activity of the gRNAs with 20 nucleotide targeting domains. As shown in Table E-14, gRNAs with shorter targeting domains have lower average activity than those with 20 nucleotide targeting domains.









TABLE E-14







Mean activity of S. aureus gRNAs with various length targeting


domains compared to gRNAs with 20 nucleotide targeting domains.









Targeting Domain
Mean Activity Compared to



Length
20 nt Targeting Domain
st. dev.












20
1
0.179


19
0.395
0.126


18
0.244
0.087


17
0.028
0.014


16
0.012
0.005


15
0.005
0.004








Claims
  • 1. A composition comprising: (a) a nucleic acid encoding a Cas9 molecule, wherein the Cas9 molecule is an S. aureus or S. pyogenes Cas9 molecule, and a nucleic acid encoding a first guide RNA (gRNA) molecule; and(b) a governing gRNA molecule that comprises a nucleotide sequence targeting domain that targets said nucleic acid encoding said Cas9 molecule and/or said nucleic acid encoding said first gRNA molecule, wherein the nucleotide sequence targeting domain is 15 to 30 nucleotides in length, and
  • 2. The composition of claim 1, wherein the nucleotide sequence targeting domain of the governing gRNA molecule is a nucleotide sequence targeting domain that provides a Cas9 molecule-mediated cleavage event in the Cas9 molecule-amino acid coding sequence of the nucleic acid sequence or in the gRNA molecule-coding sequence of the nucleic acid encoding said first gRNA molecule.
  • 3. The composition of claim 1, wherein the nucleotide sequence targeting domain of the governing gRNA molecule is a nucleotide sequence targeting domain that provides a Cas9 molecule-mediated cleavage event in a non-coding sequence of the nucleic acid encoding the Cas9 molecule.
  • 4. The composition of claim 1, wherein the nucleotide sequence targeting domain of the governing gRNA molecule is a nucleotide sequence targeting domain that provides a Cas9 molecule-mediated cleavage event in an untranslated sequence of the nucleic acid encoding the Cas9 molecule.
  • 5. The composition of claim 1, wherein the nucleotide sequence targeting domain of the governing gRNA molecule is a nucleotide sequence targeting domain that provides a Cas9 molecule-mediated cleavage event 5′ of the Cas9 molecule-coding region of the nucleic acid encoding the Cas9 molecule.
  • 6. The composition of claim 1, wherein the nucleotide sequence targeting domain of the governing gRNA molecule is a nucleotide sequence targeting domain that provides a Cas9 molecule-mediated cleavage event 3′ of the Cas9 molecule-coding region of the nucleic acid encoding the Cas9 molecule.
  • 7. The composition of claim 1, wherein the nucleotide sequence targeting domain of the governing gRNA molecule is a nucleotide sequence targeting domain that provides a Cas9 molecule-mediated cleavage event in the promoter region of the nucleic acid encoding the Cas9 molecule, wherein the promoter region is functionally linked to the Cas9 molecule amino acid coding region.
  • 8. The composition of claim 1, wherein the nucleotide sequence targeting domain of the governing gRNA molecule is a nucleotide sequence targeting domain that provides a Cas9 molecule-mediated cleavage event in an intronic sequence of the nucleic acid encoding the Cas9 molecule.
  • 9. The composition of claim 1, wherein the governing gRNA targets a naturally occurring sequence in the nucleic acid encoding the Cas9 and/or the first gRNA.
  • 10. The composition of claim 1, wherein the gRNA is a chimeric gRNA.
  • 11. The composition of claim 1, wherein the gRNA is a modular gRNA.
  • 12. The composition of claim 1, wherein the nucleic acid encoding said Cas9 molecule is carried by a first vector.
  • 13. The composition of claim 12, wherein the nucleic acid encoding said gRNA molecule is carried by a second vector.
