Crispr enzymes, methods, systems and uses thereof

Abstract

The present invention provides novel systems, methods, and compositions for the preparation and use of recombinant engineered novel Cas9 enzymes optimized for human cells for nucleic acid targeting and manipulation. The invention is based on the discovery of a novel Cas9 enzyme from the following items: streptococcus equi ATCC 33317, enterococcus hirsuti strain F1129E, streptococcus equi strain AG46, staphylococcus pseudomonas strain 19, streptococcus intermedius B196 strain G1552, streptococcus sanguis SK330, streptococcus C150, oral streptococcus subspecies oral strain RH173508, oral streptococcus SK313, staphylococcus warneri strain 691 and staphylococcus squirrel strain SNUC 2430. The present invention relates to novel Cas9 enzymes for use in human cells by codon optimization and recombinantly production, e.g., streptococcus gallate strain AM24-4, Lactobacillus curapae strain Biut2, and Streptococcus suis strain LSS83 bacteria, which novel Cas9 enzymes are codon optimized and recombinantly produced for use in human cells. In some embodiments, a novel Cas9 enzyme may be used for base editing. In some embodiments, the novel engineered Cas9 enzymes are used to treat human diseases.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 291,252 filed on December 17, 2021, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0003] Incorporation by Reference into the Sequence Listing

[0004] The contents of the file named "BEM-013WO1_SL.xml", created on November 10, 2022 and with a size of 239 kilobytes, are hereby incorporated by reference in their entirety. Background Art

[0005] Enzymes from the prokaryotic clustered, regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (CRISPR-Cas) system have been used as reprogrammable and highly specific genome editing tools for use in eukaryotic organisms. In addition to genome editing and cleavage, CRISPR-Cas9 can also be used to target effector molecules to specific sites on the genome, allowing genetic and epigenetic regulation and transcriptional modulation through a variety of mechanisms.

[0006] However, different genomes and genomic targets require multiple tools for efficient genetic engineering, and there is still a need to expand the CRISPR toolbox by discovering and engineering novel Cas proteins that can recognize and target different sequences.

[0007] Although the CRISPR-Cas9 system can be used to knock out genes or modify the expression of genes, some types of gene editing require precise modifications to the target gene, such as editing a single base within a gene. Such precise modifications remain a challenge and require a diverse gene editing toolkit to achieve precise genome modifications of multiple target genes. Summary of the invention

[0008] The identification of novel Cas9 enzymes specific for unique protospacer adjacent motifs (PAMs) allows for the expansion of available tools for gene editing. The present invention provides, among other things, engineered, non-naturally occurring novel Cas9 enzymes isolated from Streptococcus equi ATCC 33317, Enterococcus hirae strain F1129E, Streptococcus equi strain AG46, Staphylococcus pseudostrain 19, Streptococcus intermedius B196 strain G1552, Streptococcus sanguinis SK330, Streptococcus C150, Streptococcus oralis subspecies oral strain RH_1735_08, Streptococcus oralis SK313, Staphylococcus wortii strain 691, Staphylococcus squirri strain SNUC 2430, Streptococcus gallolyticus strain AM24-4, Lactobacillus kuraburgii strain Biut2, and Streptococcus suis strain LSS83.

[0009] The present invention is based in part on the surprising discovery that novel Cas9 enzymes that recognize specific PAM sequences found in different bacteria can be engineered for expression in eukaryotic cells (e.g., humans, plants, etc.). Thus, the described Cas9 enzymes and variants thereof are functional in eukaryotic organisms. The examples provided herein show the use of engineered non-natural Cas9 enzymes to target different genomic sites in human cells with different PAM recognition sequences. For example, the Cas9 engineered by Streptococcus equi ATCC33317 recognizes the consensus PAM sequence 5'-NRGNR-3', the Cas9 engineered by Enterococcus hirae strain F1129E recognizes the consensus PAM sequence 5'-NRG-3', the Cas9 engineered by Streptococcus equi AG46, Staphylococcus worderi strain 691, and Staphylococcus squirrel strain SNUC recognizes the consensus PAM sequence 5'-NRGNR-3'. 2430 engineered Cas9 recognized the consensus PAM sequence 5'-NNGR-3', Cas9 engineered by Staphylococcus aureus strain 19 recognized the consensus PAM sequence 5'-NNGRRT-3', Cas9 engineered by Streptococcus intermedius B196 strain G1552 recognized the consensus PAM sequence 5'-NNAAAA-3', Cas9 engineered by Streptococcus sanguis SK330 recognized the consensus PAM sequence 5'-NGGNG-3', Cas9 engineered by Streptococcus sp. C150 recognized the consensus PAM sequence 5'-NNGNRG-3', and Cas9 engineered by Streptococcus oralis subsp. oral strain RH_1735_ 08 engineered Cas9 recognizes the consensus PAM sequence 5'-NNAAAC-3', the Cas9 engineered by oral Streptococcus SK313 recognizes the consensus PAM sequence 5'-NNRAAG-3', the Cas9 engineered by Streptococcus gallolyticus strain AM24-4 recognizes the consensus PAM sequence 5'-NNAYAA-3', the Cas9 engineered by Lactobacillus kuraburgensis strain Biut2 recognizes the consensus PAM sequence 5'-NNGAAA-3', and the Cas9 engineered by Streptococcus suis strain recognizes the consensus PAM sequence 5'-NNAAA-3' (H = A, C or T; R = A or G).

[0010] In one aspect, provided herein is an engineered, non-naturally occurring Cas9 protein modified by: Streptococcus equi ATCC 33317 Cas9, Enterococcus hirae strain F1129E Cas9, Streptococcus equi strain AG46 Cas9, Staphylococcus aureus strain 19 Cas9, Streptococcus intermedius B196 strain G1552, Streptococcus sanguis SK330 Cas9, Streptococcus orals subsp. oral strain RH_1735_08Cas9, Streptococcus orals SK313 Cas9, Staphylococcus worderi strain 691 Cas9, Staphylococcus squirus strain SNUC 2430 Cas9, Streptococcus gallolyticus strain AM24-4 Cas9, Lactobacillus kuraburgii strain Biut2 Cas9, and Streptococcus suis strain LSS83 Cas9.

[0011] In some embodiments, the Streptococcus equi ATCC 33317 Cas9 protein has at least 80% sequence identity to

[0012]

[0013]

[0014] In some embodiments, the Enterococcus hirae strain F1129E Cas9 protein has at least 80% sequence identity to

[0015]

[0016]

[0017] In some embodiments, the S. equi strain AG46 Cas9 protein has at least 80% sequence identity to

[0018]

[0019] In some embodiments, the Staphylococcus aureus strain 19 Cas9 protein has at least 80% sequence identity to

[0020]

[0021]

[0022] In some embodiments, the S. intermedius B196 strain G1552 Cas9 protein has at least 80% sequence identity to

[0023]

[0024] In some embodiments, the Streptococcus sanguinis SK330 Cas9 protein has at least 80% sequence identity to

[0025]

[0026] In some embodiments, the Streptococcus C150 Cas9 protein has at least 80% sequence identity to

[0027]

[0028]

[0029] In some embodiments, the S. oralis subsp. oral strain RH_1735_08Cas9 protein has at least 80% sequence identity to

[0030]

[0031] In some embodiments, the S. oralis SK313 Cas9 protein has at least 80% sequence identity to

[0032]

[0033] In some embodiments, the Staphylococcus worderi strain 691 Cas9 protein has at least 80% sequence identity to

[0034]

[0035] In some embodiments, the Staphylococcus sciuri strain SNUC 2430 Cas9 protein has at least 80% sequence identity to

[0036]

[0037]

[0038] In some embodiments, the S. gallolyticus strain AM24-4 Cas9 protein has at least 80% sequence identity to

[0039]

[0040] In some embodiments, the Lactobacillus kuraburgii strain Biut2 Cas9 protein has at least 80% sequence identity to

[0041]

[0042]

[0043] In some embodiments, the S. suis strain LSS83 Cas9 protein has at least 80% sequence identity to

[0044]

[0045]

[0046] In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0047] In some embodiments, the Cas9 protein further comprises a nuclear localization sequence (NLS) and / or a FLAG, HIS or HA tag.

[0048] In some embodiments, Streptococcus equi ATCC 33317 Cas9 (Seq2Cas9) has an amino acid sequence at least 80% identical to

[0049]

[0050]

[0051] In some embodiments, the Enterococcus hirae strain F1129E Cas9 (EhiCas9) has an amino acid sequence at least 80% identical to

[0052]

[0053] (D10A mutation, bold, underlined, italic)

[0054] In some embodiments, Streptococcus equi strain AG46 (SeqCas9) has an amino acid sequence at least 80% identical to

[0055]

[0056]

[0057] In some embodiments, Staphylococcus aureus strain 19 (SsiCas9) has an amino acid sequence at least 80% identical to

[0058]

[0059]

[0060] In some embodiments, Streptococcus intermedius B196 strain G1552 (SinCas9) has an amino acid sequence at least 80% identical to

[0061]

[0062] In some embodiments, the Streptococcus sanguinis SK330 Cas9 has an amino acid sequence at least 80% identical to

[0063]

[0064] In some embodiments, the Streptococcus C150 Cas9 has an amino acid sequence at least 80% identical to

[0065]

[0066] In some embodiments, S. oralis subsp. oral strain RH_1735_08Cas9 has an amino acid sequence at least 80% identical to

[0067]

[0068] In some embodiments, the S. oralis SK313 Cas9 has an amino acid sequence at least 80% identical to

[0069]

[0070]

[0071] In some embodiments, the S. oralis SK313 Cas9 has an amino acid sequence at least 80% identical to

[0072]

[0073] In some embodiments, Staphylococcus worderi strain 691 Cas9 has an amino acid sequence at least 80% identical to

[0074]

[0075]

[0076] In some embodiments, the S. gallolyticus strain AM24-4 Cas9 has an amino acid sequence at least 80% identical to

[0077]

[0078] In some embodiments, Lactobacillus kuraburgii strain Biut2 Cas9 has an amino acid sequence at least 80% identical to

[0079]

[0080] In some embodiments, the S. suis strain LSS83 Cas9 has an amino acid sequence at least 80% identical to

[0081]

[0082]

[0083] In some embodiments, the amino acid sequence of the Cas9 protein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least 10 mutations of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0084] In some embodiments, the mutation is an amino acid substitution.

[0085] In some embodiments, the Cas9 protein has nickase activity.

[0086] In some embodiments, at least one mutation produces an inactivated Cas9 (dCas9).

[0087] In some embodiments, the Cas9 protein comprises at least one amino acid mutation in the PAM interaction domain, HNH and / or RuvC domain.

[0088] In some embodiments, the Cas9 protein further comprises a nuclear localization sequence (NLS) and / or a FLAG, HIS or HA tag.

[0089] In one aspect, provided herein is an engineered, non-naturally occurring Cas9 fusion protein comprising a Cas9 protein having at least 80% identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, and wherein the Cas9 protein is fused to a histone demethylase, a transcriptional activator, or to a deaminase.

[0090] In some embodiments, the Cas9 protein is fused to a cytosine deaminase or to an adenosine deaminase.

[0091] In some embodiments, the Cas9 protein is fused to an adenosine deaminase and has an amino acid sequence at least 80% identical to

[0092] (a)

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104] In some embodiments, the Cas9 protein is fused to a cytosine deaminase.

[0105] In some embodiments, the Streptococcus equi ATCC 33317 Cas9 protein recognizes a PAM consensus sequence comprising 5'-NRGNR-3'.

[0106] In some embodiments, Enterococcus hirae strain F1129E recognizes a PAM consensus sequence comprising 5'-NRG-3'. In some embodiments, Streptococcus equi strain AG46 recognizes a PAM consensus sequence comprising 5'-NNGR-3'.

[0107] In some embodiments, Staphylococcus worderi strain 691 recognizes a PAM consensus sequence comprising 5'-NNGR-3'.

[0108] In some embodiments, S. sciuri strain SNUC 2430 recognizes a PAM consensus sequence comprising 5'-NNGR-3'.

[0109] In some embodiments, Staphylococcus acuminata strain 19 recognizes a PAM consensus sequence comprising 5'-NNGRRT-3'.

[0110] In some embodiments, S. intermedius B196 strain G1552 recognizes a consensus PAM sequence comprising 5'-NNAAAA-3'.

[0111] In some embodiments, S. sanguis SK330 recognizes a consensus PAM sequence comprising 5'-NGGNG-3'.

[0112] In some embodiments, Streptococcus C150 recognizes a consensus PAM sequence comprising 5'-NNGNRG-3'.

[0113] In some embodiments, S. oralis subsp. oral strain RH_1735_08 recognizes a consensus PAM sequence comprising 5'-NNAAAC-3'.

[0114] In some embodiments, S. oralis SK313 recognizes a consensus PAM sequence comprising 5'-NNRAAG-3'.

[0115] In some embodiments, S. gallolyticus strain AM24-4 recognizes a consensus PAM sequence comprising 5'-NNAYAA-3'.

[0116] In some embodiments, Lactobacillus kuraburgii strain Biut2 recognizes a consensus PAM sequence comprising 5'-NNGAAA-3'.

[0117] In some embodiments, the S. suis strain recognizes a consensus PAM sequence comprising 5'-NNAAA-3' (H = A, C, or T; R = A or G).

[0118] In some embodiments, nucleic acids encoding Cas9 proteins are provided.

[0119] In some embodiments, the nucleic acid is codon-optimized for expression in mammalian cells.

[0120] In some embodiments, the nucleic acid is codon-optimized for expression in human cells.

[0121] In some embodiments, eukaryotic cells comprising a Cas9 protein are provided.

[0122] In some embodiments, the cell is a human cell. In some embodiments, the cell is a plant cell.

[0123] In one aspect, the present invention provides a method for cleaving a target nucleic acid in a eukaryotic cell, the method comprising: contacting the cell with Cas9 as described herein and an RNA guide or a nucleic acid encoding an RNA guide, wherein the RNA guide comprises a positive sequence repeat sequence and a spacer sequence capable of hybridizing to the target nucleic acid, and wherein the Cas9 protein is capable of binding to the RNA guide and causing a break in a target nucleic acid sequence that is complementary to the RNA guide.

[0124] In one aspect, the invention provides a method for altering expression of a target nucleic acid in a eukaryotic cell, the method comprising: contacting the cell with Cas9 as described herein and an RNA guide or a nucleic acid encoding an RNA guide, wherein the RNA guide comprises a positive sequence repeat sequence and a spacer sequence capable of hybridizing to the target nucleic acid, and wherein the Cas9 protein is capable of binding to the RNA guide and causing a break in a target nucleic acid sequence that is complementary to the RNA guide.

[0125] In one aspect, the present invention provides a method for altering expression of a target nucleic acid in a eukaryotic cell, the method comprising: contacting the cell with Cas9 as described herein and an RNA guide or a nucleic acid encoding an RNA guide, wherein the RNA guide comprises a positive sequence repeat sequence and a spacer sequence capable of hybridizing to the target nucleic acid, and wherein the Cas9 protein is capable of binding to the RNA guide and editing a target nucleic acid sequence that is complementary to the RNA guide.

[0126] In one aspect, the present invention provides a method for modifying a target nucleic acid in a eukaryotic cell, the method comprising: contacting the cell with Cas9 as described herein and an RNA guide or a nucleic acid encoding an RNA guide, wherein the RNA guide comprises a positive sequence repeat sequence and a spacer sequence capable of hybridizing to the target nucleic acid, and wherein the Cas9 protein is capable of binding to the RNA guide and editing a target nucleic acid sequence complementary to the RNA guide.

[0127] In some embodiments, the Cas9 protein is a deactivated Cas9 (dCas9).

[0128] In some embodiments, dCas9 is fused to a deaminase.

[0129] In some embodiments, the RNA guide comprises crRNA and tracrRNA.

[0130] In some embodiments, the RNA guide comprises a sgRNA.

[0131] In some embodiments, the sgRNA used with Seq2Cas9 comprises a scaffold comprising Sequences that are at least 80% identical.

[0132] In some embodiments, the sgRNA used with EhiCas9 comprises a scaffold comprising Sequences that are at least 80% identical.

[0133] In some embodiments, the sgRNA used with SeqCas9 comprises a scaffold comprising Sequences that are at least 80% identical.

[0134] In some embodiments, the sgRNA used with SsiCas9 comprises a scaffold comprising Sequences that are at least 80% identical.

[0135] In some embodiments, the sgRNA used with SinCas9 comprises a scaffold comprising a 5'- Sequences that are at least 80% identical.

[0136] In some embodiments, the sgRNA used with SsaCas9 comprises a scaffold comprising Sequences that are at least 80% identical.

[0137] In some embodiments, the sgRNA used with Ssc2Cas9 comprises a scaffold comprising Sequences that are at least 80% identical.

[0138] In some embodiments, the sgRNA used with Sor2Cas9 comprises a scaffold comprising Sequences that are at least 80% identical.

[0139] In some embodiments, the sgRNA used with SorCas9 comprises a scaffold comprising ID NO:51) with at least 80% identity.

[0140] In some embodiments, the sgRNA used with SwaCas9 comprises a scaffold comprising Sequences that are at least 80% identical.

[0141] In some embodiments, the sgRNA used with SscCas9 comprises a scaffold comprising Sequences that are at least 80% identical.

[0142] In some embodiments, the sgRNA used with SgaCas9 comprises a scaffold comprising a 5'-

[0143] Sequences that are at least 80% identical.

[0144] In some embodiments, the sgRNA used with LkuCas9 comprises a scaffold comprising Sequences that are at least 80% identical.

[0145] In some embodiments, the sgRNA used with SsuCas9 comprises a scaffold comprising a 5'-

[0146] 56) sequences that are at least 80% identical.

[0147] In the foregoing examples, for SEQ ID NOs 43-56: Direct repetition (italic and underlined), four rings (italic), tracrRNA (underline)

[0148] In some embodiments, the crRNA comprises a guide sequence that is between 16 nucleotides long and 26 nucleotides long.

[0149] In some embodiments, the crRNA comprises a guide sequence that is between 18 nucleotides long and 24 nucleotides long.

[0150] In some embodiments, the breaks in the target nucleic acid are single-stranded or double-stranded breaks.

[0151] In some embodiments, the breaks in the target nucleic acid are single stranded breaks.

[0152] In some embodiments, the Cas9 protein is a nuclease that cuts both strands of the target nucleic acid sequence. In some embodiments, Cas9 is a nickase that cuts one strand of the target nucleic acid sequence.

[0153] In some embodiments, the target nucleic acid is 5' to a protospacer adjacent motif (PAM) sequence.

[0154] In some embodiments, the Cas9 is operably linked to a promoter sequence for expression in a eukaryotic cell, and wherein the guide RNA is operably linked to a promoter sequence for expression in a eukaryotic cell.

[0155] In some embodiments, the eukaryotic cell is a human cell.

[0156] In some embodiments, the promoter sequence is a eukaryotic promoter or a viral promoter.

[0157] In one aspect, provided herein is an engineered, non-naturally occurring CRISPR-Cas system comprising: an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a positive sequence repeat sequence and a spacer sequence capable of hybridizing to a target nucleic acid; and a codon-optimized CRISPR-associated (Cas) protein having at least 80% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, and wherein the Cas protein is capable of binding to the RNA guide and causing a break in a target nucleic acid sequence that is complementary to the RNA guide.

[0158] In one aspect, provided herein is an engineered, non-naturally occurring CRISPR-Cas system comprising: an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a positive sequence repeat sequence and a spacer sequence capable of hybridizing to a target nucleic acid; and a codon-optimized CRISPR-associated (Cas) protein having at least 80% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14; wherein the Cas protein is fused to a deaminase, and wherein the Cas protein fusion is capable of binding to the RNA guide and editing a target nucleic acid sequence that is complementary to the RNA guide.

[0159] In one embodiment, the Cas9 protein is a deactivated Cas9 (dCas9).

[0160] In one embodiment, the RNA guide comprises crRNA and tracrRNA.

[0161] In one embodiment, the RNA guide comprises a sgRNA.

[0162] In one embodiment, the Cas protein is operably linked to a promoter sequence for expression in a eukaryotic cell, and wherein the guide RNA is operably linked to a promoter sequence for expression in a eukaryotic cell.

[0163] In some embodiments, the eukaryotic cell is a human cell.

[0164] In one embodiment, the promoter sequence is a eukaryotic promoter sequence.

[0165] In one embodiment, nucleic acids encoding the systems described herein are provided.

[0166] In one embodiment, a vector comprising a system described herein is provided.

[0167] In one embodiment, the vector is a plasmid vector or a viral vector.

[0168] In one embodiment, the viral vector is an adeno-associated viral (AAV) vector or a lentiviral vector.

[0169] In some embodiments, the viral vector is an AAV vector.

[0170] In one embodiment, more than one AAV vector is used to package the system.

[0171] In one embodiment, a method of treating a disorder or disease in a subject in need thereof comprises administering to the subject a system described herein, wherein the guide RNA is complementary to at least 10 nucleotides of a target nucleic acid, the target nucleic acid being associated with the disorder or disease; wherein the Cas protein is associated with the guide RNA; wherein the guide RNA binds to the target nucleic acid; wherein the Cas protein causes a break in the target nucleic acid, optionally wherein the Cas9 is an inactivated Cas9 (dCas9) fused to a deaminase and causes one or more base edits in the target nucleic acid, thereby treating the disorder or disease.

[0172] In some embodiments, the guide RNA is complementary to about 18 to 24 nucleotides.

[0173] In some embodiments, the guide RNA is complementary to 20 nucleotides.

[0174] In some embodiments, the base editor comprises a fusion protein.

[0175] In some embodiments, the base editor comprises an adenosine deaminase domain or a cytidine deaminase domain.

[0176] In some embodiments, provided herein is a method for editing a nucleobase of a polynucleotide, the method comprising contacting the polynucleotide with a base, the base being complexed with one or more guide RNAs, wherein the base editor comprises an adenosine deaminase domain, and wherein the one or more guide RNAs target the base editor to effect an A·T to G·C change in the polynucleotide.

[0177] In some embodiments, provided herein is a method for editing a nucleobase of a polynucleotide, the method comprising contacting the polynucleotide with a base editor, the base editor being complexed with one or more guide RNAs, wherein the base editor comprises a cytidine deaminase domain, and wherein the one or more guide RNAs target the base editor to effect a C·G to T·A change in the polynucleotide.

[0178] In some embodiments, editing results in the formation of less than 50% indels in the target polynucleotide sequence.

[0179] In some embodiments, editing produces a point mutation.

[0180] definition

[0181] In order to more readily understand the present invention, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

[0182] The articles "a" and "an" herein refer to one or more than one (ie, to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

[0183] Approximately or about: As used herein, the term "approximately" or "about" refers to a value similar to the reference value when applied to one or more values ​​of interest. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that are 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater or less than) the reference value, unless otherwise stated or apparent from the context (unless the number would exceed 100% of a possible value).

[0184] Associated with: Two events or entities are "associated with" one another, as that term is used herein, if the presence, level, and / or form of one event or entity is related to the presence, level, and / or form of another event or entity. For example, a particular entity (e.g., a polypeptide) is considered to be associated with a particular disease, disorder, or condition if the presence, level, and / or form of the entity is associated with the incidence and / or susceptibility of the disease, disorder, or condition (e.g., across a related population). In some embodiments, two or more entities are physically "associated with" one another if they interact, directly or indirectly, such that they are physically proximate to and remain in proximity to one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another, but are non-covalently associated, for example, by hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0185] Base editor: "Base editor (BE)" or "nucleobase editor (NBE)" means an agent that binds to a polynucleotide and has nucleobase modification activity. In various embodiments, the base editor includes a nucleobase modification polypeptide (e.g., a deaminase) and a polynucleotide programmable nucleotide binding domain and a guide polynucleotide (e.g., a guide RNA). In various embodiments, the agent is a biomolecular complex that includes a protein domain with base editing activity, i.e., a domain that can modify a base (e.g., A, T, C, G, or U) within a nucleic acid molecule (e.g., DNA). In some embodiments, the polynucleotide programmable DNA binding domain is fused or connected to a deaminase domain. In one embodiment, the agent is a fusion protein comprising one or more domains with base editing activity. In another embodiment, a protein domain with base editing activity is connected to a guide RNA (e.g., through an RNA binding motif on a guide RNA and fused to an RNA binding domain of a deaminase). In some embodiments, a domain with base editing activity is capable of deaminating a base within a nucleic acid molecule. In some embodiments, a base editor is capable of deaminating one or more bases within a DNA molecule. In some embodiments, the base editor is capable of deaminating cytosine (C) or adenosine (A) in DNA. In some embodiments, the base editor is capable of deaminating cytosine (C) and adenosine (A) in DNA. In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenosine base editor (ABE). In some embodiments, the base editor is an adenosine base editor (ABE) and a cytidine base editor (CBE). In some embodiments, the base editor is a Cas9 (dCas9) without nuclease activity fused to an adenosine deaminase. In some embodiments, the base editor is fused to a base excision repair inhibitor (eg, a UGI domain or a dISN domain). In some embodiments, the fusion protein includes a Cas9 nickase fused to a deaminase and a base excision repair inhibitor, such as a UGI or dISN domain. In other embodiments, the base editor is a base-free base editor. Details of base editors are described in International PCT Application Nos. PCT / 2017 / 045381 (WO2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 070632), each of which is incorporated herein by reference in its entirety.See also Komor, AC et al., “Programmable base editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, NM et al., “Programmable base editing of A·Tto G·C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); Komor, AC et al., "Improved base excision repair inhibition and bacteriophage Mu Gamprotein yields C:G-to-T:A base editors with higher efficiency and productpurity" Science Advances3:eaao4774 (2017) and Rees, HA et al., "Base editing: precision chemistry on the genome and transcriptome of living cells." Nat Rev Genet. 2018 Dec;19(12):770-788. doi:10.1038 / s41576-018-0059-1, the entire contents of which are hereby incorporated by reference.

[0186] Base editing activity: "Base editing activity" means chemically changing a base within a polynucleotide. In one embodiment, a first base is converted to a second base. In one embodiment, the base editing activity is cytidine deaminase activity, for example, converting the target C·G to T·A. In another embodiment, the base editing activity is adenosine or adenine deaminase activity, for example, converting A·T to G·C. In another embodiment, the base editing activity is cytosine or cytidine deaminase activity, for example, converting the target C·G to T·A, and adenosine or adenine deaminase activity, for example, converting A·T to G·C.

[0187] Base editor system: The term "base editor system" refers to a system for editing the nucleobases of a target nucleotide sequence. In various embodiments, the base editor (BE) system comprises (1) a polynucleotide programmable nucleotide binding domain (e.g., Cas9), a deaminase domain, and a cytidine deaminase domain, which are used to deaminate the nucleobases in the target nucleotide sequence; and (2) one or more guide polynucleotides (e.g., guide RNA) bound to the polynucleotide programmable nucleotide binding domain. In various embodiments, the base editor (BE) system includes a nucleobase editor domain selected from adenosine deaminase or cytidine deaminase, and a domain having nucleic acid sequence-specific binding activity. In some embodiments, the base editor system includes (1) a base editor (BE), which includes a polynucleotide programmable DNA binding domain and a deaminase domain, which are used to deaminate one or more nucleobases in the target nucleotide sequence; and (2) one or more guide RNAs bound to the polynucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenine or adenosine base editor (ABE). In some embodiments, the base editor is an adenine or adenosine base editor (ABE) or a cytidine base editor (CBE).

[0188] In some embodiments, the polynucleotide programmable nucleotide binding domain can target the deaminase domain to the target nucleotide sequence by non-covalent interaction or association with the deaminase domain. For example, in some embodiments, the core base editing component (e.g., deaminase component) can include another heterologous part or domain that can interact, associate or form a complex with another heterologous part or domain that is a part of the polynucleotide programmable nucleotide binding domain. In some embodiments, another heterologous part can be bound to a polypeptide, interact with a polypeptide, associate or form a complex. In some embodiments, another heterologous part can be bound to a polynucleotide, interact with a polynucleotide, associate or form a complex. In some embodiments, another heterologous part can be bound to a guide polynucleotide. In some embodiments, another heterologous part can be bound to a polypeptide joint. In some embodiments, another heterologous part can be bound to a polynucleotide joint. Another heterologous part can be a protein domain. In some embodiments, the additional heterologous portion can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif.

[0189] Biological activity: As used herein, the phrase "biological activity" refers to the property of any agent that is active in a biological system, particularly in an organism. For example, an agent that has a biological effect on an organism when administered to the organism is considered to be biologically active. In particular embodiments, when a peptide has biological activity, a portion of the peptide that shares at least one biological activity of the peptide is generally referred to as a "biologically active" portion.

[0190] Cleavage: As used herein, cleavage refers to a break in a target nucleic acid produced by a nuclease of a CRISPR system as described herein. In some embodiments, the cleavage event is a double-stranded DNA break. In some embodiments, the cleavage event is a single-stranded DNA break. In some embodiments, the cleavage event is a single-stranded RNA break. In some embodiments, the cleavage event is a double-stranded RNA break.

[0191] Complementary: As used herein, complementary refers to nucleic acid strands that form Watson-Crick base pairing, such that A bases pair with T and C bases pair with G, or form non-traditional base pairing with bases on a second nucleic acid strand. In other words, it refers to nucleic acids that hybridize to each other under appropriate conditions.

[0192] Clustered regularly spaced short palindromic repeats (Clustered Interspaced Short PalindromicRepeat, CRISPR) associated (Cas) system: As used herein, the CRISPR-Cas9 system refers to nucleic acids and / or proteins that participate in the expression of CRISPR effectors or guide the activity of CRISPR effectors, including sequences encoding CRISPR effectors, RNA guides, and other sequences and transcripts from the CRISPR locus. In some embodiments, the CRISPR system is an engineered, non-naturally occurring CRISPR system. In some embodiments, the components of the CRISPR system may include nucleic acids (e.g., vectors) encoding one or more components of the system, components in protein form, or a combination thereof.

[0193] CRISPR array: As used herein, the term "CRISPR array" refers to a nucleic acid (e.g., DNA) segment comprising CRISPR repeats and spacers, starting from the first nucleotide of the first CRISPR repeat and ending at the last nucleotide of the last (terminal) CRISPR repeat. Typically, each spacer in a CRISPR array is located between two repeats. As used herein, the term "CRISPR repeats" or "CRISPR direct repeats" or "direct repeats" refers to multiple short direct repeats that show very little or no sequence variation within a CRISPR array.

[0194] CRISPR-associated protein (Cas): As used herein, the terms "CRISPR-associated protein," "CRISPR effector," "effector," or "CRISPR enzyme" refer to a protein that performs an enzymatic activity or binds to a target site on a nucleic acid specified by an RNA guide. In various embodiments, the CRISPR effector has endonuclease activity, nickase activity, exonuclease activity, transposase activity, and / or excision activity. It should be understood that any Cas9 protein provided herein includes naturally occurring Cas9 proteins and non-naturally occurring variants thereof.

[0195] crRNA: As used herein, the term "CRISPR RNA" or "crRNA" refers to an RNA molecule comprising a guide sequence used by a CRISPR effector to target a specific nucleic acid sequence. Typically, crRNA contains a sequence that mediates target recognition and a sequence that forms a duplex with tracrRNA. In some embodiments, the crRNA:tracrRNA duplex binds to a CRISPR effector.

[0196] Ex vivo: As used herein, the term "ex vivo" refers to an event that occurs in cells or tissues that are grown outside of a multicellular organism rather than within the multicellular organism.

[0197] Functional equivalents or analogs: As used herein, the term "functional equivalent" or "functional analog" in the context of a functional derivative of an amino acid sequence refers to a molecule that retains a biological activity that is substantially similar to the biological activity (function or structure) of the original sequence. Functional derivatives or equivalents may be natural derivatives or synthetically prepared. Exemplary functional derivatives include amino acid sequences with substitutions, deletions or additions of one or more amino acids, provided that the biological activity of the protein is conserved. The substituted amino acid ideally has similar chemical and physical properties to the substituted amino acid. Ideally similar chemical and physical properties include similarities in charge, volume, hydrophobicity, hydrophilicity, etc.

[0198] Half-life: As used herein, the term "half-life" is the time required for a quantity (such as protein concentration or activity) to drop to half of its value measured at the beginning of the time period.

[0199] Improvement, increase or decrease: As used herein, the terms "improvement," "increase," or "decrease," or grammatical equivalents thereof, refer to values ​​relative to a baseline measurement, such as a measurement in the same individual prior to starting a treatment described herein, or a measurement in a control subject (or multiple control subjects) in the absence of a treatment described herein. A "control subject" is a subject having the same form of disease as the subject being treated, who is approximately the same age as the subject being treated.

[0200] Inhibition: As used herein, the terms "inhibition", "inhibit" and "inhibiting" refer to a process or method of reducing or lowering the activity and / or expression of a protein or gene of interest. Typically, inhibiting a protein or gene refers to reducing the expression or associated activity of the protein or gene by at least 10% or more, e.g., 20%, 30%, 40% or 50%, 60%, 70%, 80%, 90% or more, or reducing the expression or associated activity by more than 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold or more, as measured by one or more methods described herein or recognized in the art.

[0201] Hybridization: As used herein, the term "hybridization" refers to the reaction in which two or more nucleic acids bind to each other through hydrogen bonds between the bases of the two nucleic acids by Watson-Crick pairing, Hoogstein binding or other sequence-specific binding. A sequence that is capable of hybridizing to another sequence is called the "complement" of that sequence and is said to be "complementary" or to exhibit "complementarity."

[0202] Indel: As used herein, the term "indel" refers to the insertion or deletion of bases in a nucleic acid sequence. It usually results in a mutation and is a common form of genetic variation.

[0203] In vitro: As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as a test tube or reaction vessel, cell culture medium, etc., rather than in a multicellular organism.

[0204] In vivo: As used herein, the term "in vivo" refers to events that occur within a multicellular organism such as a human or non-human animal. In the context of a cell-based system, the term may be used to refer to events that occur within living cells (as opposed to, for example, in vitro systems).

[0205] Linker: The term "linker" refers to any means, entity or part for connecting two or more entities. In some embodiments, the linker is a covalent linker. In some embodiments, the linker is a non-covalent linker. Examples of covalent linkers include covalent bonds or linker moieties that are covalently connected to one or more of the proteins or domains to be connected. In some embodiments, the linker is a non-covalent bond, such as an organic metal bond through a metal center (such as a platinum atom). The connection can be permanent or reversible. For covalent connection, various functions can be used, such as amide groups (including carbonic acid derivatives), ethers, esters (including organic and inorganic esters), amino groups, carbamates, ureas, etc. In order to provide a connection, the domain can be modified by oxidation, hydroxylation, substitution, reduction, etc. to provide a site for coupling. Methods for conjugation are well known to those skilled in the art and are included for use in the present invention. Linker moieties include, but are not limited to, chemical linker moieties, or, for example, peptide linker moieties (linker sequences). It should be understood that modifications that do not significantly reduce the function of the RNA binding domain and the effector domain are preferred.

[0206] Mutation: As used herein, the term "mutation" has the ordinary meaning in the art, and includes, for example, point mutation, substitution, insertion, deletion, inversion, and deletion.

[0207] Oligonucleotide: As used herein, the term "oligonucleotide" generally refers to a polynucleotide of between about 5 and about 100 nucleotides of single-stranded or double-stranded DNA. Oligonucleotides are also called "oligomers" or "oligos" and can be isolated from genes or chemically synthesized.

[0208] PAM: The term "PAM" or "protospacer adjacent motif" refers to a short nucleic acid sequence (usually 2 to 6 base pairs in length) that follows a region of nucleic acid that is targeted for cleavage by CRISPR systems such as CRISPR-Cas9. A PAM is required for Cas nuclease cleavage and is typically found 3 to 4 nucleotides downstream of the cleavage site.

[0209] Polypeptide: As used herein, the term "polypeptide" refers to a continuous chain of amino acids linked together by peptide bonds. The term is used to refer to amino acid chains of any length, but one of ordinary skill in the art will understand that the term is not limited to long chains and can refer to a minimal chain including two amino acids linked together by a peptide bond. As known to those skilled in the art, polypeptides can be processed and / or modified. As used herein, the terms "polypeptide" and "peptide" are used interchangeably.

[0210] Prevent: As used herein, the terms "prevent" or "prevention" when used in connection with the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of developing a disease, disorder, and / or condition.

[0211] Protein: As used herein, the term "protein" refers to one or more polypeptides that function as discrete units. If a single polypeptide is a discrete functional unit and does not require permanent or temporary physical association with other polypeptides to form a discrete functional unit, then the terms "polypeptide" and "protein" are used interchangeably. If the discrete functional unit consists of more than one polypeptide physically associated with each other, then the term "protein" refers to multiple polypeptides that are physically coupled and function together as a discrete unit.

[0212] Reference: A "reference" entity, system, quantity, set of conditions, etc. is an entity, system, quantity, set of conditions, etc., to which a test entity, system, quantity, set of conditions, etc. described herein is compared. For example, in some embodiments, a "reference" antibody is a control antibody that has not been engineered as described herein.

[0213] RNA guide: The term RNA guide refers to an RNA molecule that facilitates the targeting of a protein described herein to a target nucleic acid. Exemplary "RNA guides" or "guide RNAs" include, but are not limited to, crRNA or a combination of crRNA and a cognate tracrRNA. The latter may be a separate RNA or fused to a single RNA using a linker (sgRNA). In some embodiments, the RNA guide is engineered to include a chemical or biochemical modification. In some embodiments, the RNA guide may include one or more nucleotides.

[0214] Subject: As used herein, the term "subject" means any subject for whom diagnosis, prognosis or treatment is desired. For example, the subject can be a mammal, such as a human or a non-human primate (such as an ape, monkey, gorilla or chimpanzee), dog, cat, guinea pig, rabbit, rat, mouse, horse, cattle or cow.

[0215] sgRNA: The term "sgRNA" or "single guide RNA" refers to a single guide RNA containing (i) a guide sequence (crRNA sequence) and (ii) a Cas9 nuclease recruitment sequence (tracrRNA).

[0216] Substantial identity: The phrase "substantial identity" is used herein to refer to a comparison between amino acid or nucleic acid sequences. As will be appreciated by one of ordinary skill in the art, two sequences are generally considered to be "substantially identical" if they contain identical residues at corresponding positions. As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, Gapped BLAST, and PSI-BLAST for amino acid sequences. Such exemplary programs are described in: Altschul et al., Basic local alignment search tool, J. Mol. Biol., 215(3):403-410, 1990; Altschul et al., Methods in Enzymology; Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener et al., (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying identical sequences, the above programs typically provide an indication of the degree of identity. In some embodiments, two sequences are considered substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the corresponding residues in the relevant stretch are identical. In some embodiments, the relevant stretch is a complete sequence. In some embodiments, the relevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues.

[0217] Target nucleic acid: As used herein, the term "target nucleic acid" refers to nucleotides (oligonucleotides or polynucleotides) of any length to which the CRISPR-Cas9 system binds, deoxyribonucleotides, ribonucleotides, or analogs thereof. The target nucleic acid may have a three-dimensional structure, may include coding regions or non-coding regions, may include exons, introns, mRNA, tRNA, rRNA, siRNA, shRNA, miRNA, ribozymes, cDNA, plasmids, vectors, exogenous sequences, endogenous sequences. The target nucleic acid may include modified nucleotides comprising methylated nucleotides or nucleotide analogs. The target nucleic acid may be interspersed with non-nucleic acid components. The target nucleic acid is not limited to single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derived nucleotide bases.

[0218] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to the amount of a therapeutic molecule (e.g., an engineered antibody described herein) that imparts a therapeutic effect to a treated subject at a reasonable benefit / risk ratio applicable to any medical treatment. The therapeutic effect can be objective (i.e., measurable by a test or marker) or subjective (i.e., the subject gives an indication of the effect or feels the effect). Specifically, a "therapeutically effective amount" refers to the amount of a therapeutic molecule or composition that effectively treats, improves or prevents a specific disease or condition, or exhibits a detectable therapeutic or preventive effect, such as by improving symptoms associated with the disease, preventing or delaying the onset of the disease, and / or also alleviating the severity or frequency of disease symptoms. A therapeutically effective amount that can be administered in a dosing regimen that can include multiple unit doses. For any particular therapeutic molecule, the therapeutically effective amount (and / or the appropriate unit dose within an effective dosing regimen) can vary, for example, depending on the route of administration, and in combination with other agents. Moreover, the specific therapeutically effective amount (and / or unit dose) for any particular subject may depend on a variety of factors, including the condition being treated and the severity of the condition; the activity of the specific agent being used; the specific ingredient employed; the age, weight, general health, sex, and diet of the subject; the time of administration, route of administration, and / or rate of excretion or metabolism of the specific therapeutic molecule employed; the duration of the treatment; and like factors well known in the medical arts.

[0219] tracrRNA: As used herein, the term "tracrRNA" or "trans-activating crRNA" refers to an RNA that contains a sequence that forms the structure required for a CRISPR-associated protein to bind to a specific target nucleic acid.

[0220] Treatment: As used herein, the term "treatment" (also "treat" and "treating") refers to the administration of a therapeutic molecule (e.g., a CRISPR-Cas therapeutic protein or system described herein) that partially or completely alleviates, ameliorates, alleviates, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or features of a disease, disorder, and / or condition. Such treatment may be for subjects who do not develop signs of the relevant disease, disorder, and / or condition and / or for subjects who only develop early signs of a disease, disorder, and / or condition. Alternatively or additionally, such treatment may be for subjects who develop one or more established signs of the relevant disease, disorder, and / or condition. BRIEF DESCRIPTION OF THE DRAWINGS

[0221] The drawings are for illustration purposes only; they are not intended to be limiting.

[0222] Figure 1A is a diagram showing the consensus PAM motif recognized by human codon-optimized S. equi ATCC 33317 Cas9. Figure 1B is a diagram showing the consensus PAM motif recognized by human codon-optimized Enterococcus hirae strain F1129E Cas9. Figure 1C is a diagram showing the consensus PAM motif recognized by human codon-optimized S. equi strain AG46 Cas9. Figure 1D is a diagram showing the consensus PAM motif recognized by human codon-optimized Staphylococcus aureus strain 19. Figure 1E is a diagram showing the consensus PAM motif recognized by human codon-optimized S. intermedius B196 strain G1552. Figure 1F is a diagram showing the consensus PAM motif recognized by human codon-optimized S. sanguinis SK330. Figure 1G is a diagram showing the consensus PAM motif recognized by human codon-optimized Streptococcus C150. Figure 1H is a diagram showing the consensus PAM motif recognized by the human codon-optimized S. oralis subsp. oral strain RH_1735_08. Fig. 1I is a diagram showing the consensus PAM motif recognized by S. oralis SK313. Figure 1J is a diagram showing the consensus PAM motif recognized by Staphylococcus worderi strain 691. Figure 1K is a diagram showing the consensus PAM motif recognized by Staphylococcus sciuri strain SNUC 2430. Figure 1Lis a diagram showing the consensus PAM motif recognized by S. gallolyticus strain AM24-4. Figure 1M is a diagram showing the consensus PAM motif recognized by Lactobacillus kuraburgii strain Biut2. Figure 1N is a diagram showing the consensus PAM motif recognized by S. suis strains (N = A, T, G or C; H = A, C or T; R = A or G).

[0223] Figure 2A is a schematic diagram showing the RNA folding structure of sgRNA for human codon-optimized Streptococcus equi ATCC33317 (Seq2Cas9) predicted using Geneious software. Figure 2A An sgRNA comprising SEQ ID NO: 198 is depicted. Figure 2B is a schematic diagram showing the RNA folding structure of sgRNA for human codon-optimized Enterococcus hirae strain F1129E Cas9 (EhiCas9) predicted using Geneious software. Figure 2B An sgRNA comprising SEQ ID NO: 44 is depicted. Figure 2C is a schematic diagram showing the RNA folding structure of the sgRNA for human codon-optimized Streptococcus equi strain AG46Cas9 (SeqCas9) predicted using Geneious software. Figure 2C An sgRNA comprising SEQ ID NO: 46 is depicted. Figure 2D is a schematic diagram showing the RNA folding structure of sgRNA for human codon-optimized Staphylococcus aureus strain 19 (SsiCas9) predicted using Geneious software. Figure 2D An sgRNA comprising SEQ ID NO: 45 is depicted. Figure 2E is a schematic diagram showing the RNA folding structure of the sgRNA for human codon-optimized Streptococcus intermedius B196 strain G1552 (SinCas9) predicted using Geneious software. Figure 2E An sgRNA comprising SEQ ID NO: 47 is depicted. Figure 2F is a schematic diagram showing the RNA folding structure of sgRNA for human codon-optimized Streptococcus sanguis SK330 (SsaCas9) predicted using Geneious software. Figure 2F An sgRNA comprising SEQ ID NO: 48 is depicted. Figure 2G is a schematic diagram showing the RNA folding structure of sgRNA for human codon-optimized Streptococcus C150 (Ssc2Cas9) predicted using Geneious software. Figure 2GAn sgRNA comprising SEQ ID NO: 49 is depicted. Figure 2H is a schematic diagram showing the RNA folding structure of the sgRNA for human codon-optimized S. oralis subsp. oral strain RH_1735_08 (Sor2Cas9) predicted using Geneious software. Figure 2H An sgRNA comprising SEQ ID NO: 50 is depicted. Fig.2I is a schematic diagram showing the RNA folding structure of sgRNA for human codon-optimized S. oralis SK313 (SorCas9) predicted using Geneious software. Fig.2I An sgRNA comprising SEQ ID NO: 51 is depicted. Figure 2J is a schematic diagram showing the RNA folding structure of the sgRNA for human codon-optimized Staphylococcus worteri strain 691 (SwaCas9) predicted using Geneious software. Figure 2J An sgRNA comprising SEQ ID NO: 52 is depicted. Figure 2K is a schematic diagram showing the RNA folding structure of sgRNA for human codon-optimized Staphylococcus sciuri strain SNUC 2430 predicted using Geneious software. Figure 2K An sgRNA comprising SEQ ID NO: 53 is depicted. Figure 2L is a schematic diagram showing the RNA folding structure of the sgRNA for human codon-optimized Streptococcus gallolyticus strain AM24-4 (SgaCas9) predicted using Geneious software. Figure 2L An sgRNA comprising SEQ ID NO: 54 is depicted. Figure 2M is a schematic diagram showing the RNA folding structure of the sgRNA for human codon-optimized Lactobacillus kuraburgensis strain Biut2 (LkuCas9) predicted using Geneious software. Figure 2M An sgRNA comprising SEQ ID NO: 55 is depicted. Figure 2N is a schematic diagram showing the RNA folding structure of sgRNA for human codon-optimized S. suis strain LSS83 predicted using Geneious software. Figure 2N An sgRNA comprising SEQ ID NO: 56 is depicted.

[0224] Figure 3AAn exemplary graph showing the results of achieving percentage of adenine to guanine base (A to G) conversion using a base editor comprising an ABE fused to the N-terminus of the Seq2Cas9 D10A mutant. The percentage of A to G conversion (y-axis) is plotted against various guide RNAs targeting an A-rich genomic test site (x-axis; Table 8) adjacent to a sequence corresponding to a PAM consensus motif (see Figure 1A ).

[0225] Figure 3B An exemplary graph showing the results of achieving percentage of adenine to guanine base (A to G) conversion using a base editor comprising an ABE fused to the N-terminus of the EhiCas9 D10A mutant. The percentage of A to G conversion (y-axis) is plotted against various guide RNAs targeting an A-rich genomic test site (x-axis; Table 9) adjacent to a sequence corresponding to a PAM consensus motif (see Figure 1B ).

[0226] Figure 3C An exemplary graph showing the results of achieving percentage of adenine to guanine base (A to G) conversion using a base editor comprising an ABE fused to the N-terminus of the SeqCas9 D10A mutant. The percentage of A to G conversion (y-axis) is plotted against various guide RNAs targeting an A-rich genomic test site (x-axis; Table 10) adjacent to a sequence corresponding to a PAM consensus motif (see Figure 1C ).

[0227] Figure 3D An exemplary graph showing the results of achieving percentage of adenine to guanine base (A to G) conversion using a base editor comprising an ABE fused to the N-terminus of the SsiCas9 D10A mutant. The percentage of A to G conversion (y-axis) is plotted against various guide RNAs targeting an A-rich genomic test site (x-axis; Table 11) adjacent to a sequence corresponding to a PAM consensus motif (see Figure 1D ).

[0228] Figure 3E An exemplary graph showing the results of achieving percentage of adenine to guanine base (A to G) conversion using a base editor comprising an ABE fused to the N-terminus of the SinCas9 D9A mutant. The percentage of A to G conversion (y-axis) is plotted against various guide RNAs targeting an A-rich genomic test site (x-axis; Table 12) adjacent to a sequence corresponding to a PAM consensus motif (see Figure 1E ).

[0229] Figure 3FAn exemplary graph showing the results of achieving percentage of adenine to guanine base (A to G) conversion using a base editor comprising an ABE fused to the N-terminus of the SsaCas9 D11A mutant. The percentage of A to G conversion (y-axis) is plotted against various guide RNAs targeting an A-rich genomic test site (x-axis; Table 13) adjacent to a sequence corresponding to a PAM consensus motif (see Figure 1F ).

[0230] Figure 3G An exemplary graph showing the results of achieving percentage of adenine to guanine base (A to G) conversion using a base editor comprising an ABE fused to the N-terminus of the Ssc2Cas9 D9A mutant. The percentage of A to G conversion (y-axis) is plotted against various guide RNAs targeting an A-rich genomic test site (x-axis; Table 14) adjacent to a sequence corresponding to a PAM consensus motif (see Figure 1G ).

[0231] Figure 3H An exemplary graph showing the results of achieving percentage of adenine to guanine base (A to G) conversion using a base editor comprising an ABE fused to the N-terminus of the Sor2Cas9 D9A mutant. The percentage of A to G conversion (y-axis) is plotted against various guide RNAs targeting an A-rich genomic test site (x-axis; Table 15) adjacent to a sequence corresponding to a PAM consensus motif (see Figure 1H ).

[0232] Fig. 3I An exemplary graph showing the results of achieving percentage of adenine to guanine base (A to G) conversion using a base editor comprising an ABE fused to the N-terminus of the SorCas9 D9A mutant. The percentage of A to G conversion (y-axis) is plotted against various guide RNAs targeting an A-rich genomic test site (x-axis; Table 16) adjacent to a sequence corresponding to a PAM consensus motif (see Fig. 1I ).

[0233] Figure 3J An exemplary graph showing the results of achieving percentage of adenine to guanine base (A to G) conversion using a base editor comprising an ABE fused to the N-terminus of a SwaCas9 mutant. The percentage of A to G conversion (y-axis) is plotted against various guide RNAs targeting an A-rich genomic test site (x-axis; Table 17) adjacent to a sequence corresponding to a PAM consensus motif (see Figure 1J ).

[0234] Figure 3KAn exemplary graph showing the results of achieving percentage of adenine to guanine base (A to G) conversion using a base editor comprising an ABE fused to the N-terminus of an SscCas9 mutant. The percentage of A to G conversion (y-axis) is plotted against various guide RNAs targeting an A-rich genomic test site (x-axis; Table 18) adjacent to a sequence corresponding to a PAM consensus motif (see Figure 1K ).

[0235] Figure 3L An exemplary graph showing the results of achieving percentage of adenine to guanine base (A to G) conversion using a base editor comprising an ABE fused to the N-terminus of an SgaCas9 mutant. The percentage of A to G conversion (y-axis) is plotted against various guide RNAs targeting an A-rich genomic test site (x-axis; Table 19) adjacent to a sequence corresponding to a PAM consensus motif (see Figure 1L ).

[0236] Figure 3M An exemplary graph showing the results of achieving percentage of adenine to guanine base (A to G) conversion using a base editor comprising an ABE fused to the N-terminus of an LkuCas9 mutant. The percentage of A to G conversion (y-axis) is plotted against various guide RNAs targeting an A-rich genomic test site (x-axis; Table 20) adjacent to a sequence corresponding to a PAM consensus motif (see Figure 1M ).

[0237] Figure 3N An exemplary graph showing the results of achieving percentage of adenine to guanine base (A to G) conversion using a base editor comprising an ABE fused to the N-terminus of an SsuCas9 mutant. The percentage of A to G conversion (y-axis) is plotted against various guide RNAs targeting an A-rich genomic test site (x-axis; Table 21) adjacent to a sequence corresponding to a PAM consensus motif (see Figure 1N ). DETAILED DESCRIPTION

[0238] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) were originally discovered as an adaptive immune system in bacteria and archaea and then engineered to generate targeted DNA breaks in living cells and organisms. During the cellular DNA repair process, a variety of DNA changes may be introduced. The diverse and expanding CRISPR toolbox enables programmable genome editing, epigenome editing, and transcriptome regulation.

[0239] According to their Cas gene organization and the sequence and structure of the constituent proteins, CRISPR-Cas systems include three main types (I, II and III). Each of the three CRISPR systems is characterized by a unique Cas gene: Cas3, a target-degrading nuclease / helicase in type I; Cas9, an RNA-binding and target-degrading nuclease in type II; Cas10, a large protein for multiple functions in type III. The three CRISPR types also differ in their associated effector complexes. Type I Cas systems are associated with cascade effector complexes, type II effector complexes consist of a single Cas9 and one or more RNA molecules, and type III interference complexes are further divided into type III-A (Csm complexes targeting DNA) and type III-B (Cmr complexes targeting RNA). Cas proteins are important components of effector complexes in all CRISPR-Cas systems.

[0240] Current genome editing technologies are mainly focused on class II CRISPR–Cas systems, which contain single-protein effector nucleases for DNA cleavage, specifically Cas9, a dual-RNA-guided nuclease that requires CRISPR RNA (crRNA) and tracrRNA and contains both HNH and RuvC nuclease domains, and Cas12a, a single-RNA-guided nuclease that requires only crRNA and contains a single RuvC domain.

[0241] Various aspects of the present invention are described in detail in the following sections. The use of sections is not intended to limit the present invention. Each section may be applicable to any aspect of the present invention. In this application, unless otherwise stated, the use of "or" means "and / or".

[0242] Engineered, non-naturally occurring Cas9 proteins

[0243] Described herein is an engineered, non-naturally occurring Cas9 protein modified with a WT Cas9 obtained from: Streptococcus equi ATCC 33317 (Seq2Cas9), Enterococcus hirae strain F1129E (EhiCas9), Streptococcus equi strain AG46 (SeqCas9), Staphylococcus aureus strain 19 (SsiCas9), Streptococcus intermedius B196 strain G1552 (SinCas9), Streptococcus sanguinis SK330 (SsaCas9), Streptococcus sp. C150 (Ssc2Cas9), Streptococcus oralis subsp. oral strain RH_1735_08 (Sor2Cas9), Streptococcus oralis SK313 (SorCas9), Staphylococcus worderi strain 691 (SwaCas9), Staphylococcus sciuri strain SNUC 2430 (SscCas9), Streptococcus gallolyticus strain AM24-4 (SgaCas9), Lactobacillus kuraburgii strain Biut2 (LkuCas9), and Streptococcus suis strain LSS83 (SsuCas9).

[0244] In some embodiments, the engineered, non-naturally occurring Cas9 proteins described herein comprise an amino acid sequence that is at least 60% (e.g., 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In some embodiments, the Cas9 protein is 80% identical to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In some embodiments, the amino acid sequence of the Cas9 protein is identical to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. Exemplary Cas9 amino acid sequences are provided in Table 1 below.

[0245] Table 1: Exemplary Cas9 amino acid sequences

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256] In some embodiments, the Cas9 protein comprises one or more mutations with respect to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. For example, the amino acid sequence of the Cas9 protein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least 10 mutations of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. Various mutations are known in the art and include, for example, amino acid substitutions.

[0257] In some embodiments, two or more catalytic domains (RuvC1, RuvCII, RuvCIII) of Cas9 are mutated to produce an inactivated or "dead" Cas9 (dCas9) lacking nucleic acid cleavage activity. In some embodiments, one or more mutations are located in the PAM interaction, HNH and or RuvC domains. In some embodiments, Cas9 is mutated to reduce DNA cleavage activity to less than about 25%, 15%, 10%, 5%, 1%, 0.1%, 0.01% or less relative to its non-mutated form.

[0258] In some embodiments, a nickase mutant version of Cas9 is provided. In some embodiments, the nickase mutant has one or more amino acid substitutions in the RuvC and / or HNH domains. Various nickase mutations associated with SpCas9 (Streptococcus pyogenes) are known, and for example include mutations at one or more amino acid positions 10, 12, 17, 762, 840, 854, 863, 982, 983, 984, 986, 987 of wild-type SpCas9. For example, aspartic acid to alanine substitution corresponding to D10A in SpCas9 results in the production of nickase. In some embodiments, Cas9 described herein has one or more mutations that result in the production of nickase. In some embodiments, Cas9 described herein has one or more mutations at amino acid positions corresponding to one or more amino acids in 10, 12, 17, 762, 840, 854, 863, 982, 983, 984, 986, 987 of SpCas9.

[0259] In some embodiments, the mutation is an aspartate to alanine substitution (D10A) in the RuvC domain of Seq2Cas9. In some embodiments, the mutation is an aspartate to alanine substitution (D10A) in the RuvC domain of EhiCas9. In some embodiments, the mutation is an aspartate to alanine substitution (D10A) in the RuvC domain of SeqCas9 (e.g., corresponding to D10A in SpCas9). In some embodiments, the mutation is an aspartate to alanine substitution (D10A) in the RuvC domain of SsiCas9. In some embodiments, the mutation is an aspartate to alanine substitution (D9A) in the RuvC domain of SinCas9. In some embodiments, the mutation is an aspartate to alanine substitution (D11A) in the RuvC domain of SsaCas9. In some embodiments, the mutation is an aspartate to alanine substitution (D9A) in the RuvC domain of Ssc2Cas9. In some embodiments, the mutation is an aspartate to alanine substitution (D9A) in the RuvC domain of Sor2Cas9. In some embodiments, the mutation is an aspartate to alanine substitution (D9A) in the RuvC domain of SorCas9. In some embodiments, the mutation is an aspartate to alanine substitution (D10A) in the RuvC domain of SwaCas9. In some embodiments, the mutation is an aspartate to alanine substitution (D10A) in the RuvC domain of SscCas9. In some embodiments, the mutation is an aspartate to alanine substitution (D10A) in the RuvC domain of SgaCas9. In some embodiments, the mutation is an aspartate to alanine substitution (D10A) in the RuvC domain of LkuCas9. In some embodiments, the mutation is an aspartate to alanine substitution (D13A) in the RuvC domain of SsuCas9.

[0260] In some embodiments, the mutation is an aspartate to glycine substitution (D10G) in the RuvC domain of Seq2Cas9. In some embodiments, the mutation is an aspartate to glycine substitution (D10G) in the RuvC domain of EhiCas9. In some embodiments, the mutation is an aspartate to glycine substitution (D10G) in the RuvC domain of SeqCas9 (e.g., corresponding to D10G in SpCas9). In some embodiments, the mutation is an aspartate to glycine substitution (D10G) in the RuvC domain of SsiCas9. In some embodiments, the mutation is an aspartate to glycine substitution (D9G) in the RuvC domain of SinCas9. In some embodiments, the mutation is an aspartate to glycine substitution (D11G) in the RuvC domain of SsaCas9. In some embodiments, the mutation is an aspartate to glycine substitution (D9G) in the RuvC domain of Ssc2Cas9. In some embodiments, the mutation is an aspartate to glycine substitution (D9G) in the RuvC domain of Sor2Cas9. In some embodiments, the mutation is an aspartate to glycine substitution (D9G) in the RuvC domain of SorCas9. In some embodiments, the mutation is an aspartate to glycine substitution (D10G) in the RuvC domain of SwaCas9. In some embodiments, the mutation is an aspartate to glycine substitution (D10G) in the RuvC domain of SscCas9. In some embodiments, the mutation is an aspartate to glycine substitution (D10G) in the RuvC domain of SgaCas9. In some embodiments, the mutation is an aspartate to glycine substitution (D10G) in the RuvC domain of LkuCas9. In some embodiments, the mutation is an aspartate to glycine substitution (D13G) in the RuvC domain of SsuCas9.

[0261] In some embodiments, such one or more mutations described herein convert Cas9 into an inactivated or "dead" version of Cas9 (dCas9). Thus, in some embodiments, the Cas9 protein comprises one or more mutations that inhibit the ability of Cas9 to cut both strands of a DNA duplex.