  • 14. A composition comprising: a first nucleic acid encoding a Cas9 molecule, wherein the Cas9 molecule is an S. aureus or S. pyogenes Cas9 molecule; and a governing gRNA molecule or a second nucleic acid encoding a governing gRNA molecule, the governing gRNA comprising a nucleotide sequence targeting domain that targets said nucleic acid encoding said Cas9 molecule and/or a nucleic acid encoding a first gRNA molecule, wherein the nucleotide sequence targeting domain is 15 to 30 nucleotides in length, andwherein the nucleotide sequence targeting domain of the governing gRNA molecule is selected from the group consisting of: a nucleotide sequence targeting domain that provides a Cas9 molecule-mediated cleavage event in the Cas9 molecule-amino acid coding sequence of the nucleic acid sequence or in the gRNA molecule-coding sequence of the nucleic acid encoding said first gRNA molecule;a nucleotide sequence targeting domain that provides a Cas9 molecule-mediated cleavage event in a non-coding sequence of the nucleic acid encoding the Cas9 molecule;a nucleotide sequence targeting domain that provides a Cas9 molecule-mediated cleavage event in an untranslated sequence of the nucleic acid encoding the Cas9 molecule;a nucleotide sequence targeting domain that provides a Cas9 molecule-mediated cleavage event 5′ of the Cas9 molecule-coding region of the nucleic acid encoding the Cas9 molecule;a nucleotide sequence targeting domain that provides a Cas9 molecule-mediated cleavage event 3′ of the Cas9 molecule-coding region of the nucleic acid encoding the Cas9 molecule;a nucleotide sequence targeting domain that provides a Cas9 molecule-mediated cleavage event in the promoter region of the nucleic acid encoding the Cas9 molecule, wherein the promoter region is functionally linked to the Cas9 molecule amino acid coding region; anda nucleotide sequence targeting domain that provides a Cas9 molecule-mediated cleavage event in an intronic sequence of the nucleic acid encoding the Cas9 molecule.
  • 15. The composition of claim 14, wherein the gRNA is a chimeric gRNA.
  • 16. The composition of claim 14, wherein the gRNA is a modular gRNA.
  • 17. The composition of claim 14, wherein the nucleic acid encoding said Cas9 molecule is carried by a first vector.
  • 18. The composition of claim 17, wherein the nucleic acid encoding said gRNA molecule is carried by a second vector.
  • 19. The composition of claim 14, further comprising at least one of a gRNA molecule that targets a sequence in the genome of the cell or subject and a nucleic acid encoding a gRNA molecule that targets a sequence in the genome of the cell.
  • 20. The composition of claim 14, wherein the nucleotide sequence targeting domain targets a nucleic acid sequence engineered into the first nucleic acid encoding a Cas9 molecule, the governing gRNA molecule, or the second nucleic acid encoding a governing gRNA molecule.
  • 21. A composition comprising: a first nucleic acid encoding a Cas9, wherein the Cas9 molecule is an S. aureus or S. pyogenes Cas9 molecule;a second nucleic acid encoding a first guide RNA (gRNA) targeted to a sequence in a genome; anda third nucleic acid encoding a governing gRNA targeted to said nucleic acid encoding said Cas9, wherein the governing gRNA comprises a nucleotide sequence targeting domain that is 15 to 30 nucleotides in length.
  • 22. The composition of claim 21, wherein the first nucleic acid is carried by a first vector.
  • 23. The composition of claim 22, wherein the second nucleic acid is carried by a second vector.
  • 24. The composition of claim 21, wherein the nucleic acid encoding the Cas9 and the nucleic acid encoding the governing gRNA are carried on the same vector.
  • 25. A composition comprising (a) a nucleic acid encoding a first guide RNA (gRNA) that targets a nucleic acid of a eukaryotic cell; and(b) a nucleic acid encoding a Cas9 molecule, wherein the Cas9 molecule is an S. aureus or S. pyogenes Cas9 molecule, and encoding a governing gRNA molecule, wherein the governing gRNA molecule comprises a nucleotide sequence targeting domain that targets(i) said nucleic acid encoding said Cas9 molecule, or(ii) said nucleic acid encoding said first gRNA,wherein the governing gRNA has a sequence that targets (i) or (ii) for cleavage, wherein the nucleotide sequence targeting domain is 15 to 30 nucleotides in length.
  • 26. The composition of claim 25, wherein the nucleotide sequence targeting domain targets a nucleic acid sequence engineered into (i) or (ii).
CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. Ser. No. 14/536,319 filed Nov. 7, 2014, now granted as U.S. Pat. No. 9,834,791, which claims the benefit of U.S. Provisional Application No. 61/901,215, filed Nov. 7, 2013, the contents of which are hereby incorporated by reference in their entirety.

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Related Publications (1)
Number Date Country
20180127783 A1 May 2018 US
Provisional Applications (1)
Number Date Country
61901215 Nov 2013 US
Divisions (1)
Number Date Country
Parent 14536319 Nov 2014 US
Child 15825616 US