[0262] In some embodiments, when co-expressed with a guide RNA, dead Cas9 generates a DNA recognition complex that can specifically interfere with transcription elongation, RNA polymerase binding, or transcription factor binding. In some embodiments, dead Cas9 is used to specifically target effector proteins of various functions to specific nucleic acid target sites.

[0263] In some embodiments, the engineered, non-naturally occurring Cas9 is codon-optimized for human cells. The engineered, non-naturally occurring Cas9 is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to SEQ ID NO. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0264] Exemplary Cas9 amino acid sequences with nuclear localization signals (NLS) and linkers are provided in Table 2 below.

[0265] Table 2: Exemplary Cas9 sequences with NLS and linkers

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277] NLS (bold), can be replaced by a different NLS

[0278] The connector (underscore) can be deleted or extended

[0279] 3xHA tag (italic), can be replaced with a different tag

[0280] In some embodiments, the recombinant engineered, non-naturally occurring, human codon-optimized Cas9 comprises a sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28. In some embodiments, the recombinant engineered, non-naturally occurring, human codon-optimized Cas9 comprises a sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28. In some embodiments, the recombinant engineered, non-naturally occurring, human codon-optimized Cas9 comprises a sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28. In some embodiments, the recombinant engineered, non-naturally occurring, human codon-optimized Cas9 comprises a sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28.

[0281] Different species exhibit codon bias (i.e., differences in codon usage between organisms), which is related to the efficiency of translation of messenger RNA (mRNA), where the usage of codons in mRNA corresponds to the abundance of tRNA species for that codon in a particular organism. Various methods in the art can be used for computer optimization, including, for example, by using software. In some embodiments, codon optimization refers to modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) of a native sequence with a codon that is more commonly used or most commonly used in the genes of the host cell, while maintaining the native amino acid sequence.

[0282] In some embodiments, the Cas9 protein described herein is codon optimized. This type of optimization is known in the art and requires mutation of exogenously derived DNA to mimic the codon preference of the intended host organism or cell while encoding the same protein. Therefore, the codons are changed, but the encoded protein remains unchanged. Codon optimization can increase the soluble protein level of a particular species and improve activity and editing efficiency. Codon optimization also leads to increased translation and protein expression.

[0283] In some embodiments, the Cas9 protein is codon optimized for expression in eukaryotic cells. In some embodiments, the Cas9 protein is codon optimized for expression in human cells.

[0284] Protospacer adjacent motif (PAM)

[0285] Each Cas endonuclease binds to its target sequence only in the presence of a specific sequence, called a protospacer adjacent motif (PAM), on the non-target (i.e., complementary DNA strand). Cas nucleases isolated from different bacterial species recognize different PAM sequences. For example, Cas9 engineered from Streptococcus equi ATCC 33317 cleaves upstream of the consensus PAM sequence 5'-NRGNR-3' (where "N" can be any nucleotide base and R is A or G), in the target, Cas9 engineered from Enterococcus hirae strain F1129E recognizes the consensus PAM sequence 5'-NRG-3', and Cas9 engineered from Streptococcus equi AG46, Staphylococcus worderi strain 691, and Staphylococcus sciuri strain SNUC recognizes the consensus PAM sequence 5'-NRGNR-3'. Cas9 engineered by 2430 recognizes the consensus PAM sequence 5'-NNGR-3', Cas9 engineered by Staphylococcus aureus strain 19 recognizes the consensus PAM sequence 5'-NNGRRT-3', Cas9 engineered by Streptococcus intermedius B196 strain G1552 recognizes the consensus PAM sequence 5'-NNAAAA-3', Cas9 engineered by Streptococcus sanguinis SK330 recognizes the consensus PAM sequence 5'-NGGNG-3', Cas9 engineered by Streptococcus sp. C150 recognizes the consensus PAM sequence 5'-NNGNRG-3', and Cas9 engineered by Streptococcus oralis subsp. oral strain RH_1735_08 Cas9 engineered by Streptococcus oralis SK313 recognizes a consensus PAM sequence of 5'-NNRAAG-3', Cas9 engineered by Streptococcus gallolyticus strain AM24-4 recognizes a consensus PAM sequence of 5'-NNAYAA-3', Cas9 engineered by Lactobacillus kuraburgii strain Biut2 recognizes a consensus PAM sequence of 5'-NNGAAA-3', and Cas9 engineered by Streptococcus suis strain recognizes a consensus PAM sequence of 5'-NNAAA-3' (N = any nucleotide base; H = A, C or T; R = A or G). Therefore, the positions in the genome that can be targeted by different Cas proteins are limited by the positions of unique PAM sequences.

[0286] In some embodiments, the target nucleic acid is 5' or upstream of the PAM sequence. Thus, the Cas9 proteins described herein exhibit activity, such as binding, cleavage, modification, or altered gene expression in the presence of a unique PAM sequence.

[0287] In some embodiments, the Cas9 proteins described herein do not bind to or exhibit activity with any other PAM sequence.

[0288] RNA guide

[0289] The RNA guide comprises a polynucleotide sequence that is complementary to the target sequence. The RNA guide hybridizes to the target nucleic acid sequence and guides sequence-specific binding of the CRISPR complex to the target nucleic acid. In some embodiments, the RNA guide has 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complementarity with the target nucleic acid sequence.

[0290] In some embodiments, the RNA guide is about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 or more nucleotides in length. In some embodiments, the RNA guide is about 18 to 24 nucleotides in length. In some embodiments, the RNA guide is complementary to about 18 to 24 nucleotides in the target nucleic acid sequence. For example, the RNA guide is complementary to about 18, 19, 20, 21, 22, 23 or 24 nucleotides in the target nucleic acid sequence. In some embodiments, the RNA guide is complementary to about 18 to 22 nucleotides. In some embodiments, the RNA guide is complementary to about 18 to 21 nucleotides. In some embodiments, the RNA guide is complementary to about 18 to 20 nucleotides. In some embodiments, the RNA guide is complementary to 20 nucleotides in the target nucleic acid sequence.

[0291] RNA guides can be designed to target any target sequence. Optimal alignment is determined using any algorithm for aligning sequences, including the Needleman-Wunsch algorithm, the Smith-Waterman algorithm, the Burrows-Wheeler algorithm, Clust1W, Clust1X, BLAST, Novoalign, SOAP, Maq, and ELAND.

[0292] In some embodiments, the RNA guide targets a unique target sequence within the genome of the cell. In some embodiments, the RNA guide is designed to lack a PAM sequence. In some embodiments, the RNA guide sequence is designed to have an optimal secondary structure using a folding algorithm including mFold or Geneious. In some embodiments, expression of the RNA guide can be under an inducible promoter, such as hormone-inducible, tetracycline- or doxycycline-inducible, arabinose-inducible, or light-inducible.

[0293] In some embodiments, the CRISPR system comprises one or more RNA guides, such as crRNA, tracrRNA, and / or sgRNA. Thus, in some embodiments, the RNA guide comprises crRNA. In some embodiments, the RNA guide comprises tracrRNA. In some embodiments, the RNA guide comprises sgRNA. In some embodiments, the CRISPR system comprises multiple RNA guides, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or more RNA guides.

[0294] In some embodiments, the RNA guide comprises crRNA. In some embodiments, the CRISPR system comprises multiple crRNAs, comprising 2 to 15 crRNAs. In some embodiments, the crRNA is a precursor crRNA (pre-crRNA) comprising a positive sequence repeat sequence, a spacer sequence, and a positive sequence repeat sequence. In some embodiments, the crRNA is a processed or mature crRNA comprising a truncated positive sequence repeat sequence.

[0295] In some embodiments, the CRISPR-associated protein cleaves the pre-crRNA to form processed or mature crRNA.

[0296] In some embodiments, the CRISPR-associated protein forms a complex with the mature crRNA, and the spacer sequence targets the complex to a complementary sequence in the target nucleic acid. In some embodiments, the RNA guide comprises a positive sequence repeat sequence and a spacer sequence that can hybridize with the target nucleic acid under appropriate conditions.

[0297] In some embodiments, the crRNA spacer length can be in the range of about 15 to 50 nucleotides. In some embodiments, the RNA guide spacer length is at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, or at least 22 nucleotides. In some embodiments, the spacer is 15 to 17 nucleotides (e.g., 15, 16, or 17 nucleotides), 17 to 20 nucleotides (e.g., 17, 18, 19, or 20 nucleotides), 20 to 24 nucleotides (e.g., 20, 21, 22, 23, or 24 nucleotides), 23 to 25 nucleotides (e.g., 23, 24, or 25 nucleotides), 24 to 27 nucleotides, 27 to 30 nucleotides, 30 to 45 nucleotides (e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 nucleotides), 30 or 35 to 40 nucleotides, 41 to 45 nucleotides, 45 to 50 nucleotides (e.g., 45, 46, 47, 48, 49, or 50 nucleotides), or longer.

[0298] In some embodiments, the RNA guide comprises a direct repeat (DR) sequence of about 16 to 26 nucleotides in length. For example, in some embodiments, the DR is about 16 nucleotides in length. In some embodiments, the DR is about 17 nucleotides in length. In some embodiments, the DR is about 18 nucleotides in length. In some embodiments, the DR is about 19 nucleotides in length. In some embodiments, the DR is about 20 nucleotides in length. In some embodiments, the DR is about 21 nucleotides in length. In some embodiments, the DR is about 22 nucleotides in length. In some embodiments, the DR is about 23 nucleotides in length. In some embodiments, the DR is about 24 nucleotides in length. In some embodiments, the DR is about 25 nucleotides in length. In some embodiments, the DR is about 26 nucleotides in length.

[0299] In some embodiments, the crRNA comprises a nucleotide guide sequence and a DR sequence. The nucleotide guide sequence may be between about 18 nucleotides long and 24 nucleotides long. Thus, in some embodiments, the nucleotide guide sequence is about 18 nucleotides long. In some embodiments, the nucleotide guide sequence is about 19 nucleotides long. In some embodiments, the nucleotide guide sequence is about 20 nucleotides long. In some embodiments, the nucleotide guide sequence is about 21 nucleotides long. In some embodiments, the nucleotide guide sequence is about 22 nucleotides long. In some embodiments, the crRNA comprises a nucleotide guide sequence of about 22 nucleotides long and a positive sequence repeat sequence of about 22 nucleotides long.

[0300] In some embodiments, the crRNA sequence can be modified into a "dead crRNA", "dead guide" or "dead guide sequence", which can form a complex with a CRISPR-associated protein and bind to a specific target without any substantial nuclease activity.

[0301] In some embodiments, crRNA can be chemically modified in the sugar phosphate backbone or base. In some embodiments, crRNA can be modified using 2'O-methyl, 2'-F or locked nucleic acid to improve nuclease resistance or base pairing. In some embodiments, crRNA can contain modified bases such as 2-thiourea diene or N6-methyladenosine.

[0302] In some embodiments, the crRNA is conjugated to other oligonucleotides, peptides, proteins, tags, dyes, or polyethylene glycol.

[0303] In some embodiments, the crRNA may comprise an aptamer or riboswitch sequence that is capable of binding to a specific target molecule due to its three-dimensional structure.

[0304] In some embodiments, trans-activating RNA (tracrRNA) associates with crRNA to promote formation of a complex with Cas9 protein. In some embodiments, the length of the tracrRNA sequence is about or no more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides. In some embodiments, the length of tracrRNA is about 70 nucleotides.

[0305] In some embodiments, the tracrRNA and crRNA are contained in a single transcript called a single guide RNA (sgRNA). In some embodiments, the sgRNA comprises a loop between the tracrRNA and the sgRNA.

[0306] In some embodiments, the length of the loop-forming sequence is 3, 4, 5 or more nucleotides. In some embodiments, the loop has the sequence GAAA, AAAG, CAAA, AAAC, UUUU, UUAUAU, UUA, UUU and / or AAUCA. In some embodiments, the loop has the sequence GAAA. In some embodiments, the loop has the sequence AAAG. In some embodiments, the loop has the sequence CAAA. In some embodiments, the loop has the sequence AAAC. In some embodiments, the loop has the sequence AAUCA. In some embodiments, the loop has the sequence UUUU. In some embodiments, the loop has the sequence UUAUAU. In some embodiments, the loop has the sequence UUA. In some embodiments, the loop has the sequence UUU. In some embodiments, the loop has the sequence AAUCA.

[0307] In some embodiments, tracrRNA and crRNA form a hairpin loop. In some embodiments, sgRNA has at least two or more hairpin bodies. In some embodiments, sgRNA has two, three, four or five hairpin bodies.

[0308] In some embodiments, the sgRNA comprises a transcription termination sequence comprising a polyT sequence comprising six nucleotides.

[0309] In some embodiments, the sgRNA comprises a

[0310] Sequences with at least 80% identity,

[0311] In some embodiments, the sgRNA comprises a

[0312] In some embodiments, the sgRNA comprises a sequence having at least 80% identity to a target of SeqCas9.

[0313] UUCAUGCCGAAUUCAAGCACCCCAUGUUUACAUGGGGUGCUUUU -3' (SEQ ID NO: Sequences with at least 80% identity,

[0314] In some embodiments, the sgRNA comprises a Sequences with at least 80% identity,

[0315] In some embodiments, the sgRNA comprises a In some embodiments, the sgRNA comprises a sequence that is at least 80% identical to a sequence targeting SsaCas9. Sequences with at least 80% identity,

[0316] In some embodiments, the sgRNA comprises a Sequences with at least 80% identity,

[0317] In some embodiments, the sgRNA comprises a Sequences with at least 80% identity,

[0318] In some embodiments, the sgRNA comprises a Sequences with at least 80% identity,

[0319] In some embodiments, the sgRNA comprises a Sequences with at least 80% identity,

[0320] In some embodiments, the sgRNA comprises a 53) sequences having at least 80% identity,

[0321] In some embodiments, the sgRNA comprises a Sequences with at least 80% identity,

[0322] In some embodiments, the sgRNA comprises a In some embodiments, the sgRNA comprises a sequence having at least 80% identity to a target SsuCas9. Sequences that are at least 80% identical.

[0323] For SEQ ID NOs 43-56: positive sequence repeat (italic and underlined), four rings (italic), tracrRNA (underline)

[0324] The guide RNA is added to the 5' end of Cas9. In some embodiments, the sgRNA comprises 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 or 56. In some embodiments, the sgRNA comprises a sequence identical to SEQ ID NO: 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 or 56.

[0325] In some embodiments, the tracrRNA is a separate transcript and is not contained in the same transcript as the crRNA sequence.

[0326] Cas9 fusion protein

[0327] In some embodiments, the Cas9 enzyme is fused to one or more heterologous protein domains. In some embodiments, the Cas9 enzyme is fused to more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more protein domains. In some embodiments, the heterologous protein domain is fused to the C-terminus of the Cas9 enzyme. In some embodiments, the heterologous protein domain is fused to the N-terminus of the Cas9 enzyme. In some embodiments, the heterologous protein domain is internally fused between the C-terminus and the N-terminus of the Cas9 enzyme. In some embodiments, the internal fusion is performed within the Cas9 RuvCI, RuvC II, RuvCIII, HNH, REC I or PAM interaction domain.

[0328] Cas9 protein can be directly or indirectly connected to another protein domain. In some embodiments, a suitable CRISPR system comprises a linker or spacer connecting Cas9 protein and a heterologous protein. Amino acid linkers or spacers are generally designed to be flexible or designed to insert a structure between two protein parts, such as an α-helix. Linkers or spacers can be relatively short or longer. Typically, a linker or spacer comprises, for example, a length of 1 to 100 (e.g., 1 to 100, 5 to 100, 10 to 100, 20 to 100, 30 to 100, 40 to 100, 50 to 100, 60 to 100, 70 to 100, 80 to 100, 90 to 100, 5 to 55, 10 to 50, 10 to 45, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20) amino acids. In some embodiments, the length of the linker or spacer is equal to or longer than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acids. Typically, longer linkers can reduce steric hindrance. In some embodiments, the linker will comprise a mixture of glycine and serine residues. In some embodiments, the linker may additionally comprise threonine, proline and / or alanine residues.

[0329] In some embodiments, the Cas9 protein is fused to a cell localization signal, an epitope tag, a reporter gene, and a protein domain having enzymatic activity, epigenetic modification activity, RNA cleavage activity, nucleic acid binding activity, and transcriptional regulation activity. In some embodiments, the Cas9 protein is fused to a nuclear localization sequence (NLS), a FLAG tag, a HIS tag, and / or a HA tag.

[0330] Suitable fusion partners include, but are not limited to, polypeptides that provide the following activities: methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity or nuclease activity, any of which can modify DNA or DNA-associated polypeptides (e.g., histones or DNA-binding proteins). In some embodiments, the Cas9 protein is fused to a histone demethylase, a transcriptional activator or a deaminase.

[0331] Other suitable fusion partners include, but are not limited to, boundary elements (e.g., CTCF), proteins and fragments thereof that provide peripheral recruitment (e.g., lamin A, lamin B, etc.), and protein docking elements (e.g., FKBP / FRB, Pill / Abyl, etc.).

[0332] In certain embodiments, Cas9 is fused to a cytidine or adenosine deaminase domain, for example, for base editing. In some embodiments, the terms "cytidine deaminase" and "cytosine deaminase" can be used interchangeably. In certain embodiments, the cytidine deaminase domain may have 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with any cytidine deaminase described herein. In some embodiments, the cytidine deaminase domain has cytidine deaminase activity (e.g., converting C to U). In certain embodiments, the adenosine deaminase domain may have 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with any adenosine deaminase described herein. In some embodiments, the adenosine deaminase domain has adenosine deaminase activity (eg, converts A to I). In some embodiments, the terms "adenosine deaminase" and "adenine deaminase" are used interchangeably.

[0333] In some embodiments, the cytidine deaminase may include all or part of an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. APOBEC is an evolutionarily conservative cytidine deaminase family. Members of this family are C to U editing enzymes. The N-terminal domain of the APOBEC-like protein is a catalytic domain, and the C-terminal domain is a pseudo-catalytic domain. More specifically, the catalytic domain is a zinc-dependent cytidine deaminase domain and is important for cytidine deamination. APOBEC family members include APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D ("APOBEC3E" now refers to this), APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, and activation-induced (cytidine or cytosine) deaminase. In some embodiments, the deaminase incorporated into the fusion protein includes all or part of an APOBEC1 deaminase. In some embodiments, the deaminase incorporated into the fusion protein includes all or part of an APOBEC2 deaminase. In some embodiments, the deaminase incorporated into the fusion protein comprises all or part of an APOBEC3 deaminase. In some embodiments, the deaminase incorporated into the fusion protein comprises all or part of an APOBEC3A deaminase. In some embodiments, the deaminase incorporated into the fusion protein comprises all or part of an APOBEC3B deaminase. In some embodiments, the deaminase incorporated into the fusion protein comprises all or part of an APOBEC3C deaminase. In some embodiments, the deaminase incorporated into the fusion protein comprises all or part of an APOBEC3D deaminase. In some embodiments, the deaminase incorporated into the fusion protein comprises all or part of an APOBEC3E deaminase. In some embodiments, the deaminase incorporated into the fusion protein comprises all or part of an APOBEC3F deaminase. In some embodiments, the deaminase incorporated into the fusion protein comprises all or part of an APOBEC3G deaminase. In some embodiments, the deaminase incorporated into the fusion protein comprises all or part of an APOBEC3H deaminase. In some embodiments, the deaminase incorporated into the fusion protein comprises all or part of an APOBEC4 deaminase. In some embodiments, the deaminase incorporated into the fusion protein comprises all or part of an activation-induced deaminase (AID). In some embodiments, the deaminase incorporated into the fusion protein comprises all or part of cytidine deaminase 1 (CDA1). It should be understood that the fusion protein can comprise a deaminase from any suitable organism (e.g., human or rat). In some embodiments, the deaminase domain of the fusion protein is from a human, chimpanzee, gorilla, monkey, cow, dog, rat or mouse. In some embodiments, the deaminase domain of the fusion protein is derived from a rat (e.g., rat APOBEC1). In some embodiments, the deaminase domain is human APOBEC1.In some embodiments, the deaminase domain is pmCDA1.Sequences of exemplary cytidine deaminases are provided below.

[0334] pmCDA1 (Sea lamprey (Petromyzon marinus))

[0335]

[0336]

[0337] Human AID:

[0338]

[0339] Human AID:

[0340]

[0341] Mouse AID:

[0342]

[0343] Canine AID:

[0344]

[0345] Cattle AID:

[0346]

[0347] Rat AID:

[0348]

[0349]

[0350] (Underline: nuclear localization sequence; double underline: nuclear export signal)

[0351] clAID (Dogs (Canis lupus familiaris)):

[0352]

[0353] btAID (cattle (Bos taurus)):

[0354]

[0355] mAID (Mus musculus):

[0356]

[0357] rAPOBEC-1 (Rattus norvegicus):

[0358]

[0359] maAPOBEC-1 (Golden rat (Mesocricetus auratus)):

[0360]

[0361] ppAPOBEC-1 (Bornean orangutan (Pongo pygmaeus)):

[0362]

[0363]

[0364] ocAPOBEC1 (Oryctolagus cuniculus):

[0365]

[0366] mdAPOBEC-1 (Monodelphis domestica):

[0367]

[0368] ppAPOBEC-2 (Bornean orangutan (Pongo pygmaeus)):

[0369]

[0370] btAPOBEC-2 (cattle):

[0371]

[0372] mAPOBEC-3-(1) (Mus musculus):

[0373]

[0374]

[0375] Mouse APOBEC-3-(2):

[0376]

[0377] Rat APOBEC-3:

[0378]

[0379] hAPOBEC-3A (Homo sapiens):

[0380] hAPOBEC-3F (Homo sapiens):

[0381]

[0382] Rhesus monkey APOBEC-3G:

[0383]

[0384]

[0385] Chimpanzee APOBEC-3G:

[0386]

[0387] (Italics: nucleic acid editing domain; underline: cytoplasmic localization signal) Green monkey APOBEC-3G:

[0388]

[0389] (Italics: nucleic acid editing domain; underline: cytoplasmic localization signal) Human APOBEC-3G:

[0390]

[0391] (Italics: nucleic acid editing domain; underline: cytoplasmic localization signal)

[0392] Human APOBEC-3F:

[0393]

[0394] (italics: nucleic acid editing domain)

[0395] Human APOBEC-3B:

[0396]

[0397] (italics: nucleic acid editing domain)

[0398] Rat APOBEC-3B:

[0399] Bovine APOBEC-3B:

[0400]

[0401] Chimpanzee APOBEC-3B:

[0402]

[0403] Human APOBEC-3C:

[0404]

[0405] (italics: nucleic acid editing domain)

[0406] Gorilla APOBEC-3C

[0407]

[0408] (italics: nucleic acid editing domain)

[0409] Human APOBEC-3A:

[0410]

[0411] (italics: nucleic acid editing domain)

[0412] Rhesus monkey APOBEC-3A:

[0413]

[0414] (italics: nucleic acid editing domain)

[0415] Bovine APOBEC-3A:

[0416]

[0417] (italics: nucleic acid editing domain)

[0418] Human APOBEC-3H:

[0419]

[0420] (italics: nucleic acid editing domain)

[0421] Rhesus monkey APOBEC-3H:

[0422] Human APOBEC-3D:

[0423]

[0424] (italics: nucleic acid editing domain)

[0425] Human APOBEC-1:

[0426]

[0427] Mouse APOBEC-1:

[0428]

[0429]

[0430] Rat APOBEC-1:

[0431]

[0432] Human APOBEC-2:

[0433]

[0434] Mouse APOBEC-2:

[0435]

[0436] Rat APOBEC-2:

[0437]

[0438] Bovine APOBEC-2:

[0439]

[0440] Sea lamprey CDA1 (pmCDAl):

[0441]

[0442] Human APOBEC3G D316R D317R:

[0443]

[0444] Human APOBEC3G A chain:

[0445]

[0446] Human APOBEC3G A chain D120R D121R:

[0447] hAPOBEC-4 (Homo sapiens):

[0448]

[0449] mAPOBEC-4 (Mus musculus):

[0450]

[0451] RQLRRILLPLYEVDDLRDAFRMLGF(SEQ ID NO:108)

[0452] rAPOBEC-4 (Rattus norvegicus):

[0453]

[0454] mfAPOBEC-4 (Macaca fascicularis):

[0455]

[0456] pmCDA-1 (sea lamprey):

[0457]

[0458] pmCDA-2 (sea lamprey):

[0459]

[0460] pmCDA-5 (sea lamprey):

[0461]

[0462] TWLDTTESMAAKMRRKLFCILVRCAGMRESGMPLHLFT(SEQ ID NO:113)

[0463] yCD (Saccharomyces cerevisiae):

[0464]

[0465] rAPOBEC-1 (δ177-186):

[0466]

[0467] rAPOBEC-1 (δ202-213):

[0468]

[0469] Mouse APOBEC-3:

[0470]

[0471] (italics: nucleic acid editing domain)

[0472] In some embodiments, adenosine deaminase may include all or part of adenosine deaminase ADAR (e.g., ADAR1 or ADAR2). In another embodiment, adenosine deaminase may include all or part of adenosine deaminase ADAT. In some embodiments, adenosine deaminase may include all or part of ADAT from Escherichia coli (Escherichia coli) (EcTadA), comprising one or more of the following mutations: D108N, A106V, D147Y, E155V, L84F, H123Y, I157F, or a corresponding mutation in another adenosine deaminase. Adenosine deaminase may be derived from any suitable organism (e.g., Escherichia coli). In some embodiments, the adenosine deaminase is from Escherichia coli, Staphylococcus aureus, Salmonella typhi, Shewanella putrefaciens, Haemophilus influenzae, Caulobacter crescentus or Bacillus subtilis. In some embodiments, the adenosine deaminase is from Escherichia coli. In some embodiments, the adenine deaminase is a naturally occurring adenosine deaminase comprising one or more mutations corresponding to any one of the mutations provided herein (e.g., mutations in ecTadA). The corresponding residues in any homologous protein can be identified by, for example, sequence alignment and determination of homologous residues. A mutation corresponding to any one of the mutations described herein (e.g., any one of the mutations identified in ecTadA) in any naturally occurring adenosine deaminase (e.g., homology to ecTadA) can be generated accordingly. In a specific embodiment, TadA is any one of the TadAs described in PCT / US2017 / 045381 (WO 2018 / 027078), which is incorporated herein by reference in its entirety. Mutations identified by multiple rounds of evolution and selection (e.g., TadA*7.10 = variant 10 from the seventh round of evolution) have ideal adenosine deaminase activity on single-stranded DNA, as shown in Table 3.

[0473] Table 3. Genotypes of TadA variants

[0474]

[0475]

[0476]

[0477] In some embodiments, TadA is provided as a monomer or dimer (eg, a heterodimer of wild-type E. coli TadA and an engineered TadA variant). In some embodiments, the adenosine deaminase is an eighth generation TadA*8 variant, as shown in Table 4 below.

[0478] Table 4: TadA8* adenosine deaminase variants

[0479]

[0480]

[0481] In some embodiments, the adenosine deaminase is a ninth generation TadA*9 variant containing an alteration at an amino acid position selected from the group consisting of 21, 23, 25, 38, 51, 54, 70, 71, 72, 72, 94, 124, 133, 138, 139, 146, and 158 of a TadA variant as shown in the reference sequence below:

[0482]

[0483] In one embodiment, the adenosine deaminase variant contains changes at two or more amino acid positions selected from the group consisting of 21, 23, 25, 38, 51, 54, 70, 71, 72, 94, 124, 133, 138, 139, 146, and 158 of the above TadA reference sequence. In another embodiment, the adenosine deaminase variant contains one or more (e.g., 2, 3, 4) changes selected from the group consisting of R21N, R23H, E25F, N38G, L51W, P54C, M70V, Q71M, N72K, Y73S, M94V, P124W, T133K, D139L, D139M, C146R, and A158K of SEQ ID NO.1. In other embodiments, the adenosine deaminase variant further comprises one or more of the following changes: Y147T, Y147R, Q154S, Y123H and Q154R. In still other embodiments, the adenosine deaminase variant comprises a combination of changes relative to the above TadA reference sequence selected from the following: E25F+V82S+Y123H, T133K+Y147R+Q154R; E25F+V82S+Y123H+Y147R+Q154R; L51W+V82S+Y123H+C146R+Y147R+Q154R; Y73S+V82S+Y123H+Y147R+Q154R; 54C+V82S+Y123H+Y147R+Q154R; N38G+V82T+Y123H+Y147R+Q154R; N72K+V82S+Y123H+D139L+Y147R+Q1 54R; E25F+V82S+Y123H+D139M+Y147R+Q154R; Q71M+V82S+Y123H+Y147R+Q154R; E25F+V82S+Y123H+T133 K+Y147R+Q154R; E25F+V82S+Y123H+Y147R+Q154R; V82S+Y123H+P124W+Y147R+Q154R; L51W+V82S+Y123 H+C146R+Y147R+Q154R; P54C+V82S+Y123H+Y147R+Q154R; Y73S+V82S+Y123H+Y147R+Q154R; N38G+V82T+ Y123H+Y147R+Q154R; R23H+V82S+Y123H+Y147R+Q154R; R21N+V82S+Y123H+Y147R+Q154R; V82S+Y123H+ Y147R+Q154R+A158K; N72K+V82S+Y123H+D139L+Y147R+Q154R; E25F+V82S+Y123H+D139M+Y147R+Q154R;M70V + V82S + M94V + Y123H + Y147R + Q154R; Q71M + V82S + Y123H + Y147R + Q154R; E25F + I76Y + V82S + Y123H + Y147R + Q154R; I76Y + V82T + Y123H + Y147R + Q154R; N38G + I76Y + V82S + Y123H + Y147R + Q154R; R23H + I76Y + V82S + Y123H + Y147R + Q154R; P54C + I76Y + V82S + Y123H + Y147R + Q154R; R21N + I76Y + V82S + Y123H + Y147R + Q154R; I76Y + V82S + Y123H + D138M + Y147R + Q154R; Y72S + I76Y + V82S + Y123H + Y147R + Q154R; E25F + I76Y + V82S + Y123H + Y147R + Q154R; I76Y + V82T + Y123H + Y147R + Q154R; N38G + I76Y + V82S + Y123H + Y147R + Q154R; R23H + I76Y + V82S + Y123H + Y147R + Q154R; P54C + I76Y + V82S + Y123H + Y147R + Q154R; R21N + I76Y + V82S + Y123H + Y147R + Q154R; I76Y + V82S + Y123H + D138M + Y147R + Q154R; Y72S + I76Y + V82S + Y123H + Y147R + Q154R; and V82S + Q154R; N72K_V82S + Y123H + Y147R + Q154R; Q71M_V82S + Y123H + Y147R + Q154R; V82S + Y123H + T133K + Y147R + Q154R; V82S + Y123H + T133K + Y147R + Q154R + A158K; M70V + Q71M + N72K + V82S + Y123H + Y147R + Q154R; N72K_V82S + Y123H + Y147R + Q154R; Q71M_V82S + Y123H + Y147R + Q154R; M70V + V82S + M94V + Y123H + Y147R + Q154R; V82S + Y123H + T133K + Y147R + Q154R; V82S + Y123H + T133K + Y147R + Q154R + A158K;and M70V+Q71M+N72K+V82S+Y123H+Y147R+Q154R. In some embodiments, the deaminase or other polypeptide sequence lacks methionine, for example when included as a component of a fusion protein. This may change the numbering of the positions. However, the skilled artisan will understand that such corresponding mutations refer to the same mutation, for example, Y73S and Y72S and D139M and D138M. ;

[0484] In some embodiments, Cas9 is fused to a nuclear localization sequence comprising NLS of SV40 large T antigen, nucleoplasmin, c-myc, hRNPA1 M9, the IBB domain from importin-α; myoma T protein, human p53, c-abl IV, influenza virus NS1, hepatitis virus delta antigen, mouse Mx1, human poly (ADP-ribose) polymerase, steroid hormone receptor (human) glucocorticoid.

[0485] In some embodiments, the Cas9 protein is fused to an epitope tag, which includes but is not limited to a hemagglutinin (HA) tag, a histidine (His) tag, a FLAG tag, a Myc tag, a V5 tag, a VSV-G tag, a SNAP tag, a thioredoxin (Trx) tag.

[0486] In some embodiments, Cas9 is fused to a reporter gene, which includes but is not limited to glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol transferase (CAT), HcRed, DsRed, cyan fluorescent protein, yellow fluorescent protein and blue fluorescent protein, green fluorescent protein (GFP), including enhanced versions or superfolded GFP, and other modified versions of reporter genes.

[0487] In some embodiments, the serum half-life of the engineered Cas9 protein is increased by fusion with heterologous proteins such as human serum albumin, transferrin, human IgG and / or sialylated peptides such as carboxyl-terminal peptide (CTP, of the β chain of chorionic gonadotropin).

[0488] In some embodiments, the serum half-life of the engineered Cas9 protein is shortened by fusion to a destabilizing domain, including but not limited to synaptin, ubiquitin, FKBP12-L106P, and / or dihydrofolate reductase.

[0489] Suitable fusion partners providing increased or reduced stability include, but are not limited to, degron sequences. Those of ordinary skill in the art readily understand degron as an amino acid sequence that controls the stability of the protein to which it belongs. For example, the stability of a protein comprising a degron sequence is at least partially controlled by a degron sequence. In some cases, a suitable degron is constitutive, so that the degron exerts its influence on protein stability independent of experimental control (i.e., the degron is not drug-induced, temperature-induced, etc.). In some cases, the degron provides a variant Cas9 polypeptide with controllable stability, so that the variant Cas9 polypeptide can be "turned on" (i.e., stable) or "turned off" (i.e., unstable, degraded) according to the desired conditions. For example, if the degron is a temperature-sensitive degron, the variant Cas9 polypeptide may function (i.e., "on", stable) below a threshold temperature (e.g., 42°C, 41°C, 40°C, 39°C, 38°C, 37°C, 36°C, 35°C, 34°C, 33°C, 32°C, 31°C, 30°C, etc.), but not function (i.e., "off", degraded) above the threshold temperature. As another example, if the degron is a drug-inducible degron, the presence or absence of the drug can convert the protein from a "off" (i.e., unstable) state to an "on" (i.e., stable) state, or vice versa. An exemplary drug-inducible degron is derived from FKBP12 protein. The stability of the degron is controlled by the presence or absence of a small molecule that binds to the degron.

[0490] Examples of suitable degrons include, but are not limited to, those controlled by Shield-1, DHFR, auxin, and / or temperature. Non-limiting examples of suitable degrons are known in the art (e.g., Dohmen et al., Science, 1994. 263(5151):1273-1276: Heat-inducible degron: a method for constructing temperature-sensitive mutants; Schoeber et al., Am J Physiol Renal Physiol. 2009 Jan;296(1):F204-1 l: Conditional fast expression and function of multimeric TRPV5 channels using Shield-1; Chu et al., Bioorg Med Chem Lett. 2008 Nov 15;18(22):5941-4: Recent progress with FKBP-derived destabilizing domains; Kanemaki, Pflugers Arch. 2012 Dec 28: Frontiers of protein expression control with conditional degrons; Yang et al., Mol. Chem. 2014 Jun;23(5):1143-115 ... Cell. 2012 Nov 30;48(4):487-8: Titivated for destruction: the methyl degron; Barbour et al., Biosci Rep. 2013 Jan 18;33(1).: Characterization of the bipartite degron that regulates subiquitin-independent degradation of thymidylate synthase; and Greussing et al., J Vis Exp. 2012 Nov 10;(69): Monitoring of ubiquitin-proteasome activity inliving cells using a Degron (dgn)-destabilized green fluorescent protein (GFP)-based reporter protein; all of which are incorporated herein by reference in their entireties).

[0491] Exemplary degron sequences have been well characterized and tested in cells and animals. Thus, inactive Cas9 is fused to the degron sequence to generate "regulatable" and "inducible" inactive Cas9 polypeptides.

[0492] Any of the fusion partners described herein can be used in any desired combination. As a non-limiting example of illustrating this point, the Cas9 fusion protein may include a YFP sequence for detection, a degradation determinant sequence for stability, and a transcriptional activator sequence for increasing target DNA transcription. In addition, the number of fusion partners that can be used in the dCas9 fusion protein is unlimited. In some cases, the Cas9 fusion protein includes one or more (e.g., two or more, three or more, four or more, or five or more) heterologous sequences.

[0493] Target nucleic acid

[0494] The target nucleic acid is a DNA molecule, an RNA molecule, which is a single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrid, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases or deoxyribonucleotides, ribonucleotides or their analogs. The target nucleic acid can have a three-dimensional structure, can include coding regions or non-coding regions, can include exons, introns, mRNA, tRNA, rRNA, siRNA, shRNA, miRNA, ribozymes, cDNA, plasmids, vectors, exogenous sequences, endogenous sequences. The target nucleic acid can include modified nucleotides, and the modified nucleotides include methylated nucleotides or nucleotide analogs. In certain embodiments, the target nucleic acid can be interspersed with non-nucleic acid components.

[0495] The target nucleic acid is recognized by the CRISPR-Cas9 system and binds to Cas9. In some embodiments, it is modified or cut or has altered expression due to the binding of Cas9. The target nucleic acid contains a specific recognizable PAM motif, such as 5'-NGG-3', 5'-NAGHC-3', 5'-NRHRRH-3' or 5'-NNAAA-3' (H = A, C or T; R = A or G).

[0496] Recombinant gene technology

[0497] In light of the present disclosure, there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are described in the literature (see, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; DNA Cloning: A Practical Approach, Vols. I and II (DN Glover, ed. 1985); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Nucleic Acid Hybridization (BD Hames and SJ Higgins, eds. (1985)); Transcription And Translation (BD Hames and SJ Higgins, eds. (1984)); Animal Cell Culture (RI Freshney, ed. (1986)); Immobilized Cells and Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984); FM Ausubel et al. (eds.), Current Protocols in Molecular Cloning (1986). Biology, John Wiley & Sons, Inc. (1994).

[0498] Recombinant expression of a gene, such as a nucleic acid encoding a polypeptide, such as an engineered Cas9 enzyme as described herein, may include constructing an expression vector comprising a nucleic acid encoding the polypeptide. Once the polynucleotide is obtained, a vector for producing the polypeptide may be produced by recombinant DNA technology using techniques known in the art. Known methods can be used to construct an expression vector comprising a polypeptide coding sequence and appropriate transcriptional control signals and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

[0499] The expression vector can be transferred to host cells by conventional techniques, and the transfected cells can then be cultured by conventional techniques to produce the polypeptide.

[0500] In some embodiments, the nucleotide sequence encoding the RNA targeting DNA and / or the Cas9 protein is operably connected to a control element, for example, a transcriptional control element, such as a promoter. The transcriptional control element can function in eukaryotic cells (e.g., mammalian cells); or prokaryotic cells (e.g., bacteria or archaeal cells). In some embodiments, the eukaryotic cell is a human cell. In some embodiments, the nucleotide sequence encoding the RNA targeting DNA and / or the novel Cas9 protein is operably connected to a plurality of control elements, which allow the encoded nucleotide sequence to be expressed in prokaryotic and eukaryotic cells.

[0501] A promoter can be a constitutively active promoter (i.e., a promoter that is constitutively in an active / "ON" state), it can be an inducible promoter (i.e., a promoter whose activity / "ON" or inactivation / "OFF" state is controlled by an external stimulus (e.g., the presence of a specific temperature, compound, or protein)), it can be a spatially restricted promoter (i.e., a transcriptional control element, an enhancer, etc.) (e.g., a tissue-specific promoter, a cell type-specific promoter, etc.), and it can be a temporally restricted promoter (i.e., a promoter that is in an "ON" state or an "OFF" state at a specific stage of embryonic development or a specific stage of a biological process (e.g., the hair follicle cycle in mice)).

[0502] Suitable promoters may be derived from viruses and therefore may be referred to as viral promoters, or they may be derived from any organism, including prokaryotic or eukaryotic organisms. Suitable promoters may be used to drive the expression of any RNA polymerase (e.g., pol I, pol II, pol III). Exemplary promoters include, but are not limited to, SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter (e.g., CMV immediate early promoter region (CMVIE)), Rous sarcoma virus (RSV) promoter, human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 September 1; 31 (17)), and / or human HI promoter (HI).

[0503] Examples of inducible promoters include, but are not limited to, T7 RNA polymerase promoter, T3 RNA polymerase promoter, isopropyl-β-D-thiogalactopyranoside (IPTG)-regulated promoter, lactose-inducible promoter, heat shock promoter, tetracycline-regulated promoter (e.g., Tet-ON, Tet-OFF, etc.), steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, etc. Thus, inducible promoters can be regulated by molecules including, but not limited to, doxycycline, RNA polymerase (e.g., T7 RNA polymerase), estrogen receptor, and / or estrogen receptor fusions.

[0504] In some embodiments, the promoter is a spatially restricted promoter (i.e., a cell type-specific promoter, a tissue-specific promoter, etc.), such that in a multicellular organism, the promoter is active (i.e., "ON") in a subset of specific cells. Spatially restricted promoters may also be referred to as enhancers, transcriptional control elements, control sequences, etc. Any convenient spatially restricted promoter may be used, and the selection of a suitable promoter (e.g., a brain-specific promoter, a promoter that drives expression in a subset of neurons, a promoter that drives expression in germline, a promoter that drives expression in lungs, a promoter that drives expression in muscles, a promoter that drives expression in pancreatic islet cells, etc.) will depend on the organism. Therefore, spatially restricted promoters can be used to regulate the expression of nucleic acids encoding subject site-directed polypeptides in a variety of different tissues and cell types, depending on the organism. Some spatially restricted promoters are also temporally restricted, such that the promoter is in an "ON" state or an "OFF" state at a specific stage of embryonic development or a specific stage of a biological process (e.g., the hair follicle cycle).

[0505] For illustrative purposes, examples of spatially restricted promoters include, but are not limited to, neuron-specific promoters, adipocyte-specific promoters, cardiomyocyte-specific promoters, smooth muscle-specific promoters, photoreceptor-specific promoters, etc. Neuron-specific spatially restricted promoters include, but are not limited to, neuron-specific enolase (NSE) promoter, aromatic amino acid decarboxylase (AADC) promoter, neurofilament promoter, synapsin promoter, thy-1 promoter, serotonin receptor promoter, tyrosine hydroxylase promoter (TH), GnRH promoter, L7 promoter, DNMT promoter, enkephalin promoter, myelin basic protein (MBP) promoter, Ca promoter. 2+ - Calmodulin-dependent protein kinase II-alpha (CamKIIa) promoter and / or CMV enhancer / platelet-derived growth factor-beta promoter.

[0506] Adipocyte-specific spatially restricted promoters include, but are not limited to, aP2 gene promoter / enhancer (e.g., the region from -5.4 kb to +21 bp of the human aP2 gene), glucose transporter-4 (GLUT4) promoter, fatty acid translocase (FAT / CD36) promoter, stearoyl-CoA desaturase-1 (SCD1) promoter, leptin promoter, and adiponectin promoter, adipose promoter and / or resistin promoter.

[0507] Cardiomyocyte-specific spatially restricted promoters include, but are not limited to, control sequences derived from the following genes: myosin light chain 2, alpha-myosin heavy chain, AE3, cardiac troponin C, and / or cardiac actin.

[0508] Smooth muscle specific spatially restricted promoters include, but are not limited to, the SM22a promoter, the smoothin promoter, and / or the α-smooth muscle actin promoter.

[0509] Photoreceptor-specific spatially restricted promoters include, but are not limited to, a rhodopsin promoter, a rhodopsin kinase promoter, a beta phosphodiesterase gene promoter, a retinitis pigmentosa gene promoter, an interphotoreceptor retinoid binding protein (IRBP) gene enhancer, and / or an IRBP gene promoter.

[0510] Gene Editing Uses of CRISPR-Cas9

[0511] The CRISPR-Cas9 system described herein can be used for gene editing, which can result in a gene silencing event or a change in expression (e.g., an increase or decrease) in the expression of a desired target gene. Therefore, in some embodiments, the CRISPR-Cas9 system described herein is used in a method for changing the expression of a target nucleic acid. In some embodiments, the CRISPR-Cas9 system described herein is used in a method for modifying a target nucleic acid in a desired target cell. In some embodiments, the present invention provides a method for site-specific modification of a target nucleic acid in a eukaryotic cell to achieve the desired modification in gene expression.

[0512] In some embodiments, the present invention provides an engineered, non-naturally occurring CRISPR-Cas system comprising: an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a positive sequence repeat sequence and a spacer sequence capable of hybridizing to a target nucleic acid; and a codon-optimized CRISPR-associated (Cas) protein having at least 80% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, and wherein the Cas protein is capable of binding to the RNA guide and causing a break in a target nucleic acid sequence that is complementary to the RNA guide.

[0513] In some embodiments, the present invention provides an engineered, non-naturally occurring CRISPR-Cas system comprising: an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a positive sequence repeat sequence and a spacer sequence capable of hybridizing to a target nucleic acid; a codon-optimized CRISPR-associated (Cas) protein having at least 80% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14; wherein the Cas protein is fused to a deaminase, and wherein the Cas protein fusion is capable of binding to the RNA guide and editing a target nucleic acid sequence that is complementary to the RNA guide.

[0514] In some embodiments, the present invention provides a method for altering expression of a target nucleic acid in a eukaryotic cell, the method comprising: contacting the cell with Cas9 as described herein and an RNA guide or a nucleic acid encoding an RNA guide, wherein the RNA guide comprises a positive sequence repeat sequence and a spacer sequence capable of hybridizing to the target nucleic acid, and wherein the Cas9 protein is capable of binding to the RNA guide and causing a break in a target nucleic acid sequence that is complementary to the RNA guide.

[0515] In some embodiments, the present invention provides a method for altering the expression of a target nucleic acid in a eukaryotic cell, the method comprising: contacting the cell with Cas9 as described herein and an RNA guide or a nucleic acid encoding an RNA guide, wherein the RNA guide comprises a positive sequence repeat sequence and a spacer sequence capable of hybridizing to the target nucleic acid, and wherein the Cas9 protein is capable of binding to the RNA guide and editing a target nucleic acid sequence that is complementary to the RNA guide.

[0516] In some embodiments, the present invention provides a method for modifying a target nucleic acid in a eukaryotic cell, the method comprising: contacting the cell with Cas9 as described herein and an RNA guide or a nucleic acid encoding an RNA guide, wherein the RNA guide comprises a positive sequence repeat sequence and a spacer sequence capable of hybridizing to the target nucleic acid, and wherein the Cas9 protein is capable of binding to the RNA guide and editing a target nucleic acid sequence complementary to the RNA guide.

[0517] Thus, in some embodiments, the Cas protein is about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In some embodiments, the Cas protein is identical to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0518] Suitable guide RNAs, Cas9 mutations, and fusion proteins for use in CRISPR-Cas9 systems and methods are described throughout this disclosure.

[0519] In one aspect, the method includes binding CRISPR-Cas9 to a target nucleic acid and achieving cleavage of the target nucleic acid. In some embodiments, the CRISPR-Cas9 system cuts the target DNA or RNA duplex by introducing double-strand breaks. In some embodiments, the CRISPR-Cas9 system cuts the target DNA or RNA by introducing single-strand breaks or nicks.

[0520] In some embodiments, the CRISPR-Cas9 method or system comprises a fusion protein with an effector that modifies a target DNA in a site-specific manner, wherein the modification activity comprises a methyltransferase activity, a demethylase activity, an acetyltransferase activity, a deacetylase activity, a kinase activity, a phosphatase activity, an ubiquitin ligase activity, a deubiquitinating activity, an adenylating activity, a deadenylating activity, a SUMOylating activity, a deSUMOylating activity, a ribosylating activity, a deribosylating activity, a myristoylation activity, a demyristoylation activity, an integrase activity, a transposase activity, a recombinase activity, a polymerase activity, a ligase activity, a helicase activity, or a nuclease activity, any of which can modify DNA or a DNA-associated polypeptide (e.g., a histone or a DNA-binding protein).

[0521] In some embodiments, the CRISPR-Cas9 method or system includes a fusion protein and an enzyme that can edit a DNA sequence by chemically modifying a nucleotide base, and the enzyme includes a deaminase that can modify adenosine or cytosine bases and act as a site-specific base editor. For example, the APOBEC1 cytidine deaminase, which generally uses RNA as a substrate, can target single-stranded and double-stranded DNA when it is fused to Cas9, thereby directly converting cytidine into uridine, and the ADAR enzyme deaminates adenosine to inosine. Therefore, "base editing" using a deaminase can programmably convert one target DNA base into another. Various base editors are known in the art and can be used in the methods and systems described herein. Exemplary base editors are described in the following: For example, Rees and Liu Nature Review Genetics, 2018, 19 (12): 770-788, the contents of which are incorporated herein. Thus, in some embodiments, the Cas9 enzymes described herein (Seq2Cas9, EhiCas9, SeqCas9, SsiCas9, SinCas9, SsaCas9, Ssc2Cas9, Sor2Cas9, SorCas9, SwaCas9, SscCas9, SgaCas9, LkuCas9, and SsuCas9) are components of a nuclear base editor. In some embodiments, the base editor is an adenine deaminase TadA8 or TadA9.

[0522] In some embodiments, base editing results in the introduction of a stop codon to silence a gene. In some embodiments, base editing alters protein function by changing the amino acid sequence.

[0523] In some embodiments, CRISPR-Cas9 methods or systems include epigenetically modifying target DNA by fusion with histones. In some embodiments, CRISPR-Cas9 systems include epigenetically modifying target DNA by fusion with epigenetic modification enzymes (e.g., reading (reader), writing (writer) or erasing (eraser) protein fusions). In some embodiments, CRISPR-Cas9 systems include fusion with histone modification enzymes to change the histone modification pattern in the selected region of the target DNA. Histone modification can occur in a variety of different ways, including methylation, acetylation, ubiquitination, phosphorylation and many different combinations, thereby causing structural changes in DNA. In some embodiments, histone modification causes transcriptional repression or activation.

[0524] In some embodiments, the CRISPR-Cas9 method or system increases or decreases transcription by fusion with a transcriptional activator or transcriptional repressor, a small molecule / drug responsive transcriptional regulator, or an inducible transcriptional regulator, thereby regulating the transcription of the target DNA. In some embodiments, the CRISPR-Cas9 system is used to control the expression of a target encoding mRNA (i.e., a protein-coding gene), wherein the combination results in increased or decreased gene expression.

[0525] In some embodiments, CRISPR-Ca s 9 Methods or systems are used to control gene regulation by editing genetic regulatory elements (eg, promoters or enhancers).

[0526] In some embodiments, CRISPR-Ca s 9 Methods or systems are used to control the expression of target non-coding RNA, including tRNA, rRNA, snoRNA, siRNA, miRNA and long ncRNA.

[0527] In some embodiments, the CRISPR-Cas9 method or system is used for targeted engineering of chromatin loop structures. Targeted engineering of chromatin loops between regulatory genomic regions provides a means for manipulating endogenous chromatin structure and enabling the formation of new enhancer-promoter connections to overcome genetic defects or suppress aberrant enhancer-promoter connections.

[0528] In some embodiments, CRISPR-Cas9 is used for live cell imaging. Fluorescently labeled Cas9 is targeted to repetitive genomic regions (e.g., centromeres and telomeres) to track native chromatin sites throughout the cell cycle and determine the differential localization of transcriptionally active and inactive regions in 3D nuclear space.

[0529] In some embodiments, the CRISPR-Cas9 method or system is used to correct a pathogenic mutation by inserting a beneficial clinical variant or a suppressor mutation.

[0530] Nucleobase editors

[0531] Disclosed herein is a novel base editor or a nuclear base editor for editing, modifying or changing a target nucleotide sequence of a polynucleotide comprising Cas9. Described herein is a nuclear base editor or a base editor comprising a polynucleotide programmable nucleotide binding domain (e.g., Cas9) and a nuclear base editing domain (e.g., adenosine deaminase). When combined with a bound guide polynucleotide (e.g., gRNA), a polynucleotide programmable nucleotide binding domain (e.g., Cas9) can specifically bind to a target polynucleotide sequence (i.e., by complementary base pairing between the base of the bound guide nucleic acid and the base of the target polynucleotide sequence), thereby positioning the base editor to the target nucleic acid sequence to be edited. In some embodiments, the target polynucleotide sequence comprises single-stranded DNA or double-stranded DNA. In some embodiments, the target polynucleotide sequence comprises RNA. In some embodiments, the target polynucleotide sequence comprises a DNA-RNA hybrid. Since most known genetic variations associated with human diseases are point mutations, a method for producing precise point mutations more efficiently and cleanly is needed. The base editing system provided herein provides a novel way to deliver genome editing without generating double-stranded DNA breaks, without requiring a donor DNA template, and without inducing excessive random insertions and deletions.

[0532] The base editors provided herein can modify specific nucleotide bases without producing a significant proportion of insertions and deletions. As used herein, the term "insertion and deletion (indel (s))" refers to the insertion or deletion of a nucleotide base in a nucleic acid. Such insertions or deletions may result in frameshift mutations in a gene coding region. In some embodiments, it is necessary to produce a base editor that effectively modifies (e.g., mutates or deaminates) a specific nucleotide in a nucleic acid without producing a large number of insertions or deletions (i.e., insertions and deletions) in the target nucleotide sequence. In certain embodiments, any one of the base editors provided herein can produce an expected modification (e.g., point mutation or deamination) with respect to a larger proportion of insertions and deletions.

[0533] In some embodiments, any of the base editing systems provided herein results in less than 50%, less than 40%, less than 30%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% indel formation in a target polynucleotide sequence.

[0534] Some aspects of the present disclosure are based on the following understanding: Any of the base editors provided herein can efficiently produce expected mutations, such as point mutations, in nucleic acids (e.g., nucleic acids within the subject's genome) without producing a large number of unexpected mutations, such as unexpected point mutations. In some embodiments, any of the base editors provided herein can produce at least 0.01% of expected mutations (i.e., at least 0.01% base editing efficiency). In some embodiments, any of the base editors provided herein can produce at least 0.01%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of expected mutations.

[0535] In some embodiments, the base editors provided herein are capable of producing a ratio of expected point mutations to indels greater than 1: 1. In some embodiments, the base editors provided herein are capable of producing at least 1.5: 1, at least 2: 1, at least 2.5: 1, at least 3: 1, at least 3.5: 1, at least 4: 1, at least 4.5: 1, at least 5: 1, at least 5.5: 1, at least 6: 1, at least 6.5: 1, at least 7: 1, at least 7.5: 1, at least 8: 1, at least 8.5: 1, at least 9: 1, at least 10: 1, at least 11: 1, at least 12: 1, A ratio of expected point mutations to indels of at least 13:1, at least 14:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, at least 40:1, at least 50:1, at least 100:1, at least 200:1, at least 300:1, at least 400:1, at least 500:1, at least 600:1, at least 700:1, at least 800:1, at least 900:1, or at least 1000:1 or greater.

[0536] Any suitable method can be used to determine the number of expected mutations and indels, for example, as described in International PCT Application Nos. PCT / 2017 / 045381 (WO2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 070632); Komor, AC et al., "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage" Nature 533, 420-424 (2016); Gaudelli, NM et al., "Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage" Nature 551, 464-471 (2017); and Komor, AC et al., "Improved base excisionrepair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A baseeditors with higher efficiency and product purity" Science Advances 3:eaao4774 (2017); the entire contents of these are incorporated herein by reference.

[0537] In some embodiments, in order to calculate the insertion and deletion frequency, the sequencing reads are scanned to accurately match the two 10-bp sequences flanking the window where the insertion and deletion may occur. If an exact match is not found, the read is excluded from the analysis. If the length of this insertion and deletion window matches the reference sequence exactly, the read is classified as not containing insertion and deletion. If the insertion and deletion window is two or more bases longer or shorter than the reference sequence, the sequencing reads are classified as insertion and deletion respectively. In some embodiments, the base editor provided herein can limit the formation of insertion and deletion in a nucleic acid region. In some embodiments, the region is located at a nucleotide targeted by a base editor or a region within 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides of a nucleotide targeted by a base editor.

[0538] The number of indels formed at the target nucleotide region may depend on the amount of time that the nucleic acid (e.g., nucleic acid in the cell genome) is exposed to the base editor. In some embodiments, the target nucleotide sequence (e.g., nucleic acid in the cell genome) is exposed to the base editor for at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days or at least 14 days, the number and ratio of indels are determined. It should be understood that the features of the base editors described herein can be applied to any one of the fusion proteins, or methods using the fusion proteins provided herein.

[0539] Therapeutic applications

[0540] The CRISPR-Cas9 methods or systems described herein may have a variety of therapeutic applications. Thus, in some embodiments, a method of treating a disorder or disease in a subject in need thereof is provided, the method comprising administering to the subject a CRISPR-Cas9 system comprising Cas9 as described herein, wherein the guide RNA is complementary to at least 10 nucleotides of a target nucleic acid, the target nucleic acid being associated with the disorder or disease; wherein the Cas protein is associated with the guide RNA; wherein the guide RNA binds to the target nucleic acid; wherein the Cas protein causes a break in the target nucleic acid, optionally wherein the Cas9 is an inactivated Cas9 (dCas9) fused to a deaminase and causes one or more base edits in the target nucleic acid, thereby treating the disorder or disease.

[0541] In some embodiments, the CRISPR-Cas9 method or system can be used to treat various diseases and disorders, such as genetic disorders (e.g., single gene diseases), diseases treatable by nuclease activity, various cancers, etc.

[0542] In some embodiments, the CRISPR methods or systems described herein can be used to edit target nucleic acids to modify target nucleic acids (e.g., by inserting, deleting, or mutating one or more nucleic acid residues). For example, in some embodiments, the CRISPR system described herein comprises an exogenous donor template nucleic acid (e.g., a DNA molecule or an RNA molecule), which comprises a desired nucleic acid sequence. After resolving a cleavage event induced by the CRISPR system described herein, the molecular machinery of the cell will use an exogenous donor template nucleic acid to repair and / or resolve the cleavage event. Alternatively, the molecular machinery of the cell can use an endogenous template to repair and / or resolve the cleavage event. In some embodiments, the CRISPR system described herein can be used to change the target nucleic acid, resulting in insertions, deletions, and / or point mutations). In some embodiments, the insertion is a scarless insertion (i.e., inserting the expected nucleic acid sequence into the target nucleic acid, resulting in no additional unexpected nucleic acid sequence after resolving the cleavage event). The donor template nucleic acid can be a double-stranded or single-stranded nucleic acid molecule (e.g., DNA or RNA). In some embodiments, the CRISPR method or system described herein comprises a nucleobase editor. For example, in some embodiments, the Cas9 protein described herein is fused to a polypeptide having nucleobase editing activity.

[0543] In one aspect, the CRISPR methods or systems described herein can be used to treat diseases caused by overexpression of RNA, toxic RNA, and / or mutant RNA (e.g., splicing defective or truncated).

[0544] In some embodiments, the CRISPR methods or systems described herein can also target trans-acting mutations that affect RNA-dependent functions that cause various diseases.

[0545] In some embodiments, the CRISPR methods or systems described herein can also be used to target mutations in cis-acting splicing codes that can lead to splicing defects and disease.

[0546] The CRISPR methods or systems described herein can also be used for antiviral activity, particularly against RNA viruses. CRISPR-associated proteins can target viral RNA using appropriate RNA guides selected to target viral RNA sequences.

[0547] The CRISPR methods or systems described herein can also be used to treat cancer in a subject (e.g., a human subject). For example, the CRISPR-associated proteins described herein can be programmed with crRNAs targeting RNA molecules with abnormalities (e.g., including point mutations or alternative splicing) found in cancer cells to induce cell death (e.g., by apoptosis) in cancer cells.

[0548] In addition, the CRISPR method or system described herein can also be used to treat infectious diseases of subjects. For example, the CRISPR-associated proteins described herein can be programmed with crRNAs targeting RNA molecules expressed by infectious agents (e.g., bacteria, viruses, parasites, or protozoa) to target and induce cell death in infectious agent cells. The CRISPR system can also be used to treat diseases in which intracellular infectious agents infect cells of host subjects. By programming CRISPR-associated proteins to target RNA molecules encoded by infectious agent genes, cells infected with infectious agents can be targeted and cell death can be induced.

[0549] In addition, in vitro RNA sensing assays can be used to detect specific RNA substrates. CRISPR-associated proteins can be used for RNA-based sensing in living cells. Application examples are diagnostics by sensing disease-specific RNA, etc.

[0550] In applications where it is desired to insert a polynucleotide sequence into a target DNA sequence, a polynucleotide comprising a donor sequence to be inserted is also provided to the cell. "Donor sequence" or "donor polynucleotide" means a nucleic acid sequence to be inserted at a cleavage site induced by a site-directed modification polypeptide. The donor polynucleotide will contain sufficient homology with the genomic sequence at the cleavage site, for example, with a nucleotide sequence flanking the cleavage site (e.g., within about 50 bases or less of the cleavage site, such as within about 30 bases, within about 15 bases, within about 10 bases, within about 5 bases, or closely flanking the cleavage site) having 70%, 80%, 85%, 90%, 95% or 100% homology to support homology-directed repair between it and the genomic sequence with which it has homology. Approximately 25, 50, 100 or 200 nucleotides or more than 200 nucleotides (or any integer value between 10 and 200 or more nucleotides) with sequence homology between the donor and the genomic sequence will support homology-directed repair. The donor sequence can be of any length, such as 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 500 nucleotides or more, 1000 nucleotides or more, 5000 nucleotides or more, etc.

[0551] The donor sequence is usually different from the genomic sequence it replaces. On the contrary, the donor sequence can contain at least one or more single base changes, insertions, deletions, inversions or rearrangements relative to the genomic sequence, as long as there is enough homology to support homology-directed repair. In certain embodiments, the donor sequence includes a non-homologous sequence flanked by two homologous regions, so that the homology-directed repair between the target DNA region and the two flanking sequences causes the insertion of the non-homologous sequence at the target region. The donor sequence can also include a vector backbone, which contains sequences that are not homologous to the DNA region of interest and are not intended to be inserted into the DNA region of interest. Typically, the homologous regions of the donor sequence will have at least 50% sequence identity with the genomic sequence that needs to be recombined. In certain embodiments, there is 60%, 70%, 80%, 90%, 95%, 98%, 99% or 99.9% sequence identity. Depending on the length of the donor polynucleotide, any value between 1% and 100% sequence identity can be present.

[0552] Compared to the genomic sequence, the donor sequence may include certain sequence differences, such as restriction sites, nucleotide polymorphisms, selectable markers (e.g., drug resistance genes, fluorescent proteins, enzymes, etc.), etc., which may be used to assess the successful insertion of the donor sequence at the cleavage site, or in some cases may be used for other purposes (e.g., to indicate expression at the targeted genomic locus). In some cases, if located in the coding region, such nucleotide sequence differences will not change the amino acid sequence, or will cause silent amino acids to change (i.e., changes that do not affect the structure or function of the protein). Alternatively, these sequence differences may include flanking recombination sequences, such as FLP, loxP sequences, etc., which may be activated at a later time to remove the marker sequence.

[0553] The donor sequence can be provided to the cell as single-stranded DNA, single-stranded RNA, double-stranded DNA or double-stranded RNA. It can be introduced into the cell in linear or circular form. If introduced in linear form, the end of the donor sequence can be protected by methods known to those skilled in the art (for example, to prevent exonucleolytic degradation). For example, one or more dideoxynucleotide residues are added to the 3' end of the linear molecule and / or a self-complementary oligonucleotide is connected to one or both ends. Other methods for protecting exogenous polynucleotides from degradation include but are not limited to adding terminal amino groups and using modified internucleotide bonds, such as thiophosphates, phosphoramidates and O-methyl ribose or deoxyribose residues. As an alternative to protecting the ends of linear donor sequences, sequences of extra length can be included outside the homology region, and the homology region can be degraded without affecting recombination. The donor sequence can be introduced into the cell as a part of a vector molecule, and the vector molecule has additional sequences, such as genes for replication origin, promoters and encoding antibiotic resistance. In addition, the donor sequence can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by a virus (e.g., adenovirus, AAV), as described above for nucleic acids encoding a DNA-targeting RNA and / or a site-directed modifying polypeptide and / or a donor polynucleotide.

[0554] According to the above method, the DNA region of interest, i.e., "genetically modified", can be cut and modified in vitro. In certain embodiments, when a selectable marker is inserted into a DNA region of interest, by separating the genetically modified cells from the remaining cell population, the cell population can be enriched to include genetically modified cells. Before enrichment, "genetically modified" cells may only account for about 1% or more (e.g., 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 15% or more, or 20% or more) of the cell population. The separation of "genetically modified" cells can be achieved by any convenient separation technique suitable for the selectable marker used. For example, if a fluorescent marker has been inserted, cells can be separated by fluorescence-activated cell sorting, and if a cell surface marker has been inserted, cells can be separated from heterogeneous populations by affinity separation techniques, such as magnetic separation, affinity chromatography, "panning" or other convenient techniques with affinity reagents attached to a solid matrix. The technology that provides accurate separation includes fluorescence activated cell sorter, and its complexity varies, such as multiple color channels, low angle and obtuse angle light scattering detection channels, impedance channels, etc. Cells can be selected for inactivated cells by using dyes (such as propidium iodide) associated with inactivated cells. Any technology that does not excessively damage the vitality of genetically modified cells can be adopted. In this way, a cell composition with a high enrichment of cells including modified DNA is achieved. "Highly enriched" means that genetically modified cells will be 70% or more, 75% or more, 80% or more, 85% or more, 90% or more of the cell composition, such as about 95% or more or 98% or more of the cell composition. In other words, the composition can be a composition of substantially pure genetically modified cells.

[0555] Genetically modified cells produced by the methods described herein can be used immediately. Alternatively, cells can be frozen at liquid nitrogen temperatures and stored for a long time, thawed and can be reused. In this case, cells will typically be frozen in 10% dimethyl sulfoxide (DMSO), 50% serum, 40% buffered medium or other solutions as commonly used in the art to store cells at this freezing temperature, and thawed in a manner known in the art for thawing frozen cultured cells.

[0556] Genetically modified cells can be cultured in vitro under various culture conditions. The cells can be expanded in culture, i.e., grown under conditions that promote their proliferation. The culture medium can be liquid or semisolid, for example, containing agar, methylcellulose, etc. The cell population can be suspended in an appropriate nutrient medium, such as Iscove's modified DMEM or RPMI 1640, usually supplemented with fetal bovine serum (about 5% to 10%),

[0557] L-glutamine, thiols, particularly 2-mercaptoethanol and antibiotics, such as penicillin and streptomycin. The culture may contain growth factors to which regulatory T cells respond. As defined herein, growth factors are molecules that promote cell survival, growth and / or differentiation in culture or intact tissues through specific effects on transmembrane receptors. Growth factors include polypeptide and non-polypeptide factors.

[0558] Cells that have been genetically modified in this manner can be transplanted into a subject for purposes such as gene therapy, for example, to treat a disease or as an antiviral, antipathogen or anticancer therapeutic, for the production of genetically modified organisms in agriculture, or for biological research. The subject can be a newborn, an adolescent or an adult. Of particular interest are mammalian subjects. Mammalian species that can be treated with this method include canines and felines; equines; bovines; sheep; etc., as well as primates, particularly humans. Animal models, particularly small mammals (e.g., mice, rats, guinea pigs, hamsters, lagomorphs (e.g., rabbits), etc.) can be used for experimental studies.

[0559] The cells may be provided to the subject alone or in combination with a suitable substrate or matrix, for example, to support their growth and / or organization in the tissue to which they are transplanted. Typically, at least 1×10 3 cells, such as 5×103 cells, 1×10 4 cells, 5×10 4 cells, 1×10 5 cells, 1×10 6 Cells may be introduced into a subject by any of the following routes: parenteral, subcutaneous, intravenous, intracranial, intraspinal, intraocular, or into the cerebrospinal fluid. Cells may be introduced by injection, catheter, etc. Cells may also be introduced into embryos (e.g., blastocysts) for the purpose of producing transgenic animals (e.g., transgenic mice).

[0560] The number of times a treatment is administered to a subject may vary. The introduction of genetically modified cells into a subject may be a one-time event; however, in some cases, such treatment may bring improvements for a limited time and require a series of ongoing repeated treatments. In other cases, multiple administrations of genetically modified cells may be required before an effect is observed. The exact regimen depends on the disease or condition, the stage of the disease, and the parameters of the individual subject being treated.

[0561] In other aspects of the invention, the DNA-targeting RNA and / or the site-directed modifying polypeptide and / or the donor polynucleotide are used to modify the cellular DNA in vivo, and are also used for purposes such as gene therapy, for example, for treating diseases or as antiviral agents, antipathogen agents or anticancer agents, for producing genetically modified organisms in agriculture, or for biological research. In these in vivo embodiments, the DNA-targeting RNA and / or the site-directed modifying polypeptide and / or the donor polynucleotide are directly administered to the individual. The DNA-targeting RNA and / or the site-directed modifying polypeptide and / or the donor polynucleotide can be administered by any of the many well-known methods used in the art for administering peptides, small molecules and nucleic acids to subjects. The DNA-targeting RNA and / or the site-directed modifying polypeptide and / or the donor polynucleotide can be incorporated into a variety of formulations. More specifically, the DNA-targeting RNA and / or the site-directed modifying polypeptide and / or the donor polynucleotide of the present invention can be formulated into a pharmaceutical composition by combining with an appropriate pharmaceutically acceptable carrier or diluent.

[0562] A pharmaceutical formulation is a composition comprising one or more DNA-targeting RNAs and / or site-directed modifying polypeptides and / or donor polynucleotides in a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" can be a pharmaceutically acceptable carrier approved by a federal or state regulatory agency or approved in the U.S.

[0563] The term "vehicle" refers to a diluent, adjuvant, excipient or carrier with which the compound of the invention is formulated for administration to a mammal. Such pharmaceutical vehicles may be lipids, such as liposomes, such as liposomal dendrimers; liquids, such as water and oils, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc., saline; gum arabic, gelatin, starch paste, talc, keratin, colloidal silicon dioxide, urea, etc. In addition, adjuvants, stabilizers, thickeners, lubricants and colorants may be used. Pharmaceutical compositions may be formulated into solid, semisolid, liquid or gaseous preparations, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols. Thus, administration of the DNA targeting RNA and / or site-directed modifying polypeptide and / or donor polynucleotide can be achieved in a variety of ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intratracheal, intraocular, etc. The active agent can be systemic after administration or can be localized using topical administration, intramural administration, or using an implant, which serves to retain the active dose at the site of implantation. The active agent can be formulated to be immediately active or can be formulated to be sustained release.

[0564] For some conditions, particularly central nervous system conditions, it may be necessary to formulate agents to cross the blood-brain barrier (BBB). A strategy for delivering drugs across the blood-brain barrier (BBB) ​​requires disrupting the BBB through permeability means such as mannitol or leukotrienes or biochemically by using vasoactive substances such as bradykinin. The potential to target specific agents to brain tumors using BBB opening is also an option. When the composition is administered by intravascular injection, a BBB disruptor can be co-administered with the therapeutic composition of the present invention. Other strategies for passing the BBB may require the use of endogenous transport systems, including caveolin-1-mediated transcytosis, carrier-mediated transporters such as glucose and amino acid carriers, receptor-mediated insulin or transferrin transcytosis, and active efflux transporters such as p-glycoprotein. Active transport moieties can also be conjugated to therapeutic compounds used in the present invention to facilitate transport across the endothelial wall of blood vessels.

[0565] Alternatively, drug delivery of therapeutic agents behind the BBB can be by local delivery, such as by intrathecal delivery.

[0566] Typically, an effective amount of DNA-targeting RNA and / or site-directed modified polypeptide and / or donor polynucleotide is provided. As discussed above for the in vitro method, the effective amount or effective dose of the DNA-targeting RNA and / or site-directed modified polypeptide and / or donor polynucleotide in vivo is an amount that induces the amount of recombination observed between two homologous sequences relative to a negative control, such as an increase of 2 times or more in cells contacted with an empty vector or an unrelated polypeptide. The amount of recombination can be measured by any convenient method, for example, as described above and known in the art. The calculation of the effective amount or effective dose of the DNA-targeting RNA and / or site-directed modified polypeptide and / or donor polynucleotide to be administered is within the skill of those of ordinary skill in the art, and is conventional for those skilled in the art. The final amount to be administered will depend on the nature of the route of administration and the disorder or condition to be treated.

[0567] The effective amount given to a particular patient will depend on a variety of factors, some of which vary from patient to patient. A competent clinician will be able to determine an effective amount of a therapeutic agent to be administered to a patient to stop or reverse the progression of a disease state as needed. Using LD50 animal data and other information available for the agent, a clinician can determine the maximum safe dose for an individual based on the route of administration. For example, assuming that the therapeutic composition is administered to a larger body fluid, the dose administered intravenously may be greater than the dose administered intrathecally. Similarly, compositions that are rapidly cleared from the body can be administered at higher doses or in repeated doses to maintain therapeutic concentrations. Using ordinary skills, a competent clinician will be able to optimize the dose of a particular therapeutic agent during a routine clinical trial.

[0568] For inclusion in a medicament, the DNA-targeting RNA and / or site-directed modifying polypeptide and / or donor polynucleotide can be obtained from a suitable commercial source. As a general suggestion, the total pharmaceutically effective amount of the DNA-targeting RNA and / or site-directed modifying polypeptide and / or donor polynucleotide administered parenterally per dose will be within a range that can be measured by a dose response curve.

[0569] Therapies based on RNA targeting DNA and / or site-directed modified polypeptides and / or donor polynucleotides, i.e., preparations of RNA targeting DNA and / or site-directed modified polypeptides and / or donor polynucleotides for therapeutic administration, must be sterile. Sterility is easily achieved by filtration through a sterile filtration membrane (e.g., a 0.2 μm membrane). The therapeutic composition is typically placed in a container with a sterile access port, such as an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle. Therapies based on RNA targeting DNA and / or site-directed modified polypeptides and / or donor polynucleotides can be stored in unit or multidose containers (e.g., sealed ampoules or vials) as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, a 10-mL vial is filled with 5 ml of a sterile filtered 1% (w / v) aqueous solution of the compound, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized compound with antibacterial water for injection.

[0570] Depending on the desired formulation, the pharmaceutical composition may include a pharmaceutically acceptable, non-toxic diluent carrier, which is defined as a carrier commonly used to formulate a pharmaceutical composition for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, buffered water, normal saline, PBS, Ringer's solution, glucose solution and Hank's solution. In addition, the pharmaceutical composition or formulation may include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, excipients, etc. The composition may also include additional substances that approach physiological conditions, such as pH adjusters and buffers, toxicity regulators, wetting agents and detergents.

[0571] The composition can also include any of a variety of stabilizers, such as antioxidants. When the pharmaceutical composition includes a polypeptide, the polypeptide can be compounded with various well-known compounds that enhance the in vivo stability of the polypeptide or otherwise enhance its pharmacological properties (e.g., increase the half-life of the polypeptide, reduce its toxicity and improve solubility or absorption). Examples of such modifiers or complexing agents include sulfates, gluconates, citrates, and phosphates. The nucleic acid or polypeptide of the composition can also be compounded with molecules that enhance its in vivo properties. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese) and lipids.

[0572] Pharmaceutical compositions can be administered for preventive and / or therapeutic treatment. The toxicity and therapeutic efficacy of the active ingredient can be determined according to standard pharmaceutical procedures in cell culture and / or experimental animals, including, for example, determining LD50 (a dose lethal to 50% of the population) and ED50 (a dose effective for the treatment of 50% of the population). The dose ratio between toxic effects and therapeutic effects is the therapeutic index and the therapeutic index can be expressed as the ratio LD50 / ED50. Therapies that preferably exhibit a large therapeutic index are preferred.

[0573] The data obtained from cell culture and / or animal studies can be used to formulate a range of dosages for human use. The dosage of the active ingredient is generally within a circulating concentration range that includes a low toxicity ED50. The dosage may vary within this range depending on the dosage form employed and the route of administration utilized.

[0574] The components used to formulate pharmaceutical compositions are preferably of high purity and substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, usually at least analytical grade, and more usually at least pharmaceutical grade). In addition, compositions for in vivo use are typically sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is typically substantially free of any potentially toxic agents, particularly any endotoxins that may be present during the synthesis or purification process. Compositions for parenteral administration are also sterile, substantially isotonic, and prepared under GMP conditions.

[0575] Delivery System

[0576] The CRISPR system described herein or its components, its nucleic acid molecules and / or nucleic acid molecules encoding or providing its components, CRISPR-related proteins or RNA guides can be delivered by various delivery systems such as vectors (e.g., plasmids and delivery vectors). Exemplary embodiments are described below. The CRISPR system (e.g., including Cas9 containing the nuclear base editor described herein) can be encoded on a nucleic acid contained in a viral vector. The viral vector may include a lentivirus, an adenovirus, a retrovirus, and an adeno-associated virus (AAV). The viral vector can be selected according to the application. For example, AAV is commonly used for in vivo gene delivery due to its mild immunogenicity. Adenoviruses are commonly used as vaccines because they induce strong immunogenic responses. The packaging capacity of viral vectors can limit the size of the base editor that can be packaged into the vector. For example, the packaging capacity of AAV is about 4.5kb, containing two 145 base reverse terminal repeats (ITRs).

[0577] AAV is a small, single-stranded DNA-dependent virus belonging to the family Parvoviridae. The 4.7 kb wild-type (wt) AAV genome consists of two genes encoding four replication proteins and three capsid proteins, respectively, and is flanked on both sides by 145-bp inverted terminal repeats (ITRs). Virions are composed of three capsid proteins, Vp1, Vp2, and Vp3, which are produced from the same open reading frame in a 1:1:10 ratio but result from differential splicing (Vp1) and alternative translation start sites (Vp2 and Vp3, respectively). Vp3 is the most abundant subunit in the virion and is involved in receptor recognition on the cell surface, defining the tropism of the virus. A phospholipase domain that plays a role in viral infectivity has been identified at the unique N-terminus of Vp1.

[0578] Similar to wt AAV, recombinant AAV (rAAV) utilizes cis-acting 145-bp ITRs to flank the vector transgene cassette, providing packaging of up to 4.5 kb for exogenous DNA. After infection, rAAV can express the fusion protein of the present invention and persist without integrating into the host genome by existing in a free form in a circular head-to-tail concatemer. Although there are many examples of successful use of rAAV in this system in vitro and in vivo, the limited packaging capacity limits the use of AAV-mediated gene delivery when the length of the gene coding sequence is equal to or greater than the size of the wt AAV genome.

[0579] The small packaging capacity of AAV vectors makes the delivery of a large number of genes exceeding this size and / or the use of large physiological regulatory elements challenging. These challenges can be solved by, for example, dividing the protein to be delivered into two or more fragments, wherein the N-terminal fragment is fused to the split intein-N, and the C-terminal fragment is fused to the split intein-C. These fragments are then packaged into two or more AAV vectors. As used herein, "intein" refers to a self-splicing protein intron (e.g., peptide) connecting the flanking N-terminal and C-terminal exons (e.g., fragments to be connected). The use of certain inteins for connecting heterologous protein fragments is described in, for example, Wood et al., J.Biol.Chem.289(21); 14512-9(2014). For example, when fused to a separate protein fragment, intein IntN and IntC recognize each other, splice themselves and simultaneously connect the flanking N-terminal extein and C-terminal extein of the protein fragment they are fused to, thereby reconstructing the full-length protein from these two protein fragments. Other suitable inteins will be apparent to those skilled in the art.

[0580] In some embodiments, the length of the CRISPR system of the present invention can vary. In some embodiments, the length of the protein fragment ranges from 2 amino acids to about 1000 amino acids. In some embodiments, the length of the protein fragment ranges from about 5 amino acids to about 500 amino acids. In some embodiments, the length of the protein fragment ranges from about 20 amino acids to about 200 amino acids. In some embodiments, the length of the protein fragment ranges from about 10 amino acids to about 100 amino acids. Suitable protein fragments of other lengths will be apparent to those skilled in the art.

[0581] In some embodiments, a portion or fragment of a nuclease (e.g., Cas9) is fused to an intein. The nuclease may be fused to the N-terminus or C-terminus of an intein. In some embodiments, a portion or fragment of a fusion protein is fused to an intein and fused to an AAV capsid protein. Inteins, nucleases, and capsid proteins may be fused together in any arrangement (e.g., nuclease-intein-capsid, intein-nuclease-capsid, capsid-intein-nuclease, etc.). In some embodiments, the N-terminus of an intein is fused to the C-terminus of a fusion protein, and the C-terminus of an intein is fused to the N-terminus of an AAV capsid protein.

[0582] In one embodiment, a dual AAV vector is generated by splitting a large transgene expression cassette into two separate halves (5' and 3' ends, or head and tail), wherein each half of the cassette is packaged in a single AAV vector (<5 kb). Reassembly of the full-length transgene expression cassette is then achieved by co-infection of the same cell with two dual AAV vectors, followed by: (1) homologous recombination (HR) between the 5' and 3' genomes (dual AAV overlapping vector); (2) ITR-mediated tail-to-head tandem of the 5' and 3' genomes (dual AAV trans-splicing vector); or (3) a combination of these two mechanisms (dual AAV hybrid vector). The use of dual AAV vectors in vivo results in the expression of full-length proteins. The use of a dual AAV vector platform represents an efficient and feasible gene transfer strategy for transgenes of size >4.7 kb.

[0583] The disclosed strategies for designing CRISPR systems including Cas9 described herein can be used to generate CRISPR systems that can be packaged into viral vectors. The use of RNA or DNA virus-based systems to deliver base editors utilizes highly evolved processes for targeting viruses to specific cells in culture or in the host and transporting viral payloads to the nucleus or host cell genome. Viral vectors can be directly applied to cells in culture, patients (in vivo), or they can be used to treat cells in vitro, and modified cells can be optionally applied to patients (ex vivo). Traditional virus-based systems can include retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, and herpes simplex virus vectors for gene transfer. Retrovirus, lentivirus, and adeno-associated virus gene transfer methods can be integrated into the host genome, which generally results in long-term expression of the inserted transgene. In addition, high transduction efficiency has been observed in many different cell types and target tissues.

[0584] The tropism of retroviruses can be altered by incorporating exogenous envelope proteins, thereby expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Therefore, the choice of retroviral gene transfer system may depend on the target tissue. Retroviral vectors contain cis-acting long terminal repeats with the ability to package up to 6kb to 10kb of exogenous sequences. The minimal cis-acting LTR is sufficient to replicate and package the vector, which is then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT / US94 / 05700).

[0585] Retroviral vectors, particularly lentiviral vectors, may require polynucleotide sequences less than a given length to be efficiently integrated into target cells. For example, a retroviral vector greater than 9 kb in length may result in low viral titers compared to retroviral vectors of smaller sizes. In some aspects, the CRISPR system of the present disclosure (e.g., comprising Cas9 disclosed herein) is of sufficient size to enable efficient packaging and delivery to target cells by retroviral vectors. In some cases, the size of Cas9 allows efficient packaging and delivery even when expressed with guide nucleic acids and / or other components of a targetable nuclease system.

[0586] In applications where transient expression is preferred, adenovirus-based systems can be used. Adenovirus-based vectors are able to have very high transduction efficiencies in many cell types and do not require cell division. High titers and high levels of expression have been achieved using such vectors. This vector can be mass-produced in a relatively simple system. Adeno-associated virus ("AAV") vectors can also be used to transduce cells with target nucleic acids, e.g., for in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectors is described in many publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat and Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989).

[0587] Therefore, the CRISPR system described herein (e.g., including Cas9 disclosed herein) can be delivered with a viral vector. One or more components of the base editor system can be encoded on one or more viral vectors. For example, the base editor and the guide nucleic acid can be encoded on a single viral vector. In other cases, the base editor and the guide nucleic acid are encoded on different viral vectors. In either case, the base editor and the guide nucleic acid can be operably linked to a promoter and a terminator.

[0588] The combination of components encoded on the viral vector can be determined by the cargo size limitations of the chosen viral vector.

[0589] Non-viral delivery of base editors

[0590] Non-viral delivery methods for CRISPR are also available. An important category of non-viral nucleic acid vectors is nanoparticles, which can be organic or inorganic. Nanoparticles are well known in the art. Any suitable nanoparticle design can be used to deliver genome editing system components or nucleic acids encoding such components. For example, organic (e.g., lipids and / or polymers) nanoparticles may be suitable for use as delivery vehicles in certain embodiments of the present disclosure. Exemplary lipids for nanoparticle formulations and / or gene transfer are shown in Table 5 (below).

[0591] Table 5

[0592]

[0593]

[0594] Table 6 lists exemplary polymers for gene transfer and / or nanoparticle formulations.

[0595] Table 6

[0596]

[0597]

[0598] Table 7 summarizes the delivery methods for polynucleotides encoding Cas9 described herein.

[0599] Table 7

[0600]

[0601]

[0602] On the other hand, delivery of nucleic acids encoding genome editing system components or such components (e.g., nucleic acid binding proteins (e.g., Cas9 or variants thereof) optionally fused to biologically active polypeptides (e.g., nucleobase editors), and gRNAs targeting genomic nucleic acid sequences of interest) can be achieved by delivering ribonucleoproteins (RNPs) to cells. RNPs include nucleic acid binding proteins, such as Cas9, complexed with targeting gRNAs. RNPs can be delivered to cells using known methods, such as electroporation, nuclear transfection, or cationic lipid-mediated methods, for example, as reported by Zuris, JA et al., 2015, Nat. Biotechnology, 33(1): 73-80. RNPs are advantageous for use in CRISPR base editing systems, particularly for cells that are difficult to transfect, such as primary cells. In addition, RNPs can also alleviate difficulties that may arise in protein expression in cells, particularly when eukaryotic promoters (e.g., CMV or EF1A that can be used for CRISPR plasmids) are poorly expressed. Advantageously, the use of RNPs does not require delivery of exogenous DNA to cells. Furthermore, since RNPs, which include a nucleic acid binding protein and gRNA complex, degrade over time, the use of RNPs has the potential to limit off-target effects. In a similar manner to plasmid-based technologies, RNPs can be used to deliver binding proteins (e.g., Cas9 variants) and direct homology-directed repair (HDR).

[0603] The promoter for driving the CRISPR system (e.g., comprising Cas9 as described herein) can comprise AAV ITR. This is advantageous for eliminating the need for additional promoter elements that may take up space in the vector. The released additional space can be used to drive the expression of other elements, such as guide nucleic acids or selectable markers. ITR activity is relatively weak, so it can be used to reduce the potential toxicity caused by overexpression of the selected nuclease.

[0604] Any suitable promoter can be used to drive the expression of Cas9 and guide nucleic acid when appropriate. For universal expression, promoters that can be used include CMV, CAG, CBh, PGK, SV40, ferritin heavy chain or light chain, etc. For brain or other CNS cell expression, suitable promoters may include: synapsin I for all neurons, CaMKIIα for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc. For hepatocyte expression, suitable promoters include albumin promoters. For lung cell expression, suitable promoters may include SP-B. For endothelial cells, suitable promoters may include ICAM. For hematopoietic cells, suitable promoters may include IFNβ or CD45. For osteoblasts, suitable promoters may include OG-2.

[0605] In some cases, the Cas9 of the present disclosure is of a sufficiently small size to allow a single promoter to drive expression of a base editor and a compatible guide nucleic acid within the same nucleic acid molecule. For example, a vector or viral vector can include a first promoter operably linked to a nucleic acid encoding a base editor and a second promoter operably linked to a guide nucleic acid.

[0606] Promoters used to drive expression of guide nucleic acids can include: Pol III promoters, such as U6 or H1, using PolII promoters and intronic cassettes to express gRNA adeno-associated viruses (AAV).

[0607] Adeno-associated virus (AAV), lentivirus, adenovirus or other plasmid or viral vector types can be used to deliver Cas9 described herein with or without one or more guide nucleic acids, particularly using formulations and doses from, for example, U.S. Pat. No. 8,454,972 (formulations, doses of adenovirus), U.S. Pat. No. 8,404,658 (formulations, doses of AAV) and U.S. Pat. No. 5,846,946 (formulations, doses of DNA plasmids) and clinical trials and publications involving lentivirus, AAV and adenovirus. For example, for AAV, the route of administration, formulation and dose can be as in U.S. Pat. No. 8,454,972 and in clinical trials involving AAV. For adenovirus, the route of administration, formulation and dose can be as in U.S. Pat. No. 8,404,658 and in clinical trials involving adenovirus. For plasmid delivery, the route of administration, formulation and dose can be as in U.S. Pat. No. 5,846,946 and in clinical studies involving plasmids. The dosage can be based on or extrapolated to an average of 70 kg individuals (e.g., adult males), and can be adjusted for patients, subjects, and mammals of different weights and species. The frequency of administration is within the range of medical or veterinary practitioners (e.g., physicians, veterinarians), depending on common factors, including age, sex, general health, other conditions of the patient or subject, and the specific condition or symptom being addressed. The viral vector can be injected into the tissue of interest. For cell type-specific base editing, the expression of the base editor and the optional guide nucleic acid can be driven by a cell type-specific promoter.

[0608] AAV may be superior to other viral vectors for in vivo delivery. In some cases, AAV has low toxicity, which may be due to the purification method that does not require ultracentrifugation of cell pellets that can activate immune responses. In some cases, AAV is less likely to cause insertional mutagenesis because it does not integrate into the host genome.

[0609] The packaging limit of AAV is 4.5 or 4.75 Kb. Constructs larger than 4.5 or 4.75 Kb can result in significantly reduced viral yields. For example, SpCas9 is quite large, with the gene itself exceeding 4.1 Kb, which makes it difficult to package into AAV. Therefore, embodiments of the present disclosure include the use of a disclosed Cas9 that is shorter in length than conventional Cas9.

[0610] The AAV can be AAV1, AAV2, AAV5, or any combination thereof. The type of AAV can be selected based on the cells to be targeted; for example, AAV serotypes 1, 2, 5, or mixed capsid AAV1, AAV2, AAV5, or any combination thereof can be selected to target brain or neuronal cells; AAV4 can be selected to target cardiac tissue. AAV8 can be used for delivery to the liver. A list of certain AAV serotypes for these cells can be found in Grimm, D. et al., J. Virol. 82:5887-5911 (2008)).

[0611] Lentiviruses are complex retroviruses that are able to infect and express their genes in both mitotic and post-mitotic cells. The most common lentivirus is the human immunodeficiency virus (HIV), which uses the envelope glycoproteins of other viruses to target a variety of cell types.

[0612] Lentivirus can be prepared as follows. After cloning pCasES10 (containing a lentiviral transfer plasmid backbone), low-passage (p=5) HEK293FT was inoculated into a T-75 flask in DMEM containing 10% fetal bovine serum and without antibiotics the day before transfection to achieve 50% fusion. After 20 hours, the culture medium was replaced with OptiMEM (serum-free) medium, and transfection was completed after 4 hours. Cells were transfected with 10 μg of lentiviral transfer plasmid (pCasES10) and the following packaging plasmids: 5 μg of pMD2.G (VSV-g pseudotype) and 7.5 μg of psPAX2 (gag / pol / rev / tat). Transfection can be performed in 4 mL of OptiMEM using a cationic lipid delivery agent (50 μl of Lipofectamine 2000 and 100 ul Plus reagent). After 6 hours, the culture medium was replaced with DMEM containing 10% fetal bovine serum without antibiotics. These methods use serum during cell culture, but serum-free methods are preferred.

[0613] Lentivirus can be purified as follows. Harvest the viral supernatant after 48 hours. First clear the supernatant of debris and filter through a 0.45μm low protein binding (PVDF) filter. Then spin them in an ultracentrifuge at 24,000rpm for 2 hours. Resuspend the viral pellet in 50μl DMEM at 4°C overnight. Then aliquot them and freeze immediately at -80°C.

[0614] In another embodiment, a minimal non-primate lentiviral vector based on equine infectious anemia virus (EIAV) is also contemplated. In another embodiment, A lentiviral gene therapy vector based on equine infectious anemia virus that expresses the vasoinhibitory proteins endostatin and angiostatin is intended to be delivered via subretinal injection. In another embodiment, the use of a self-inactivating lentiviral vector is contemplated.

[0615] Any RNA of the system, such as a guide RNA or an mRNA encoding Cas9, can be delivered in the form of RNA. The mRNA encoding Cas9 can be generated using in vitro transcription. For example, Cas9 mRNA can be synthesized using a PCR box comprising the following elements: a T7 promoter, an optional kozak sequence (GCCACC), a nuclease sequence, and a 3'UTR (such as a 3'UTR from a β-globin-polyA tail). The box can be used for transcription of a T7 polymerase. A guide polynucleotide (e.g., gRNA) can also be transcribed in vitro using a box comprising a T7 promoter, followed by a sequence "GG" and a guide polynucleotide sequence.

[0616] To enhance expression and reduce potential toxicity, the Cas9 sequence and / or the guide nucleic acid can be modified to include one or more modified nucleosides, for example using pseudo-U or 5-methyl-C.

[0617] The disclosure in some embodiments includes methods for modifying cells or organisms. The cell may be a prokaryotic cell or a eukaryotic cell. The cell may be a mammalian cell. The mammalian cell may be a non-human primate, a cattle, a pig, a rodent, or a mouse cell. Introducing modifications into cells by the base editors, compositions, and methods disclosed herein may cause cells and the progeny of cells to be altered to improve the production of biological products (such as antibodies, starch, alcohol, or other desired cell outputs). Introducing modifications into cells by the methods disclosed herein may cause cells and the progeny of cells to include changes that alter the biological products produced.

[0618] The system may comprise one or more different vectors. In one aspect, Cas9 is codon optimized for expression in a desired cell type, preferably a eukaryotic cell, preferably a mammalian cell or a human cell.

[0619] Generally, codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) of a native sequence with a more commonly used or most commonly used codon in the genes of the host cell, while maintaining the native amino acid sequence. Different species show specific biases to certain codons of specific amino acids. Codon bias (differences in codon usage between organisms) is generally related to the translation efficiency of messenger RNA (mRNA), which is considered to depend on the properties of the codons to be translated and the availability of specific transfer RNA (tRNA) molecules, etc. The advantage of selected tRNA in a cell generally reflects the most commonly used codons in peptide synthesis. Therefore, genes can be customized based on codon optimization to achieve optimal gene expression in a given organism. Codon usage tables are readily available, for example, in the "Codon Usage Database" at www.kazusa.orjp / codon / (accessed July 9, 2002), and these tables can be modified in a variety of ways. See, Nakamura, Y. et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28: 292 (2000). Computer algorithms for codon optimization of specific sequences for expression in specific host cells are also available, such as Gene Forge (Aptagen; Jacobus, Pa.) can also be provided. In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more, or all codons) in the sequence encoding the engineered nuclease correspond to the most commonly used codons for a specific amino acid.

[0620] Packaging cells are usually used to form viral particles that can infect host cells. Such cells include 293 cells for packaging adenovirus, and psi.2 cells or PA317 cells for packaging retrovirus. The viral vectors used in gene therapy are usually produced by producer cell lines that package nucleic acid vectors into viral particles. The vector usually contains the minimum viral sequences required for packaging and subsequent integration into the host, and other viral sequences are replaced by expression cassettes of polynucleotides to be expressed. The missing viral functions are usually provided in trans by the packaging cell line. For example, AAV vectors used in gene therapy usually have only ITR sequences from the AAV genome, which are necessary for packaging and integration into the host genome. Viral DNA can be packaged in a cell line that contains helper plasmids encoding other AAV genes, i.e., rep and cap, but lacks ITR sequences. The cell line can also be infected with adenovirus as an aid. Helper viruses can promote the replication of AAV vectors and the expression of AAV genes from helper plasmids. In some cases, due to the lack of ITR sequences, helper plasmids are not packaged in large quantities. Contamination with adenovirus can be reduced, for example, by heat treatment, to which adenovirus is more sensitive than AAV.

[0621] Pharmaceutical composition

[0622] Other aspects of the present disclosure relate to pharmaceutical compositions comprising a CRISPR system (e.g., including Cas9 disclosed herein). As used herein, the term "pharmaceutical composition" refers to a composition formulated for pharmaceutical use. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises additional agents (e.g., for specific delivery, increased half-life or other therapeutic compounds).

[0623] As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium, calcium or zinc stearate or stearic acid), or solvent encapsulating material, involved in carrying or transporting a compound from one part of the body (e.g., a delivery site) to another part (e.g., an organ, tissue, or part of the body). A pharmaceutically acceptable carrier is "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissues of the subject (e.g., physiologically compatible, sterile, physiological pH, etc.).

[0624] Some non-limiting examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, sodium methylcellulose, ethylcellulose, sodium microcrystalline cellulose and cellulose acetate; (4) astragalus powder; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) diols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) fillers, such as polypeptides and amino acids; (23) serum alcohols, such as ethanol; and (23) other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, colorants, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives and antioxidants may also be present in the formulation. Terms such as "excipient", "carrier", "pharmaceutically acceptable carrier", "vehicle" and the like are used interchangeably herein.

[0625] The pharmaceutical composition may include one or more pH buffer compounds to maintain the pH of the formulation at a predetermined level reflecting physiological pH, such as in the range of about 5.0 to about 8.0. The pH buffer compound used in the aqueous liquid formulation can be an amino acid or an amino acid mixture, such as histidine or an amino acid mixture (such as histidine and glycine). Alternatively, the pH buffer compound is preferably a medicament that maintains the pH of the formulation at a predetermined level (such as in the range of about 5.0 to about 8.0) and does not chelate calcium ions. Illustrative examples of such pH buffer compounds include, but are not limited to, imidazole and acetate ions. The pH buffer compound can be present in any amount suitable for maintaining the pH of the formulation at a predetermined level.

[0626] The pharmaceutical composition may also include one or more osmotic regulators, i.e., compounds that adjust the osmotic properties (e.g., tension, osmotic pressure, and / or osmotic pressure) of the formulation to a level acceptable to the blood flow and blood cells of the recipient individual. An osmotic regulator may be an agent that does not chelate calcium ions. An osmotic regulator may be any compound that regulates the osmotic properties of the formulation known or available to those skilled in the art. Those skilled in the art may empirically determine the suitability of a given osmotic regulator for the formulation of the present invention. Illustrative examples of suitable types of osmotic regulators include, but are not limited to, salts such as sodium chloride and sodium acetate; sugars such as sucrose, dextrose, and mannitol; amino acids such as glycine; and mixtures of one or more of these agents and / or agent types. An osmotic regulator may be present in any concentration sufficient to regulate the osmotic properties of the formulation.

[0627] In some embodiments, the pharmaceutical composition is formulated for delivery to a subject, e.g., for gene editing. Suitable routes of administration of the pharmaceutical compositions described herein include, but are not limited to, topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseous, periocular, intratumoral, intracerebral, and intraventricular administration.

[0628] In some embodiments, the pharmaceutical compositions described herein are topically administered to the diseased site. In some embodiments, the pharmaceutical compositions described herein are administered to a subject by injection, by catheter, by suppository, or by implant, which is a porous, non-porous, or gel-like material, including a membrane (e.g., a silicone rubber membrane) or a fiber.

[0629] In other embodiments, the pharmaceutical compositions described herein are delivered in a controlled release system. In one embodiment, a pump can be used (see, e.g., Langer, 1990, Science 249: 1527-1533; Sefton, 1989, CRC Crit. Ref. Biomed. Eng. 14: 201; Buchwald et al., 1980, Surgery 88: 507; Saudek et al., 1989, N. Engl. J. Med. 321: 574). In another embodiment, a polymeric material can be used. (See, e.g., Medical Applications of Controlled Release (Langer and Wise, eds., CRC Press, Boca Raton, Fla., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball, eds., Wiley, New York, 1984); Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61. See also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105.) Other controlled-release systems are discussed, e.g., in Langer, supra.

[0630] In certain embodiments, pharmaceutical composition is formulated as a composition suitable for intravenous or subcutaneous administration to a subject (e.g., people) according to routine procedures. In certain embodiments, the pharmaceutical composition for administration by injection is a sterile isotonic solution used as a solubilizer and a local anesthetic (e.g., lidocaine), to relieve pain at the injection site. Generally, components are separated or mixed together and provided in unit dosage form, e.g., as a dry lyophilized powder or anhydrous concentrate in an airtight container, the airtight container such as an ampoule or a sachet indicating the amount of an active agent. When the drug is administered by infusion, an infusion bottle containing sterile pharmaceutical grade water or saline can be distributed. When the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed before administration.

[0631] Pharmaceutical compositions for systemic administration can be liquids, such as sterile saline, lactated Ringer's solution or Hank's solution. In addition, the pharmaceutical composition can be in solid form and redissolved or suspended immediately before use. Lyophilized forms are also contemplated. The pharmaceutical composition can be contained in lipid particles or vesicles (such as liposomes or microcrystals), which are also suitable for parenteral administration. The particles can have any suitable structure, such as a single layer or multiple layers, as long as the composition is contained therein. The compound can be embedded in a "stable plasmid-lipid particle" (SPLP) containing a fusion lipid dioleoylphosphatidylethanolamine (DOPE), a low level (5 to 10 mol%) of cationic lipids, and stabilized by a polyethylene glycol (PEG) coating (Zhang YP et al., Gene Ther. 1999, 6: 1438-47). Positively charged lipids such as N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-methylammonium sulfate or "DOTAP" are particularly preferred for such particles and vesicles. The preparation of such lipid particles is well known. See, e.g., U.S. Pat. Nos. 4,880,635; 4,906,477; 4,911,928; 4,917,951; 4,920,016; and 4,921,757; each of which is incorporated herein by reference.

[0632] For example, the pharmaceutical compositions described herein can be administered or packaged as unit doses. When used in relation to the pharmaceutical compositions of the present disclosure, the term "unit dose" refers to physically discrete units suitable as unit doses for subjects, each unit containing a predetermined amount of active material calculated to produce the desired therapeutic effect associated with the required diluent; i.e., carrier or vehicle.

[0633] In addition, the pharmaceutical composition can be provided as a pharmaceutical kit, which comprises (a) a container containing a lyophilized compound of the invention and (b) a second container containing a pharmaceutically acceptable diluent (e.g., a sterile diluent for reconstituting or diluting the lyophilized compound of the invention). Optionally, associated with such container may be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of drugs or biological products, which notice reflects the approval obtained by the manufacturer, use or sale agency for human administration.

[0634] On the other hand, articles containing materials useful for treating the above-mentioned diseases are included. In some embodiments, the article comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container can be formed of various materials such as glass or plastic. In some embodiments, the container holds a composition effective for treating the diseases described herein and can have a sterile access port. For example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle. The active agent in the composition is a compound of the present invention. In some embodiments, a label on the container or a label associated with the container indicates that the composition is used to treat a selected disease. The article may further include a second container, which includes a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. The article may further include other materials required from a commercial and user's point of view, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0635] In some embodiments, the CRISPR system (e.g., comprising Cas9 described herein) is provided as part of a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises any fusion protein provided herein (e.g., including a nucleobase editor comprising LubCas9 described herein). In some embodiments, the pharmaceutical composition comprises any one of the complexes provided herein. In some embodiments, the pharmaceutical composition comprises a ribonucleoprotein complex comprising a nuclease (e.g., Cas9) guided by RNA that forms a complex with gRNA and a cationic lipid. In some embodiments, the pharmaceutical composition comprises gRNA, a nucleic acid programmable DNA binding protein, a cationic lipid, and a pharmaceutically acceptable excipient. The pharmaceutical composition may optionally comprise one or more additional therapeutically active substances.

[0636] Reagent test kit

[0637] In one aspect, the present invention provides a kit comprising any one or more of the elements disclosed in the above methods and compositions. In some embodiments, the kit comprises a vector system and instructions for using the kit. In some embodiments, the vector system comprises one or more insertion sites for inserting a guide sequence, wherein when expressed, the guide sequence guides the CRISPR complex to perform sequence-specific binding with a target sequence in a eukaryotic cell, wherein the CRISPR complex comprises a CRISPR enzyme compounded with the following items: (1) a guide sequence hybridized with a target sequence, and (2) a sequence hybridized with a tracrRNA sequence; and / or (b) a second regulatory element, which is operably linked to an enzyme encoding sequence encoding the CRISPR enzyme comprising a nuclear localization sequence. Elements may be provided individually or in combination and may be provided in any suitable container (such as a vial, bottle, or tube). In some embodiments, the kit includes instructions in one or more languages, such as instructions in more than one language.

[0638] In some embodiments, the kit comprises a nucleobase editor. For example, in some embodiments, the kit comprises a nucleobase editor comprising a Cas9 enzyme described herein (ScoCas9, Seq2Cas9, EhiCas9, SeqCas9, SsiCas9, SinCas9, SsaCas9, Ssc2Cas9, Sor2Cas9, SorCas9, SwaCas9, SscCas9, SgaCas9, LkuCas9, and SsuCas9).

[0639] In some embodiments, the kit includes one or more reagents for use in the method utilizing one or more elements described herein. The reagent can be provided in any suitable container. For example, the kit can provide one or more reaction or storage buffers. The reagent can be provided in a form that can be used for a specific assay, or in a form that needs to add one or more other components before use (for example, in a concentrate or lyophilized form). The buffer can be any buffer, including but not limited to sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer and combinations thereof. In some embodiments, the buffer is alkaline. In some embodiments, the pH of the buffer is about 7 to about 10. In some embodiments, the kit includes one or more oligonucleotides corresponding to the guide sequence for insertion into the vector, so as to operably connect the guide sequence and the regulatory element. In some embodiments, the kit includes a homologous recombination template polynucleotide.

[0640] All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In addition, materials, methods and examples are only illustrative and are not intended to be limiting. Unless otherwise defined, all technical terms and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs. Although methods and materials similar to or equivalent to the methods and materials described herein can be used to practice or test the present invention, suitable methods and materials are described herein.

[0641] Examples

[0642] The following examples describe some of the preferred modes for preparing and implementing the present invention. However, it should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0643] Example 1. Screening, discovery and optimization of new Cas9 enzymes

[0644] This example describes a screen to discover novel Cas9 enzymes. As described herein, novel Cas9 enzymes were isolated and optimized from the following bacteria using this screening method: Streptococcus equi ATCC 33317, Enterococcus hirae strain F1129E, Streptococcus equi strain AG46, Staphylococcus pseudostrain 19, Streptococcus intermedius B196 strain G1552, Streptococcus sanguinis SK330, Streptococcus C150, Streptococcus oralis subsp. oralis strain RH_1735_08, Streptococcus oralis SK313, Staphylococcus wortii strain 691, Staphylococcus sciuri strain SNUC 2430, Streptococcus gallolyticus strain AM24-4, Lactobacillus kuraburgii strain Biut2, and Streptococcus suis strain LSS83.

[0645] In the search for novel Cas9 enzymes that recognize novel PAM sequences, a bioinformatics screen was used to search for additional enzymes to expand the targeting range of CRISPR. The screen utilized seed sequences of Cas9 from either Lachnospira LubCas9 or Streptococcus asteroides subsp. ScoCas9. Bioinformatics analysis was performed using the tblastn variant of BLAST, with an e-value threshold of 1e-6 considered for BLAST hits. In brief, loci selected for testing were those that remained intact in the presence of Cas9 proteins from other species. The loci selected had more than 3 spacers within the CRISPR array, more than 1 kb of endogenous sequence at the 5' end of Cas9, and more than 300 nt of endogenous sequence at the 3' end of the CRISPR array. Using this approach, novel Cas9 enzymes were identified from different bacterial species and codon-optimized for expression in human cells. The novel engineered Cas9 enzymes were then recombinantly produced and tested. Example 2. Identification of novel 3' PAM consensus motif of Cas9 enzyme: Streptococcus equi ATCC 33317, Enterococcus hirae strain F1129E, Streptococcus equi strain AG46, Staphylococcus aureus strain 19, Streptococcus intermedius B196 strain G1552, Streptococcus sanguinis SK330, Streptococcus spp. C150, oral Streptococcus subsp. oral strain RH_1735_08, Streptococcus oralis SK313, Staphylococcus worderi strain 691, Staphylococcus sciuri strain SNUC 2430, Streptococcus gallolyticus strain AM24-4, Lactobacillus kuraburgii strain Biut2, and Streptococcus suis strain LSS83

[0646] This example illustrates the identification of protospacer adjacent motif (PAM) sequences for human codon-optimized Cas9 originally isolated from the following bacteria: Streptococcus equi ATCC 33317, Enterococcus hirae strain F1129E, Streptococcus equi strain AG46, Staphylococcus aureus strain 19, Streptococcus intermedius B196 strain G1552, Streptococcus sanguis SK330, Streptococcus sp. C150, Streptococcus orals subsp. oral strain RH_1735_08, Streptococcus orals SK313, Staphylococcus worteri strain 691, Staphylococcus sciuri strain SNUC 2430, Streptococcus gallolyticus strain AM24-4, Lactobacillus kuraburgii strain Biut2, and Streptococcus suis strain LSS83.

[0647] The recognition of PAM sequences by human codon-optimized Cas9 was tested using an in vitro PAM recognition assay. A pool of plasmids with random PAM sequences was incubated with Cas9 isolated from different bacteria. Uncut plasmids were purified and sequenced to identify the specific PAM motif that was cleaved. For example, Cas9 engineered from Streptococcus equi ATCC 33317 recognized the consensus PAM sequence 5'-NRGNR-3'( Figure 1A ), Cas9 engineered by Enterococcus hirae strain F1129E recognizes the consensus PAM sequence 5'-NRG-3'( Figure 1B ), by Streptococcus equi AG46 ( Figure 1C ), Staphylococcus waldenii strain 691 ( Figure 1J ), and Staphylococcus sciuri strain SNUC 2430 ( Figure 1K ) engineered Cas9 recognizes the consensus PAM sequence 5'-NNGR-3', and the Cas9 engineered by Staphylococcus aureus strain 19 recognizes the consensus PAM sequence 5'-NNGRRT-3' ( Figure 1D ), Cas9 engineered by Streptococcus intermedius B196 strain G1552 recognizes the consensus PAM sequence 5'-NNAAAA-3'( Figure 1E ), the Cas9 engineered by Streptococcus sanguinis SK330 recognizes the consensus PAM sequence 5'-NGGNG-3'( Figure 1F ), the Cas9 engineered by Streptococcus C150 recognizes the consensus PAM sequence 5'-NNGNRG-3'( Figure 1G ), Cas9 engineered from S. oralis subsp. oral strain RH_1735_08 recognizes the consensus PAM sequence 5'-NNAAAC-3'( Figure 1H ), Cas9 engineered by S. oralis SK313 recognizes the consensus PAM sequence 5'-NNRAAG-3'( Fig. 1I ), the Cas9 engineered by Streptococcus gallolyticus strain AM24-4 recognizes the consensus PAM sequence 5'-NNAYAA-3'( Figure 1L ), Cas9 engineered by Lactobacillus kuraburgensis strain Biut2 recognizes the consensus PAM sequence 5'-NNGAAA-3'( Figure 1M ), and the Cas9 engineered by Streptococcus suis strain recognizes the consensus PAM sequence 5'-NNAAA-3'( Figure 1N )(H=A, C or T; R=A or G).

[0648] Example 3. Prediction of the presence of Streptococcus asteroides, Sharpea isolate RUG017, Micrococcus vermiformis, Ezhaki New strains of Ezakiella peruensis, Lactobacillus fermentum strain AF15-40LB and Peptophilus Marseille-P3761 RNA folding structure of sgRNA for Cas9 enzyme

[0649] This example shows the predicted RNA folding structure of an exemplary sgRNA comprising crRNA and tracrRNA for use with the novel Cas9 enzyme.

[0650] Small RNA sequencing was performed on RNA derived from an E. coli strain heterologously expressing the Cas9 Crispr locus. Briefly, RNA was isolated from stationary-phase bacteria by first resuspending E. coli in Trizol and then homogenizing the bacteria with zirconium oxide / silica beads in a homogenizer for three 1-minute cycles. Total RNA was purified from the homogenized samples, treated with DNase and 3' dephosphorylated with T4 polynucleotide kinase, and rRNA was depleted. RNA libraries were prepared from rRNA-depleted RNA and size-selected for small RNAs.

[0651] For RNA sequencing, transcripts were poly-A tailed with Escherichia coli Poly(A) polymerase, ligated to 5' RNA adapters using T4 RNA ligase 1 and reverse transcribed, and cDNA was PCR amplified using barcoded primers and sequenced on MiSeq. Reads for each sample were identified according to the associated barcode and aligned to the reference sequence using BWA. Transcript sequences were extracted using paired-end alignment using the Picard tool, and sequences were analyzed using Geneious software.

[0652] RNA folding is based on predictions from Geneious 11.1.2 software. A single sgRNA transcript fuses crRNA to tracrRNA that mimics the dual RNA structure required for guide site-specific Cas9 activity. The predicted RNA folding structure of the chimeric sgRNA for use with Seq2Cas9 from Streptococcus equi ATCC 33317 is shown in Figure 2A The sgRNA for use with EhiCas9 from E. hirae strain F1129E is shown in Figure 2BThe sgRNA for use with SeqCas9 from S. equi strain AG46 is shown in Figure 2C The sgRNA for use with SsiCas9 from Staphylococcus aureus strain 19 is shown in Figure 2D The sgRNA for use with SinCas9 of S. intermedius B196 strain G1552 is shown in Figure 2E The sgRNA for use with SsaCas9 from Streptococcus sanguis SK330 is shown in Figure 2F The sgRNA for use with Ssc2Cas9 from Streptococcus C150 is shown in Figure 2G The sgRNA for use with Sor2Cas9 from S. oralis subsp. oral strain RH_1735_08 is shown in Figure 2H The sgRNA for use with Sor2Cas9 from S. oralis subsp. oral strain RH_1735_08 is shown in Fig.2I The sgRNA for use with SwaCas9 from Staphylococcus worderi strain 691 is shown in Figure 2J The sgRNA for use with SsiCas9 from S. sciuri strain SNUC 2430 is shown in Figure 2K The sgRNA for use with SgaCas9 from S. gallolyticus AM24-4 is shown in Figure 2L The sgRNA for use with LkuCas9 from Lactobacillus kuraburgensis strain Biut2 is shown in Figure 2M , and SsuCas9 from Streptococcus suis strain LSS83 is shown in Figure 2N middle.

[0653] Example 4. Fusion of Cas9 enzyme to the N-terminus of adenine base editor (ABE) or cytidine base editor (CBE) Performing base editing

[0654] This example illustrates the base conversion efficiency of Cas9 enzymes fused to an adenine base editor (ABE) or a cytidine base editor (CBE).

[0655] Briefly, 25,000 HEK293T cells were seeded per 96-well. 100 ng of Cas9 expression plasmid and 100 ng of guide expression plasmid were transfected 24 hours after plating. Cells were harvested 5 days after transfection and DNA was extracted.

[0656] Deep sequencing was performed to characterize A to G conversion in HEK293T cells. Exemplary targets were amplified using two rounds of PCR regions to add Illumina adapters as well as unique barcodes to the target amplicon. PCR products were run on a 2% gel and gel extracted. Samples were pooled, quantified, and cDNA libraries were prepared and sequenced on MiSeq. The percentage of A to G conversion was determined by deep sequencing of N-terminal and C-terminal TadA8 fusion constructs.

[0657] Table 8 shows exemplary RNA guide sequences for use with Seq2Cas9. Figure 3A An exemplary graph showing the results of achieving the percentage of adenine to guanine base (A to G) conversion using a base editor comprising an ABE fused to the N-terminus of the Seq2Cas9 D10A mutant.

[0658] Table 8. Guide RNA sequences and PAM sequences used with Seq2Cas9

[0659]

[0660] Table 9 shows exemplary guide RNA sequences for use with EhiCas9. Figure 3B An exemplary graph showing the results of indel frequency and percentage of adenine to guanine base (A to G) conversion achieved with a base editor comprising an ABE fused to the N-terminus of the EhiCas9 D10A mutant.

[0661] Table 9. Guide RNA sequences and PAM sequences used with EhiCas9

[0662]

[0663]

[0664] Table 10 shows exemplary guide RNA sequences for use with SeqCas9. Figure 3C An exemplary graph showing the results of achieving the percentage of adenine to guanine base (A to G) conversion using a base editor comprising an ABE fused to the N-terminus of the SeqCas9 D10A mutant.

[0665] Table 10. Guide RNA sequences and PAM sequences for use with SeqCas9

[0666]

[0667] Table 11 shows exemplary guide RNA sequences for use with SsiCas9. Figure 3DAn exemplary graph showing the results of achieving the percentage of adenine to guanine base (A to G) conversion using a base editor comprising an ABE fused to the N-terminus of the SsiCas9 D10A mutant.

[0668] Table 11. Guide RNA sequences and PAM sequences for use with SSiCas9

[0669]

[0670] Table 12 shows exemplary guide RNA sequences for use with SinCas9. Figure 3E An exemplary graph showing the results of achieving the percentage of adenine to guanine base (A to G) conversion using a base editor comprising an ABE fused to the N-terminus of the SinCas9 D9A mutant.

[0671] Table 12. Guide RNA sequences and PAM sequences for use with SinCas9

[0672]

[0673] Table 13 shows exemplary guide RNA sequences for use with SsaCas9. Figure 3F An exemplary graph showing the results of achieving the percentage of adenine to guanine base (A to G) conversion using a base editor comprising an ABE fused to the N-terminus of the SsaCas9 D11A mutant.

[0674] Table 13. Guide RNA sequences and PAM sequences for use with SsaCas9

[0675]

[0676] Table 14 shows exemplary guide RNA sequences for use with Ssc2Cas9. Figure 3G An exemplary graph showing the results of indel frequency and percentage of adenine to guanine base (A to G) conversion achieved with a base editor comprising an ABE fused to the N-terminus of the Ssc2Cas9 D9A mutant.

[0677] Table 14. Guide RNA sequences and PAM sequences for use with Ssc2Cas9

[0678]

[0679] Table 15 shows exemplary guide RNA sequences for use with Sor2Cas9. Figure 3HAn exemplary graph showing the results of indel frequency and percentage of adenine to guanine base (A to G) conversion achieved with a base editor comprising an ABE fused to the N-terminus of the Sor2Cas9 D9A mutant.

[0680] Table 15. Guide RNA sequences and PAM sequences for use with Sor2Cas9

[0681]

[0682] Table 16 shows exemplary guide RNA sequences for use with SorCas9. Fig. 3I An exemplary graph showing the results of achieving the percentage of adenine to guanine base (A to G) conversion using a base editor comprising an ABE fused to the N-terminus of the SorCas9 D9A mutant.

[0683] Table 16. Guide RNA sequences and PAM sequences for use with SorCas9

[0684]

[0685]

[0686] Table 17 shows exemplary guide RNA sequences for use with SwaCas9. Figure 3J An exemplary graph showing the results of achieving the percentage of adenine to guanine base (A to G) conversion using a base editor comprising an ABE fused to the N-terminus of a SwaCas9 mutant.

[0687] Table 17. Guide RNA sequences and PAM sequences for use with SwaCas9

[0688]

[0689] Table 18 shows exemplary guide RNA sequences for use with SscCas9. Figure 3K An exemplary graph showing the results of achieving the percentage of adenine to guanine base (A to G) conversion using a base editor comprising an ABE fused to the N-terminus of an SscCas9 mutant.

[0690] Table 18. Guide RNA sequences and PAM sequences for use with SscCas9

[0691]

[0692] Table 19 shows exemplary guide RNA sequences for use with SgaCas9. Figure 3LAn exemplary graph showing the results of achieving the percentage of adenine to guanine base (A to G) conversion using a base editor comprising an ABE fused to the N-terminus of an SgaCas9 mutant.

[0693] Table 19. Guide RNA sequences and PAM sequences for use with SgaCas9

[0694]

[0695] Table 20 shows exemplary guide RNA sequences for use with LkuCas9. Figure 3M An exemplary graph showing the results of achieving the percentage of adenine to guanine base (A to G) conversion using a base editor comprising an ABE fused to the N-terminus of an LkuCas9 mutant.

[0696] Table 20. Guide RNA sequences and PAM sequences for use with LkuCas9

[0697]

[0698] Table 21 shows exemplary guide RNA sequences for use with SsuCas9. Figure 3N An exemplary graph showing the results of achieving the percentage of adenine to guanine base (A to G) conversion using a base editor comprising an ABE fused to the N-terminus of an SsuCas9 mutant.

[0699] Table 21. Guide RNA sequences and PAM sequences for use with SsuCas9

[0700]

[0701] Table 22. Summary of Cas9 orthologs, predicted PAM, seed sequence, and A to G editing efficiency

[0702]

[0703] Table 23 discloses exemplary Cas9 adenosine or adenine sequences for base editing function.

[0704] Table 23. Sequences of exemplary Cas9 adenosine or adenine base editors

[0705]

[0706]

[0707]

[0708]

[0709]

[0710]

[0711]

[0712]

[0713]

[0714]

[0715]

[0716]

[0717] Connector (underlined, no italics or bold)

[0718] TadA8(ABE) (italics and underlined)

[0719] Nickase mutations (bold and italic)

[0720] 3xHA tag (italic), can be replaced with a different tag

[0721] Equivalents and scope

[0722] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments of the invention described herein.The scope of the present invention is not intended to be limited to the above description, but rather is set forth in the claims that follow.

Claims

1. This article provides an engineered, non-naturally occurring Cas9 protein, which is modified by: Streptococcus equi ATCC33317 Cas9, Enterococcus seais strain F1129E Cas9, Streptococcus equi strain AG46 Cas9, Staphylococcus pseudostaphylococcus strain 19Cas9 , Streptococcus intermedius B196 strain G1552, Streptococcus sanguinis SK330 Cas9, Streptococcus sp. C150 Cas9, Streptococcus oralis subsp. oral strain RH_1735_08 Cas9, Streptococcus oralis SK313 Cas9, Staphylococcus wolverii strain 691 Cas9, Staphylococcus squirrel strain SNUC 2430 Cas9, Streptococcus gallolyticum strain AM24-4Cas9, Lactobacillus kulaguensis strain Biut2 Cas9, or S. suis strain LSS83 Cas9.

2. The Cas9 protein of claim 1, wherein the Streptococcus equi ATCC 33317 Cas9 has at least 80% sequence identity with MEKSYSIGLDIGTNSVGWSVITDDYKVPAKKMRVLGNTDKKYIKKNLLGALLFDSGETAEATRLKRTARRRYTRRKNRLRYLQEIFAKEMAKVDESFFQRLDESFLTDDDKTFDSHPIFGNKAEEDAYHQKFPTIYHLRKHLADSQEKADLRLIYLALAHMIKFRGHFLYDNFND DNFDWRNIDIQKRYEEFIETYDSTLGESYLADISVDAASILEEKVSKTERLENLLKYYPTEKKTTFFGNLIKLILGQQAKFKAIFNLEDEISLQFATPTYDEDLEELLGKIDNGDSYSELFVAAQNLYNTILLASFLKTDNKSAKAPLSTSMIERYENHKKDLAKLKDFVKKNCP DQYHDIFRDKSKNGYAGYIDNGVSQNDFYTFIGKCLEESLKKDKGAQYFLDKIDRDDFLRKQRTFDNGAFPYQIHLQEMHAILRRQGDYYPFLKENQDKIEKILTFRIPYYVGPLARKDSRFAWANYRSDEAITPWNFDEVVDKEKSAEKFITRMTLNDLYLPEEKVLPKHSLIY ETFTVYNELTNIKYVNDQGNAIHFDSELKEKIFNQLFKENRKVSKKTLIDFLNNSEGIYTDKLVGIDEEVKYLNASLGTYHDLKKILESFMDDEINEKIIEDIIQTLTLFEDIEMKRQRLQKYDDIFTPKQLKELARRNYTGWGRLSYKLINGIRNKENNKTILDYLKNGNRNFMQ LINDDRLSFKQIIIDARKIEKLDNIESVVYNLPGSPAIKKGILQSIKIVDELVKVMGHNPDNIVIEMARENQTTNQGKNRSQQRLKRLQDSMSNFKDSSISLKDVDNSDLQNDRLFLYYIQNGKDMYTGEELDIDHLSDYDIDHIIPQSFIKDNSIDNRVLTSSAKNRGKSDDVP GRDVVLKMKPFWKKLYDVKLISKRKFDNLTKSEHGGLTESDKAGFIKRQLVETRQITKYVAQILDGRFNTKRDDNNKVIRDVKVITLKSSLVSQFRKDFGFYKVREINDYHHAHDAYLNAVVGTAILKKYPKLAPEFVYGEYKKCDVRKLIAKSGDKSEIGKATAKYFFYSNLMN FFKRVIRYSNGMIVVRPVIEYSKDTGEIAWDKEKDFKTVCKVLSCPQVNIVKKVEKQSHGLDRGKPKGFYNANPSPKPKKGSKVNLVPIKANLNPKNYGGYAGISNSYAVLVDATIEKGAKKKLTRIQEFQGISIIDREKYEKNKVEFLKGLGYKEIYSIITLPKYSLFELADGS RRMLASILSTNNKRGEIHKGNELVLPAKYIPLLYHANRIHNTFETGHREYVEKHIAEFKEIAEIILEFNNKYVNAKKNSSIIEKALESFDSFSLDEICDSFVGKLKKNNTKKNSGLFELVSLGSASDFEFLETKVPRYRDYTPSSLLNATLIHQSITGLYETRIDLSKLGEE(SEQ ID NO:1) 3. The Cas9 protein of claim 1, wherein the Enterococcus seais strain F1129E Cas9 has at least 80% sequence identity with MTKDYTIGLDIGTNSVGWAVLTDDYQLMKRKMSVHGNTEKKKIKKNFWGARLFDEGQTAEFRRTKRTNRRRLARRKYRLSKLQDLFAEELCKQDDCFFVRLEESFLVPEEKQYKPASIFPTLEEEKEYYQKYPTIYHLRQKLVDSTEKEDLRLVYLALAHLLKYRGH FLFEGDLDTENTSIEESFRVFLEQYSKQSDQPLIVHQPVLTILTDKLSKTKKVEEILKYYPTEKINSFFAQCLKLIVGNQANFKRIFDLEAEVKLQFSKETYEEDLESLLEKIGDEYLDIFLQAKKVHDAILLSEIISSTVKHTKAKLSSGMVERYERHKADLAKFKQ FVKENVPQKSTAFFKDTTKNGYAGYIKGKTTQEEFYKFVKKELSGVVGSEPFLEKIDQETFLLKQRTYTNGVIPHQVHLIELKAIIDQQKQHYPFLEEAGPKIIALFKFRIPYYVGPLAKEQEASSFAWIERKTAEKIHPWNFSEVVDIEKSAMRFIQRMTKQDTYL PTEKVLPKNSLLYQKYMIFNELTKVSYKDERGVKQYFSGDEKQQIFKQLFQKERGKITVKKLQNFLYTHYHIENAQIFGIEKAFNASYSTYHDFMKLAKTNQKAMQEWLEQPEMEPIFEDIVKILTIFEDRQMIKHQLSKYQEVFGEKLLKEFARKHYTGWGRFSAKL IHGIRDRKTNKTILDYLINDDDVPANRNRNLMQLINDEHLSFKEEIAKATVFSKHKSLVDVIQDLPGSPAIKKGIWQSLKIVEELIAIIGYKPKNIVIEMARENQKTHRTKPRLKALENGLKQIGSTLLKEQPTDNKALQKERLYLYYLQNGRDMYTGEPLEIENLH QYEVDHIIPRSFIVDNSIDNKVLVARKQNQKKRDDVPKKQIVNEQRIFWNQLKEAKLISPKKYAYLTKIELTPEDKARFIQRQLVETRQITKHVANILHQSFHQEEEGTDCDGVQIITLKATLTSQFRQTFGLYKVREINPHHHAHDAYLNGFIANVLLKRYPKLAPE FVYGKYVKYSLARENKATAKKEFYSNILKFFESDEPFCDENGEIYWEKSHHLPRIKKVLSSHQVNVVKKVEQQKGGFYKETVNSKEKPDKLIERKNNWDVTKYGGFGSPVIAYAIAFVYAKGKTQKKTRAIEGITIMEQAAFEKDPTTFLKEKGFPQVTEFIKLPKY TLFEFDNGRRRFLASHKESQKGNPFILSDQLVTLLYHAQHYDKITYQESFDYVNTHLSDFSAILTEVLAFAEKYTLADKNIERIQELYEENKYGEISMIAQSFLQLLQFNAIGAPADFKFFGVTIPRKRYTSLTEIWDATIIYQSVTGLYETRIRMGDLWAGEQ(SEQ ID NO:2).

4. The Cas9 protein of claim 1, wherein the Streptococcus equi strain AG46 Cas9 has at least 80% sequence identity with MTNGKILGLLDIGVASVGVGIIEAKTGKVIHANSRLFSAANAENNAERRGFRGARRLTRRKKHRVKRVRDLFEKYDISTDFRNLNLNPYELRVKGLSEQLTNEELFAALRTIAKRRGISYLDDAEDDSTGSSDYAKSIDENR RLLKSMTPGQIQLERLEKYGQLRGNFTVYDENGEAHRLINVFSTSDYKNEARKILETQSNYNKQITDEFIEDYIEILTQKRKYYHGPGNEKSRTDYGRFRTDGTTLENIFGILIGKCSFYPEEYRASKASYTAQEFNFLNDL NNLKVPTETGKLSTEQKEYLVDFAKNSKTLGASKLLKEIAKLVDGDVKDISGYREDSKGKPDLHTFEPYRKLKFNLTTVDIDNLSRDILDKLANILTLNTEREGIEDAINRNLPEQFTKEQISEIVQIRKSQSSAFNKGWHS FSAKLMNELIPELYATSEEQMTILTRLEKFKATKKSSKNTKTIDEKEITDEIYNPVVAKSVRQTIKIINAAVKKYGDFDKIVIEMPRDKNADDEKKFIDKKNKENKKEKDDSLKRAAYLYNGTDKLPDDVFHGNKQLATKIR LWYQQGERCLYSGKPILIQDLVHNSNNFEIDHILPLSLSFDDSLANKVLVYAWTNQEKGQKTPYQVIDSMDAAWSFREMKDYVLKQKGIGKKKREYLLTTENIDKIEVKKKFIERNLVDTRYASRVVLNSLQTALKELGKD TKVSVVRGQFTSQLRRKWKIDKSRETYHHHAVDALIIAASSQLKLWQKHENLMFENYGENQVVNKETGEILSISDDEYKELVFQPPYQGFVNTISSKAFEDEILFSYQVDSKFNRKVSDATIYSTRKAKLGKDKKEETYVLG KIKDIYSQDGFDTFIKRYKKDKTQFLMYQKDPLTWENVIEVILRDYPTTKKSEDGKNDVKCNPFEEYRRENGLICKYSKKGKGTPIKSLKYYDKKLGNHISITPKESKNDVVLQSLNPWRADLYFNPDTLKYELMGLKYSDL SFEKGTGKYHISQEKYDEIKEKEGIGQNSEFKFTLYRNDLILIKDTESGEQEIYRFLSRTMPNVKHYVELKPYDKEKFNGGQELIKSLGEADKVGRCLKGLSKPGISIYKVRTDVLGNKFFVKKEGDKPKLDFKNNKK(SEQ IDNO:3) 5. The Cas9 protein of claim 1, wherein the Staphylococcus pseudostaphylococcus strain 19 Cas9 has at least 80% sequence identity with MNNSYILGLLDIGITSVGYGIIEYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRLQRVKKMLFDYKLLNEDSEISGINPYEARVKGLSEKLSDEEFSAALLHLAKRRGVHNVSDVEEDTGNEL STKEQIARNNKALEDKYVAELQLERLKEQGEVRGAANRFKTSDYIKEAKQLLKTQSDYHKIDETFIETYISLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNN LVIARDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNFKIYHDIKGITSRKEILENADLLNQIAEILTIYQSAEDIQEELAELEPKLTQEEIEQISNLTGYTGTHRLS LKAINLILDELWNTSDNQMTIFNRLKLVPKKVDLSQQKEIPTSLVDDFILSPVVKRSFIQSIKVINAIIKKFGLPKDIIIELAREKNSKEAQKFINEMQKRNRQTNERIEKIIKETGKEKAKFLIEKIKLHD MQEGKCLYSLESIPLEDLLNNPYHYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDAKISYETFKKHILNLSKGKGRVSKKKKEYLLEERDINRFSVQKDFINRNLVDTRYATRELMNLL RSYFRVNDLDVKVKSINGGFTSFLRTKWKFKKERNQGYKHHAEDALVIANADFIFKEWKKLDTTNKVMENQTVEEQQAENMPGIETDDEYKEIFVIPRQIQSIKDFKDYKYSHRVDKKPNRELVNDTLYSTR KDDKGNTLIINNIKGLYDKDNDKLKNLIKKSPEKLLMYHHDPQTYQKLKTIMEQYSNEKNPLYKYHEETGNYLTKYSKKDNGPIIKKVKYYGKKLNAHLDITNDYSNSQNKIVKLSLKPYRFDVYLDNGGYK FVTVKNLDVIKKEGFFKIDSNAYEKAKSEKKIDENAVFIASFYNNDLIKIDGELYRIVGVNNDTRNVVELNMIPITYKEYLENINDKRTPRILKTISQKTYSIEKYSTDILGNLYKVKSKKKPQMIMKG(SEQ IDNO:4) 6. In some embodiments, the Streptococcus intermedius B196 strain G1552 Cas9 protein has at least 80% sequence identity with MNGLVLGLDIGIASVGVGILNKETGEIIHVNSRIFPAATADSNVERRGFRQGRRLGRRKKHRSARLNDLFEEFGFITDFSAVPLNLNPYALRVKGLSEELTNEELFIALKNIIKRRGISYLDDASEDGETASNEYGKAVEE NRKLLADKTPGQIQLERFEKYGQVRGDFTVVENGENHRLINVFSTSAYKKEAERILRRQQEFNVRISDEFIEAYLTILTGKRKYYHGPGNEKSRTDYGRFRTDGTTLDNIFGILIGKCTFYPDEYRAAKASYTAQEFNLLN DLNNLTVPTETKKLRPEQKRQIVEYARTAKTLGTPTLLKYIAKLVDGSIDDIKGYRIDKSDKPEMHTFDAYRKMRTLELVDVDILSRETLDDDLAYILTLNTESEGILEALNSKMPGTFTKEQIDELIQFRKKNSAVFGKGW HNFSLKLMNELISELYETSEEQMTILTRLGKQRSREISKRTKYIDEKELTDEIYNPVVAKSVRQAIKIINLATKKYGIFDNIVIEMARESNEDDEKKAIQNVQKANEDEKKAAMEKAADLYNGKKELPDSIFHGHKELATK IRLWHQQGERCLYTGKNISIHDLIHNPHQYEIDHILPLSLSFDDGLANKVLVLATANQEKGQRTPFQALDSMDDAWSYIEFKQYVRNSKSLSNKKKDYLLTEEDISKIEVKQKFIERNLVDTRYSSRVVLNTLQEFYKTND FDTKISVVRGQFTSQLRRKWKIEKSRDTYHHHAVDALIIAASSQLRLWKKQNNPLISYREGQLVDPETGEILSLLTDDEYKELVFRPPYDYFVDTLKSKSFEDSILFSYQVDSKYNRKISDATIYGTRKAQLGKDKQEETYV LGKIKDIYSQKGYEDFIKRYKKDTTQFLMYHKDPQTFAKVIEEILKTYPDKELNEKGKEIPCNPFEKYRQENGPIRKYSKKGKGPEIKSLKYYDNKLGNHIDITPVNSQNQVVLQSLKPWRTDVYFNPQTSKYELMGLKYS DLRFEKGSGSYGISPEKYNKVKAKEGVDEDSEFKFTLYKNDLILIKDTETGEQQLFRYGSRNDTSKHYVELKPYEKAKFEGNQQLMNLLGTVAKGGQCLKGINKPNLSIYKVKTDVLGNKHFIKKEGDQPQLNFKKKI(SEQ ID NO:5) 7. The Cas9 protein of claim 1, wherein the Streptococcus sanguis SK330 Cas9 has at least 80% sequence identity with MENKNYSIGLDIGTNSVGWAVITDDYKVPSKKMKVFGNTDKHFIKKNLIGALLFDEGATAEDRRLKRTARRRYTRRKNRLRYLQEIFSEEISKLDSSFFHRLDDSFLVPKDKRGSKYPIFATLEEEKEYHKKFPTIYHLRKHLADSKEKTDLRLIYLALAHMIKYRGHFLYEES FDIKNNDIQKIFNEFISIYDNTFEGSSLSGQNAQVEAIFTDKISKSAKRERVLKLFSDEKSTSLFSEFLKLIVGNQADFKKHFDLEEKAPLQFSKDTYDEDLENLLGQIGDGFTDLFLVAKKLYDAILLSGILTVTDPSTKAPLSASMIERYESHQKDLAALKQFIKNNLPKRYN EVFSDQSKDGYAGYIDGKTTQEAFYKYIKNLLSKFEGADYFLDKIEREDFLRKQRTFDNGSIPHQIHLQEMNAILRRQGEHYPFLKENREKIEKILTFRIPYYVGPLARGNRDFAWLTRNSDQAIRPWNFEEIVDKASSAEEFINKMTNYDLYLPEEKVLPKHSLLYETFAVYN ELTKVKFIAEGLRDYQFLDSGQKKQIVNQLFKEKRKVTEKDIIHYLHNVDGYDGIELKGIEKQFNASLSTYHDLLKIIKDKEFMDDPKNEEILENIVHTLTIFEDREMIKQRLAQYDSIFDEKVIKALTRRHYTGWGKLSAKLINGICDKQTGDTILDYLIDDGKINRNFMQLIN DDGLSFKEIIQKAQVVGKTDDVKQVVQELPGSPAIKKGILQSIKIVDELVKVMGYAPESIVIEMARENQTTARGKKNSQQRYKRIEDSLKNLAPGLDSNILKENPTDNIQLQNDRLFLYYLQNGKDMYTGKPLDIDQLSSYDIDHIIPQAFIKDDSIDNRVLTSSKDNRGKSDN VPSLEVVQKRKAFWQQLLDSKLISERKFNNLTKAERGGLDERDKVGFIRRQLVETRQITKHVAQILDASFNTEVNEKNQKIRTVKIITLKSNLVSNFRKEFELYKVREINDYHHAHDAYLNAVVAKAILKKYPKLEPEFVYGDYQKYDLKRYISRFKPSKEIEKATEKYFFYSNL LNFFKEEVHYADGIIVKRENIEYSKDTGEIAWNKEKDFATIKKVLSYPQVNIVKKTEIQTHGLDRGKPKGLFNSNPSPKPSEDSKENLVPIKQGLDPRKYGGYAGISNSYAVLVKAIVEKGAKKQQKTILEFQGISILDKINFENNKENYLLKKRYIEILSTITLPKYSLFEFP DGTRRRLASILSTNNKRGEIHKGNELVLPGKYTTLLYHAKNINKKLEPEHLEYVEKHRNDFAKLLECVLNFNDKYVGALKNGERIRQAFTDWETVDIEKLCFSFIGPENSKNAGLFELTSQGSASDFEFLGVKIPRYRDYAPSSLLKATLIHQSITGLYETRIDLSKLGED(SEQ ID NO:6) 8. The Cas9 protein of claim 1, wherein the Streptococcus C150 Cas9 has at least 80% sequence identity with MSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQAENNLERRTNRQGRRLTRRKKHRRVRLNHLFEESGLITDFTKVSINLNPYQLRVKGLTAELSNEELFIALKNMVKHRGISYLDDASDDGNSSVGDYAQIVKEN SKQLETKTPGQIQLERYQKYGQLRGDFTVEEDGRKHRLINVFPTSAYHAEALRILQTQQEFNPQITDEFINSYLEILTGKRKYYHGPGNEKSRTDYGKYTTKKDAQGQYITLNNIFGILIGKCTFYPEEYRAAKASYTAQEFN LLNDLNNLTVPTETKKLSEEQKYQIITYVKNEKAMGPAKLFKYIAKLLSCDVADIKGYRIDKSDKAEIHTFEAYRKMKTLETLDVKKMAREELDKLAYVLTLNTEREGIQEALDHEFADGTFSQEQVDELVQFRKANSSIFGK GWHSFSVKLMMELIPELYATSEEQMTILTRLGKQKTTSSSNKTKYIDEKQLTEEIYNPVVAKSVRQAIKIVNAAIKKYGDFDNIVIEMARETNEDDEKKAIQKIQKANKAEKDAAMRKAANQYNGKAELPHSVFHGHKQLATK IRLWHQQGERCLYTGKTISIHDLINNPNQFEIDHILPLSITFDDSLANKVLVYATANQEKGQRTPYQALDSMDDAWSFRELKAFVRESKALSNKKKEYLLTEEDISKFDVRKKFIERNLVDTRYASRVVLNALQEHFRTHKID TKVSVVRGQFTSQLRRHWGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKNTLVSYSEDQLLDIETGELISDDEYKESVFKAPYQHFVDTLKSKEFEDSILFSYQVDSKFNRKISDATIYATRKAKLDKEKKEYTYTLGKIKD IYALGTKTPSKTGFYKFLDLYKKDKSQFLMYQKDRRTWDEVIEKILEQYRPFKEKDKNGKEVDFNPFEKYRIENGPIRKYSRKGNGPEIKSLKYYDNLLGRFVDITPSESKNPVALLSLNPWRTDVYYNTETRKYEFLGLKYA DLCFEKGGSYGISKVKYNKIREKEGIGKNSEFKFTLYKNDLILIKDTETNRQQIFRFWSRTGKDNPKSFEKHKLELKPYEKTRFEKGEELKVLGKVPPSSNRLQKNMQIENLSIYKVRTDVLGNQHIIKNEGDKPKLDF(SEQ ID NO:7) 9. The Cas9 protein of claim 1, wherein the Streptococcus oralis subsp. oralis strain RH_1735_08 Cas9 has at least 80% sequence identity with MNGLVLGLDIGIASVGVGILEKNSGKIIHANSRIFPAATADNNVERRKNRQARRLHRRKKHRGVRLQDIFEDYGLLTDFSKVSINLNPYRLRVDGLDQQLTNEELFIALKNIVKRRGISYLDDASEDGGTVSSDYGKAVE ENRKLLAEKTPGQIQLERFEKYGQVRGDFTVVENGEKRRLINVFSTSAYRKEAERILRKQQEFNSKITDEFIEDCLKILTGKRKYYHGPGNEKSRTDYGRFRTDGTTLDNIFGILIGKCTFYPNEYRASKASHTAQEFNLL NDLNNLTVPTETKKLSEEQKKVIVEYAKEAKTLGASTLLKYIAKMIDASVDQISGYRVDVNNKPEMHTFEVYRKMQSLETISVGELSRNVLDELAHILTLNTEREGIEEAINTKLKDSFSQDQVLELVQFRKNNSSLFSRG WHNFSLKLMMELIPELYETSEEQMTILTRLGKQKSKETSKRTKYIDEKELTEEIYNPVVAKSVRQAIKIINEATKKYGIFDNIVIEMARENNEEDAKKDYIKRQKANQDEKNAAMEKAAFQYNGKKELPDNIFHGHKELAT KIRLWHQQGEKCLYTGKNIPISDLIHNQYKYEIDHILPLSLSFDDSLSNKVLVLATANQEKGQRTPFQALDSMDDGWSYREFKSYVKESKLLGNKKKEYLLTEEDISKIEVKQKFIERNLVDTRYSSRVVLNALQDFYKA HKFDTTISVVRGQFTSQLRRKWGLEKSRETYHHHAVDALIIAASSQLRLWKKQNNPLIAYKEGQFVDSETGEILSLSDDEYKELVFKAPYDHFVDTLRSKTFEDSILFSYQVDSKYNRKISDATIYATRKAKLDKDKSEET YVLGKIKDIYSQAGYDAFIKIYNKDKSKFLMYHKDPQTFEKVIEEILRTYPSKELNDKNKEIPCNPFEKYRQENGPIRKYSKKGNGPEIKCLKYYDNKLGNYIDITPDGSDNQVVLQSIAPWRTDVYYNHKTGKYEFLGLK YSDLYFEKGTGKYKISKEKYDNIKKIEGVVETSEFKFTLYKNDLILIKDVEKGQEQLFRFLSRNNKGKHQVQLKPMNKSDFEKGEKLIDIFGTVPNSTTQCVKGLNKSNISIFKVKTDVLGKKHIIKKEGDEPKLKF(SEQ ID NO:8) 10. The Cas9 protein of claim 1, wherein the oral Streptococcus SK313 Cas9 has at least 80% sequence identity with MNGLVLGLDIGIASVGVGILEKNSGKIVHASSRIFPAATADNNVERRKNRQARRLHRRKKHRGARLKDLFEYYGLLTDFSKVSINLNPYRLRVDGLDQQLTNEELFIALKNIVKRRGISYLDDASEDGGTVSSDYGKAVEEN RKLLAEQTPGQIQLERFEKYGQLRGDFTVVENSEKCRLINVFSTSAYKKEAERILRKQQEFNNQITDEFIEDYLKILTGKRKYYHGPGNEKSRTDYGRFRTDGATLDNIFGILIGKCTFYPNEYRASKASYTAQEFNLLNDL NNLTVPTETKKLSEEQKKTIIEYAKSAKTLGASTLLKYIAKMIDASVDQIRGYRVDVNNKPEMHTFEVYRKMQSLETISVGELSRNILDELAHILTLNTEREGIEEAINTKLRDSFSQDQVLELVQFRKNNSSLFSKGWHNF SLKLMMELIPELYETSEEQMTILTRLGKQKSKETSKRTKYIDEKEVTEEIYNPVVAKSVRQAIKIINEATKKHGIFDNIVIEMARENNEEDAKKDYIKRQKANQDEKYAAMEKAAFQYNGKKELPDNIFHGHKELATKIRLWH QQGEKCLYTGKSIPISDLIHNQYKYEIDHILPLSLSFDDSLSNKVLVLATANQEKGQRTPFQALDSMDDAWSYREFKSYVKESKLLGNKKKEYLLTEEDISKIEVKQKFIERNLVDTRYSSRVVLNALQDFYKNHNFDTTIS VVRGQFTSQLRRKWGLEKSRETYHHHAVDALIIAASSQLRLWKKQNNPLIAYKEGQFVDSQTGEIISLTDDEYKELVFKAPYDHFVDTLSSKTFEDSILFSYQVDSKFNRKISDATIYATRKAKLDKEKKEYTYTLGKIKDI YSLGTKTPSKTGFYKFLDLYNKDKSQFLMFQKDRKTWDEVIEKIMEQYRPFKEYDKAGKLVDFNPFEKYRQENGPIRKYSKKGNGPEIKSLKYYDILLGKHKNITPEGSRNTVALLSLNPWRTDVYYNMETKKYEFLGLKYA DLPFEEGGAYGISTETYNELREKEGIGKNSEFKFTLYKNDLILIKDTETNCQQFFRFWSRTGKDNPKSFEKHKIELKPYEKAKFEKGEELEVLGKVPPSSNQFQKNMQIENLSIYKVKTDVLGNKHFIKKEGDKPKLKF(SEQ ID NO:9) 11. The Cas9 protein of claim 1, wherein the Staphylococcus wouleri strain 691 Cas9 has at least 80% sequence identity with MKEKYILGLLDLGITSVGYGIINFETKKIIDAGVRLFPEANVDNNEGRRSKRGSRRLKRRRIHRLDRVKSLLTEYNLINREQIPTSNNPYQIRVKGLSEILSKDELAIALLHLAKRRGIHNINVSSEDEDASN ELSTKEQINRNNKLLKNKYVCEVQLQRLKEGQIRGEKNRFKTTDILKEIDQLLKVQKDYHNLDIDFINQYKEIVETRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELRSVKYAYSADLFNALNDL NNLIIQRNDSEKLEYHEKYHIIENVFKQKKKPTLKQIAKEIGVNPEDIKGYRITKSGTPQFTEFKLYHDLKSIVFDKSILENEAILDQIAEILTIYQDEESIKEELNKLPEILNEQDKAEIAKLTGYNVTHR LSLKCIHLINEELWQTSRNQMEIFNYLNIKPNKVDLSEQNKIPKDLVDEFILSPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQKKNEATRKRINEIIGQTGNQNGKRIVEKIRLH DQQEGKCLYSLESIPLMDLLNNPQNYEVDHIIPRSVAFDNSIHNKVLVKQIENSKKGNRTPYQYLNSSDANLSYNQFKQHILNLSKSKDRISKKKKDYLLEERDINKFEVQKEFINRNLVDTRYATRELTSY LKAYFSANNMDVKVKTINGSFTNHLRKVWRFDKYRNHSYKHHAEDALIIANADFLFKENKKLQNANKILEKPTIENDTQKVTVEKEEDYNNMFETPKLVEDIKQYRDYKFSHRVDKKPNRQLIKDTLYSTRM KDEHNYIVQTITDIYGKDNTNLKKQFNKNPEKFLMYQNDPKTFEKLSIIMKQYSDEKNPLAKYYEETGEYLTKYSKKNNGPIVKKIKLLGNKVGNHLDVTNKYENSTKKLVKLSIKNYRFDVYLTEKGYKFV TIAYLNVFKKDNYYYIPKDLYQELKAKKKIKDTDQFIASFYKNDLIKLNGDLYKIIGVNSDDRNIIELDYYDIKYKDYCEINNIKGEPRIKKTIGKKTESIEKLTTDVLGNLYLHTTEKAPQLIFKRGL(SEQ IDNO:10) 12. The Cas9 protein of claim 1, wherein the Staphylococcus squiraris strain SNUC 2430 Cas9 has at least 80% sequence identity with MKRNYILGLLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRLQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEVEFSAALLHLAKRRGVHNVNEVEEDTGNEL STKEQISRNSKALEEKYVAELQLERLKTDGEVRGPNNRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNN LVIARDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKELLVNEEDIKGYRVTSIGKPEFTNFKIYHDIKGITERKEVLENAELLDQIAEILTIYQSSEDVQEELANLNSELTQEEIEQISNLKGYTGTHNLS LKAINLILDELWHTNDNQMAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQMNERIEEIIRTTGKENAKYLIEKIKLHD MQEGKCLYSLESIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRELMNLL RSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQTVEEKQAESMPEIETEQEYKEIFITPHQIQHIKGFKDYKYSHRVDKKPNRELINDTLYSTR KDDKGNTLIVNNLNGLYDKDNDKLKKLMNKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGKNLKAHLDITDDYPNSRNKVVKLSVKPYRFDVYLDNDIYK FVTVKNLDVIKKEDYYEVNSKCYKEAKKLKKISDQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMINITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKQKPQMIMKG(SEQ IDNO:11) 13. The Cas9 protein of claim 1, wherein the Streptococcus gallolyticum strain AM24-4 Cas9 has at least 80% sequence identity with MTNGKILGLDIGIASVGVGIIEAKTGKVVHANSRLFSAANAENNTERRGFRGSRRLNRRRKKHRVKRVRDLFEKHEIVTDFRNLNLSPYELRVKGLTEQLTNEELFAALRTISKRRGISYLDDAEDDSTGSTDYAKSIDENR RLLKNKTPGQIQLERLEKYGQLRGNFTVYDENGEAHRLINVFSTSDYEKEARKILETQADYNKKITAEFIDDYVEILTQKRKYYHGPGNEKSRTDYGRFRTDGTTLENIFGILIGKCNFYPDEYRASKASYTAQEYNFLNDL NNLKVPTETGKLPTEQKESLVEFAKNTATLGPSKLLKEIAKILDCKVDEIKGYREDDKGKPDLHTFEPYRKLKFNLESINIDDLSREVIDKLADILTLNTEREGIEDAIKRNLPNQFTEEQISEIIKVRKSQSTAFNKGWHS FSAKLMNELIPELYATSDEQMTILTRLEKFKVNKKSSKNTKTIDEKEVTDEIYNPVVAKSVRQTIKIINAAVKKYGDFDKIVIEMPRDKNADDEKKFIDKRNKENKKEKDDALKRAAYLYNSSDKLPDEVFHGNKQLETKIR LWYQQGERCLYSGKPIPIHDLVHNSNNFEIDHILPLSLSFDDSLANKVLVYDWANQEKGQKTPYQVIDSMDAAWSFREMKDYVLKQKGLGKKKRDYLLTTENIDKIEVKKKFIERNLIDTRYASRVVLNSLQSALRELCKD TKVSVIRGQFTSQLRRKWKIDKSRETYHHHAVDALIIAASSQLKLWEKQDNLMFIDYGNNQVVDKETGEILSVSDDEYKELVFQPPYQGFVNTISSKGFEDEILFSYQVDSKYNRKVSDATIYSTRKAKIGKDKKEETYVLG KIKDIYSQNGFDTFIKKYNKDKTQFLMYQKDPLTWENVIEVILRDYPTTKKSEDGKNDVKCNPFEEYRRENGLICKYSKKGKGTPIKSLKYYDKKLGNCIDITPEESRNKVILQSINPWRADVYFNPETLKYELMGLKYSDL SFEKGTGKYHISQEKYDAIKEKEGIGKKSEFKFTLYRNDLILIKDTASGEQEIYRFLSRTMPNVNHYAELKPYDKEKFDGGQELMEVFGKVANGGQCLKSLNKSNISIYKVRTDVLGNKYFVKKEGDKPKLNFKNNKK(SEQ IDNO:12) 14. The Cas9 protein of claim 1, wherein the Lactobacillus kurabacillus strain Biut2 Cas9 has at least 80% sequence identity with MKRVNEDYILGLLDIGTNSCGWAVTDKKNNLLKLRGKTAIGSHLFEEGHTAADRRGFRTTRRRLKRRKWRLRLLEEIFAEPMAKVDPGFFVRLHQSWVSPLDKDRKKYNAIVFPTAKEDQAFYKHYATIYHLRDELMTQDRQFDLREIFLAIHHIVKYRGNFLQDTPVKDFEASKI EVGPILSHINNAFAEKIVEDQDPIELNVANAADIEDVIRGKDAEKTVYKLDKVKKIAKLLTDSTAKEEKNVAKQIANAIMGYKTQFETILDKEIDKSDKAQWEFKLSDADADDKLDALLPDLDETDQTVVAEIEKLFSAITLSTIVDENKSLSQSMVEKYKKHKKDYKKLKKYIN TLQDQTKAKKLLLAYDLYVNNRHGRLLEAKKTFEDKKKKKALTKDEFYKIVKDNLDDSDLAHEIQQEIAADNFMPKQRTNSNGVIPFQLHQIELDKIIANQGKYYPFLAAENPVEDHRKQAPYKLDELVRFRVPYYVGPMITADEQEKTSGKSFAWMVRKEDGQITPWNFEQKVD RQESANKFIKRMTIKDTYLLSEDVLPANSLLYQRFEVLNELNNIRINGSRISVDLKQQIFNDLFEEKKTVTEKSLTSYLKQNLHLPTVEIKGLADPTKFNSSLASYYHLKSLHVFDKELADPQYQKDFEKIIEYSSIFEDKKIFQDKLHAEFKWLTPEQFKAISTWRLQGWGRLS RKLLVELHDTNGQNIMEQLWDSQKNFMQIVTEPDFKDAIAKENQNVTRANGVEEILADAYTSPANKKAIRQVVKVVADVVKAAGGKKPAQFAIEFTRDPDKNPQLSHIRGTKLLKAYQETAGELVDQKLTDSLKEAMTSRKLLKDKYFLYFMQAGRDAYTGQKINIDEVSTNYQI DHILPQSFIKDDSFDNRVLTATPLNAEKSDDVPYKRFANNYVSDMKMTVGEMWKHWQKAGIINKHKLGNLLLDPDRLNKFQKSGFINRQLVETSQIIKLVSVILQNKYPDAEIITVKAGDNSALRQRLNLYKSRDVNDYHHAIDAYLSIICGNFLYQVYPKYRPYFVYGKYKKFS QDPDLQKEVIKHFKGFTFMWPLLQKDNSERKAPEKIKENNSDRIVFYKHPDIFDKLRKAYNYKYMLVSRETTTENSGLFDVTIYPRGERDLAKTRKLIPKSNGLDPKIYGGYSGNTDAYMVIVKIDKGKESIYKVIGVPMRALASLNRAKKQGNYKEELHQVLEPQIMFDKNGKP KRSVKGFRIIKDHVPFKQVVLDGDKKFMLNSSTYEINAKQLTLTPETMRIVTDNKKGEDQDQLLVKAYDEILQKVDQYLPLFDVNKFRNSLHLGRAKFLDLAVNDKKITLTNILNGLHDNLVTPDLKNIGIKTPLGKLQVPSGIVLSSEAILIFQSPTGLFEKRVRIADL(SEQ ID NO:13) 15. The Cas9 protein of claim 1, wherein the Streptococcus suis strain LSS83 Cas9 has at least 80% sequence identity with MSNGKILGLDIGIASVGVGVIDAQTGEIIHASSRIFPSANAANNAERRTFRGSRRLIRRKKHRIKRLDDLFNDFHINLDGEMSTDNPYVLRVKGLSQKLTVEELYISIKNIMKRRGISYLDDAESDNEAGRSDYAKAIER NRQLLTSKTPGEIQLERLEKYGQLRGNFTIIDEEGQSQQIINVFSTSDYVKEVEKILDCQKMYHKFISDEFCDKLIELLREKRKYYVGPGNEKSRTDYGIYRTDGTTLENLFGILIGKCTFYPDQYRSSRASYTAQEFNFL NDLNNLTVPTETKKLSQEQKEFLVNYAKETSVLGAGKILQQIAKLADCKVEDIRGYRLDNKDKPELHTFETYRAMKGLVPLVDIGVLSREQLDILADILTLNTDFEGIREALKKQLPNVFDEKQVKGLASFRKSKSQLFAK GWHNLSQKIMLEVIPELYATSDEQMTILTRLGKFEKSSVAEYPSSINVDEITDEIYNPVVAKSIRQTIKIINASIKKWDEFDQIVIEMPRDRNEDEEKKRIADGQKANAKEKADSILRAAELYCAGKVLPDYVYNGHNQLA TKIRLWYQQGERCIYTGQPISIHDLIHNQNQYEIDHILPLSLTFDDSLSNKVLVLATANQEKAQRTPYNYLKSATSAWSYREFKDYVTKRKGIGKKKCEYLTFEEDINGFEVRSKFIQRNLVDTRYASKVILNALQDYFK ISGIQTKVSVVRGQFTSQLRHKWGIEKTRETYHHHAVDALIIAASSQLRLWKKQESPLVVDYQEGRQVDLETGEILELTDEQYKELVYQPPYQGFVNTISSSAFDNEILFSYQVDSKVNRKISDATIYATRNAQLGKDKTE GIYVLGKIKDIYTQAGYEAFLKRYTKDKTSFLMYHKDLDTWEKVIEIILRDYREYDEKGKEIGNPFERYRRENGYVKKYSRKGNGTAIKSLKYYDNKLGNHIDITPENSRNAVVLQSLKPWRTDVYFNKETGKYEFLGIKY SDLSFEKGTGEYGISQEKYDSIKIAEGVAKKSIFKFTLYKQDLLFIKDIENNFGKLLRFTSKNDTSKHYVELKPYDKNKFGTEEPLLPVLGNVAKSGQCIKGLNKSNISIYKVRTDILGYRHFIKQEGEHPQLKFKK(SEQ ID NO:14) 16. The Cas9 protein according to any one of claims 1 to 15, comprising SEQ ID Nos: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical amino acid sequences.

17. The Cas9 protein according to any one of the preceding claims, further comprising a nuclear localization sequence (NLS) and / or a FLAG, HIS or HA tag, and / or a deaminase.

18. The protein of claim 17, wherein the Streptococcus equi ATCC 33317Cas9 has an amino acid sequence that is at least 80% identical to MPAAKRVKLD GSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILA DECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSG PKKKRKVGTMEKSYSIGLAIGTNSVGWSVITDDYKVPAKKMRVLGNTDKKYIKKNLLGALLFDSGETAEATRLKRTARRRYTRRKNRLRYLQEIFAKEMAKVDESFFQRLDESFLTDDDKTFDSHPIFGNKAEEDAYHQKFPTIYHLRKHLADSQEKADLRLIYLALAHMIKFRGHF LYDNFNDDNFDWRNIDIQKRYEEFIETYDSTLGESYLADISVDAASILEEKVSKTERLENLLKYYPTEKKTTFFGNLIKLILGQQAKFKAIFNLEDEISLQFATPTYDEDLEELLGKIDNGDSYSELFVAAQNLYNTILLASFLKTDNKSAKAPLSTSMIERYENHKKDLAKLKDFVK KNCPDQYHDIFRDKSKNGYAGYIDNGVSQNDFYTFIGKCLEESLKKDKGAQYFLDKIDRDDFLRKQRTFDNGAFPYQIHLQEMHAILRRQGDYYPFLKENQDKIEKILTFRIPYYVGPLARKDSRFAWANYRSDEAITPWNFDEVVDKEKSAEKFITRMTLNDLYLPEEKVLPKHSLI YETFTVYNELTNIKYVNDQGNAIHFDSELKEKIFNQLFKENRKVSKKTLIDFLNNSEGIYTDKLVGIDEEVKYLNASLGTYHDLKKILESFMDDEINEKIIEDIIQTLTLFEDIEMKRQRLQKYDDIFTPKQLKELARRNYTGWGRLSYKLINGIRNKENNKTILDYLKNGNRNFMQL INDDRLSFKQIIIDARKIEKLDNIESVVYNLPGSPAIKKGILQSIKIVDELVKVMGHNPDNIVIEMARENQTTNQGKNRSQQRLKRLQDSMSNFKDSSISLKDVDNSDLQNDRLFLYYIQNGKDMYTGEELDIDHLSDYDIDHIIPQSFIKDNSIDNRVLTSSAKNRGKSDDVPGRDV VLKMKPFWKKLYDVKLISKRKFDNLTKSEHGGLTESDKAGFIKRQLVETRQITKYVAQILDGRFNTKRDDNNKVIRDVKVITLKSSLVSQFRKDFGFYKVREINDYHHAHDAYLNAVVGTAILKKYPKLAPEFVYGEYKKCDVRKLIAKSGDKSEIGKATAKYFFYSNLMNFFKRVIR YSNGMIVVRPVIEYSKDTGEIAWDKEKDFKTVCKVLSCPQVNIVKKVEKQSHGLDRGKPKGFYNANPSPKPKKGSKVNLVPIKANLNPKNYGGYAGISNSYAVLVDATIEKGAKKKLTRIQEFQGISIIDREKYEKNKVEFLKGLGYKEIYSIITLPKYSLFELADGSRRMLASILST NNKRGEIHKGNELVLPAKYIPLLYHANRIHNTFETGHREYVEKHIAEFKEIAEIILEFNNKYVNAKKNSSIIEKALESFDSFSLDEICDSFVGKLKKNNTKKNSGLFELVSLGSASDFEFLETKVPRYRDYTPSSLLNATLIHQSITGLYETRIDLSKLGEEKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO: 29).

19. The protein of claim 17, wherein the Enterococcus seais strain F1129E Cas9 has an amino acid sequence that is at least 80% identical to MPAAKRVKLD GSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILA DECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSG PKKKRKV GT MTKDYTIGL A IGTNSVGWAVLTDDYQLMKRKMSVHGNTEKKKIKKNFWGARLFDEGQTAEFRRTKRTNRRRLARRKYRLSKLQDLFAEELCKQDDCFFVRLEESFLVPEEKQYKPASIFPTLEEEKEYYQKYPTIYHLRQKLVDSTEKEDLRLVYLALAHLLKYRGHFLFEGDLDTE NTSIEESFRVFLEQYSKQSDQPLIVHQPVLTILTDKLSKTKKVEEILKYYPTEKINSFFAQCLKLIVGNQANFKRIFDLEAEVKLQFSKETYEEDLESLLEKIGDEYLDIFLQAKKVHDAILLSEIISSTVKHTKAKLSSGMVERYERHKADLAKFKQFVKENVPQKS TAFFKDTTKNGYAGYIKGKTTQEEFYKFVKKELSGVVGSEPFLEKIDQETFLLKQRTYTNGVIPHQVHLIELKAIIDQQKQHYPFLEEAGPKIIALFKFRIPYYVGPLAKEQEASSFAWIERKTAEKIHPWNFSEVVDIEKSAMRFIQRMTKQDTYLPTEKVLPKNSL LYQKYMIFNELTKVSYKDERGVKQYFSGDEKQQIFKQLFQKERGKITVKKLQNFLYTHYHIENAQIFGIEKAFNASYSTYHDFMKLAKTNQKAMQEWLEQPEMEPIFEDIVKILTIFEDRQMIKHQLSKYQEVFGEKLLKEFARKHYTGWGRFSAKLIHGIRDRKTNK TILDYLINDDDVPANRNRNLMQLINDEHLSFKEEIAKATVFSKHKSLVDVIQDLPGSPAIKKGIWQSLKIVEELIAIIGYKPKNIVIEMARENQKTHRTKPRLKALENGLKQIGSTLLKEQPTDNKALQKERLYLYYLQNGRDMYTGEPLEIENLHQYEVDHIIPRS FIVDNSIDNKVLVARKQNQKKRDDVPKKQIVNEQRIFWNQLKEAKLISPKKYAYLTKIELTPEDKARFIQRQLVETRQITKHVANILHQSFHQEEEGTDCDGVQIITLKATLTSQFRQTFGLYKVREINPHHHAHDAYLNGFIANVLLKRYPKLAPEFVYGKYVKYSL ARENKATAKKEFYSNILKFFESDEPFCDENGEIYWEKSHHLPRIKKVLSSHQVNVVKKVEQQKGGFYKETVNSKEKPDKLIERKNNWDVTKYGGFGSPVIAYAIAFVYAKGKTQKKTRAIEGITIMEQAAFEKDPTTFLKEKGFPQVTEFIKLPKYTLFEFDNGRRRF LASHKESQKGNPFILSDQLVTLLYHAQHYDKITYQESFDYVNTHLSDFSAILTEVLAFAEKYTLADKNIERIQELYEENKYGEISMIAQSFLQLLQFNAIGAPADFKFFGVTIPRKRYTSLTEIWDATIIYQSVTGLYETRIRMGDLWAGEQKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA(SEQID NO:30) 20. The protein of claim 17, wherein the Streptococcus equi strain AG46 Cas9 has an amino acid sequence that is at least 80% identical to MPAAKRVKLD GSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILA DECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSG PKKKRKV GT MTNGKILGLAIGVASVGVGIIEAKTGKVIHANSRLFSAANAENNAERRGFRGARRLTRRKKHRVKRVRDLFEKYDISTDFRNLNLNPYELRVKGLSEQLTNEELFAALRTIAKRRGISYLDDAEDDSTGSSDYAKSIDENRRL LKSMTPGQIQLERLEKYGQLRGNFTVYDENGEAHRLINVFSTSDYKNEARKILETQSNYNKQITDEFIEDYIEILTQKRKYYHGPGNEKSRTDYGRFRTDGTTLENIFGILIGKCSFYPEEYRASKASYTAQEFNFLNDLNNL KVPTETGKLSTEQKEYLVDFAKNSKTLGASKLLKEIAKLVDGDVKDISGYREDSKGKPDLHTFEPYRKLKFNLTTVDIDNLSRDILDKLANILTLNTEREGIEDAINRNLPEQFTKEQISEIVQIRKSQSSAFNKGWHSFSAK LMNELIPELYATSEEQMTILTRLEKFKATKKSSKNTKTIDEKEITDEIYNPVVAKSVRQTIKIINAAVKKYGDFDKIVIEMPRDKNADDEKKFIDKKNKENKKEKDDSLKRAAYLYNGTDKLPDDVFHGNKQLATKIRLWYQQG ERCLYSGKPILIQDLVHNSNNFEIDHILPLSLSFDDSLANKVLVYAWTNQEKGQKTPYQVIDSMDAAWSFREMKDYVLKQKGIGKKKREYLLTTENIDKIEVKKKFIERNLVDTRYASRVVLNSLQTALKELGKDTKVSVVRG QFTSQLRRKWKIDKSRETYHHHAVDALIIAASSQLKLWQKHENLMFENYGENQVVNKETGEILSISDDEYKELVFQPPYQGFVNTISSKAFEDEILFSYQVDSKFNRKVSDATIYSTRKAKLGKDKKEETYVLGKIKDIYSQD GFDTFIKRYKKDKTQFLMYQKDPLTWENVIEVILRDYPTTKKSEDGKNDVKCNPFEEYRRENGLICKYSKKGKGTPIKSLKYYDKKLGNHISITPKESKNDVVLQSLNPWRADLYFNPDTLKYELMGLKYSDLSFEKGTGKYH ISQEKYDEIKEKEGIGQNSEFKFTLYRNDLILIKDTESGEQEIYRFLSRTMPNVKHYVELKPYDKEKFNGGQELIKSLGEADKVGRCLKGLSKPGISIYKVRTDVLGNKFFVKKEGDKPKLDFKNNKKKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO: 31).

21. The protein of claim 17, wherein the Streptococcus intermedius B196 strain G1552 Cas9 has an amino acid sequence that is at least 80% identical to MPAAKRVKLD GSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILA DECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSG PKKKRKV GT MNGLVLGL A IGIASVGVGILNKETGEIIHVNSRIFPAATADSNVERRGFRQGRRLGRRKKHRSARLNDLFEEFGFITDFSAVPLNLNPYALRVKGLSEELTNEELFIALKNIIKRRGISYLDDASEDGETASNEYGKAVEENRKLLADKT PGQIQLERFEKYGQVRGDFTVVENGENHRLINVFSTSAYKKEAERILRRQQEFNVRISDEFIEAYLTILTGKRKYYHGPGNEKSRTDYGRFRTDGTTLDNIFGILIGKCTFYPDEYRAAKASYTAQEFNLLNDLNNLTVPTE TKKLRPEQKRQIVEYARTAKTLGTPTLLKYIAKLVDGSIDDIKGYRIDKSDKPEMHTFDAYRKMRTLELVDVDILSRETLDDLAYILTLNTESEGILEALNSKMPGTFTKEQIDELIQFRKKNSAVFGKGWHNFSLKLMNE LISELYETSEEQMTILTRLGKQRSREISKRTKYIDEKELTDEIYNPVVAKSVRQAIKIINLATKKYGIFDNIVIEMARESNEDDEKKAIQNVQKANEDEKKAAMEKAADLYNGKKELPDSIFHGHKELATKIRLWHQQGERC LYTGKNISIHDLIHNPHQYEIDHILPLSLSFDDGLANKVLVLATANQEKGQRTPFQALDSMDDAWSYIEFKQYVRNSKSLSNKKKDYLLTEEDISKIEVKQKFIERNLVDTRYSSRVVLNTLQEFYKTNDFDTKISVVRGQ FTSQLRRKWKIEKSRDTYHHHAVDALIIAASSQLRLWKKQNNPLISYREGQLVDPETGEILSLLTDDEYKELVFRPPYDYFVDTLKSKSFEDSILFSYQVDSKYNRKISDATIYGTRKAQLGKDKQEETYVLGKIKDIYSQKG YEDFIKRYKKDTTQFLMYHKDPQTFAKVIEEILKTYPDKELNEKGKEIPCNPFEKYRQENGPIRKYSKKGKGPEIKSLKYYDNKLGNHIDITPVNSQNQVVLQSLKPWRTDVYFNPQTSKYELMGLKYSDLRFEKGSGSYG ISPEKYNKVKAKEGVDEDSEFKFTLYKNDLILIKDTETGEQQLFRYGSRNDTSKHYVELKPYEKAKFEGNQQLMNLLGTVAKGGQCLKGINKPNLSIYKVKTDVLGNKHFIKKEGDQPQLNFKKKIKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO: 32).

22. The protein of claim 17, wherein the Staphylococcus pseudostaphylococcus strain 19Cas9 has an amino acid sequence that is at least 80% identical to MPAAKRVKLDGS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILA DECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSG PKKKRKV GT MNNSYILGLAIGITSVGYGIIEYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRLQRVKKMLFDYKLLNEDSEISGINPYEARVKGLSEKLSDEEFSAALLHLAKRRGVHNVSDVEEDTGNELS TKEQIARNNKALEDKYVAELQLERLKEQGEVRGAANRFKTSDYIKEAKQLLKTQSDYHKIDETFIETYISLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVI ARDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNFKIYHDIKGITSRKEILENADLLNQIAEILTIYQSAEDIQEELAELEPKLTQEEIEQISNLTGYTGTHRLSLKAI NLILDELWNTSDNQMTIFNRLKLVPKKVDLSQQKEIPTSLVDDFILSPVVKRSFIQSIKVINAIIKKFGLPKDIIIELAREKNSKEAQKFINEMQKRNRQTNERIEKIIKETGKEKAKFLIEKIKLHDMQEGKC LYSLESIPLEDLLNNPYHYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDAKISYETFKKHILNLSKGKGRVSKKKKEYLLEERDINRFSVQKDFINRNLVDTRYATRELMNLLRSYFRVN DLDVKVKSINGGFTSFLRTKWKFKKERNQGYKHHAEDALVIANADFIFKEWKKLDTTNKVMENQTVEEQQAENMPGIETDDEYKEIFVIPRQIQSIKDFKDYKYSHRVDKKPNRELVNDTLYSTRKDDKGNTLI INNIKGLYDKDNDKLKNLIKKSPEKLLMYHHDPQTYQKLKTIMEQYSNEKNPLYKYHEETGNYLTKYSKKDNGPIIKKVKYYGKKLNAHLDITNDYSNSQNKIVKLSLKPYRFDVYLDNGGYKFVTVKNLDVIK KEGFFKIDSNAYEKAKSEKKIDENAVFIASFYNNDLIKIDGELYRIVGVNNDTRNVVELNMIPITYKEYLENINDKRTPRILKTISQKTYSIEKYSTDILGNLYKVKSKKKPQMIMKGKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO: 33).

23. The protein of claim 17, wherein the Streptococcus sanguis SK330 Cas9 has an amino acid sequence that is at least 80% identical to MPAAKRVKLD GSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILA DECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSG PKKKRKV GT MENKNYSIGLAIGTNSVGWAVITDDYKVPSKKMKVFGNTDKHFIKKNLIGALLFDEGATAEDRRLKRTARRRYTRRKNRLRYLQEIFSEEISKLDSSFFHRLDDSFLVPKDKRGSKYPIFATLEEEKEYHKKFPTIYHLRKHLADSKEKTDLRLIYLALAHMIKYRGHFLYEESFD IKNNDIQKIFNEFISIYDNTFEGSSLSGQNAQVEAIFTDKISKSAKRERVLKLFSDEKSTSLFSEFLKLIVGNQADFKKHFDLEEKAPLQFSKDTYDEDLENLLGQIGDGFTDLFLVAKKLYDAILLSGILTVTDPSTKAPLSASMIERYESHQKDLAALKQFIKNNLPKRYNEVF SDQSKDGYAGYIDGKTTQEAFYKYIKNLLSKFEGADYFLDKIEREDFLRKQRTFDNGSIPHQIHLQEMNAILRRQGEHYPFLKENREKIEKILTFRIPYYVGPLARGNRDFAWLTRNSDQAIRPWNFEEIVDKASSAEEFINKMTNYDLYLPEEKVLPKHSLLYETFAVYNELTKV KFIAEGLRDYQFLDSGQKKQIVNQLFKEKRKVTEKDIIHYLHNVDGYDGIELKGIEKQFNASLSTYHDLLKIIKDKEFMDDPKNEEILENIVHTLTIFEDREMIKQRLAQYDSIFDEKVIKALTRRHYTGWGKLSAKLINGICDKQTGDTILDYLIDDGKINRNFMQLINDDGLSF KEIIQKAQVVGKTDDVKQVVQELPGSPAIKKGILQSIKIVDELVKVMGYAPESIVIEMARENQTTARGKKNSQQRYKRIEDSLKNLAPGLDSNILKENPTDNIQLQNDRLFLYYLQNGKDMYTGKPLDIDQLSSYDIDHIIPQAFIKDDSIDNRVLTSSKDNRGKSDNVPSLEVVQ KRKAFWQQLLDSKLISERKFNNLTKAERGGLDERDKVGFIRRQLVETRQITKHVAQILDASFNTEVNEKNQKIRTVKIITLKSNLVSNFRKEFELYKVREINDYHHAHDAYLNAVVAKAILKKYPKLEPEFVYGDYQKYDLKRYISRFKPSKEIEKATEKYFFYSNLLNFFKEEVH YADGIIVKRENIEYSKDTGEIAWNKEKDFATIKKVLSYPQVNIVKKTEIQTHGLDRGKPKGLFNSNPSPKPSEDSKENLVPIKQGLDPRKYGGYAGISNSYAVLVKAIVEKGAKKQQKTILEFQGISILDKINFENNKENYLLKKRYIEILSTITLPKYSLFEFPDGTRRRLASIL STNNKRGEIHKGNELVLPGKYTTLLYHAKNINKKLEPEHLEYVEKHRNDFAKLLECVLNFNDKYVGALKNGERIRQAFTDWETVDIEKLCFSFIGPENSKNAGLFELTSQGSASDFEFLGVKIPRYRDYAPSSLLKATLIHQSITGLYETRIDLSKLGEDKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO: 34).

24. The protein of claim 17, wherein the Streptococcus C150 Cas9 has an amino acid sequence that is at least 80% identical to MPAAKRVKLD GSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILA DECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSG PKKKRKV GT MSDLVLGL A IGIGSVGVGILNKVTGEIIHKNSRIFPAAQAENNLERRTNRQGRRLTRRKKHRRVRLNHLFEESGLITDFTKVSINLNPYQLRVKGLTAELSNEELFIALKNMVKHRGISYLDDASDDGNSSVGDYAQIVKENSKQLETKTPG QIQLERYQKYGQLRGDFTVEEDGRKHRLINVFPTSAYHAEALRILQTQQEFNPQITDEFINSYLEILTGKRKYYHGPGNEKSRTDYGKYTTKKDAQGQYITLNNIFGILIGKCTFYPEEYRAAKASYTAQEFNLLNDLNNLTV PTETKKLSEEQKYQIITYVKNEKAMGPAKLFKYIAKLLSCDVADIKGYRIDKSDKAEIHTFEAYRKMKTLETLDVKKMAREELDKLAYVLTLNTEREGIQEALDHEFADGTFSQEQVDELVQFRKANSSIFGKGWHSFSVKLM MELIPELYATSEEQMTILTRLGKQKTTSSSSNKTKYIDEKQLTEEIYNPVVAKSVRQAIKIVNAAIKKYGDFDNIVIEMARETNEDDEKKAIQKIQKANKAEKDAAMRKAANQYNGKAELPHSVFHGHKQLATKIRLWHQQGERC LYTGKTISIHDLINNPNQFEIDHILPLSITFDDSLANKVLVYATANQEKGQRTPYQALDSMDDAWSFRELKAFVRESKALSNKKKEYLLTEEDISKFDVRKKFIERNLVDTRYASRVVLNALQEHFRTHKIDTKVSVVRGQFT SQLRRHWGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKNTLVSYSEDQLLDIETGELISDDEYKESVFKAPYQHFVDTLKSKEFEDSILFSYQVDSKFNRKISDATIYATRKAKLDKEKKEYTYTLGKIKDIYALGTKTPSK TGFYKFLDLYKKDKSQFLMYQKDRRTWDEVIEKILEQYRPFKEKDKNGKEVDFNPFEKYRIENGPIRKYSRKGNGPEIKSLKYYDNLLGRFVDITPSESKNPVALLSLNPWRTDVYYNTETRKYEFLGLKYADLCFEKGGSYG ISKVKYNKIREKEGIGKNSEFKFTLYKNDLILIKDTETNRQQIFRFWSRTGKDNPKSFEKHKLELKPYEKTRFEKGEELKVLGKVPPSSNRLQKNMQIENLSIYKVRTDVLGNQHIIKNEGDKPKLDFKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO: 35).

25. The protein of claim 17, wherein the Streptococcus oralis subsp. oralis strain RH_1735_08Cas9 has an amino acid sequence that is at least 80% identical to MPAAKRVKLD GSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILA DECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSG PKKKRKV GT MNGLVLGLAIGIASVGVGILEKNSGKIIHANSRIFPAATADNNVERRKNRQARRLHRRKKHRGVRLQDIFEDYGLLTDFSKVSINLNPYRLRVDGLDQQLTNEELFIALKNIVKRRGISYLDDASEDGGTVSSDYGKAVEEN RKLLAEKTPGQIQLERFEKYGQVRGDFTVVENGEKRRLINVFSTSAYRKEAERILRKQQEFNSKITDEFIEDCLKILTGKRKYYHGPGNEKSRTDYGRFRTDGTTLDNIFGILIGKCTFYPNEYRASKASHTAQEFNLLNDL NNLTVPTETKKLSEEQKKVIVEYAKEAKTLGASTLLKYIAKMIDASVDQISGYRVDVNNKPEMHTFEVYRKMQSLETISVGELSRNVLDELAHILTLNTEREGIEEAINTKLKDSFSQDQVLELVQFRKNNSSLFSRGWHNF SLKLMMELIPELYETSEEQMTILTRLGKQKSKETSKRTKYIDEKELTEEIYNPVVAKSVRQAIKIINEATKKYGIFDNIVIEMARENNEEDAKKDYIKRQKANQDEKNAAMEKAAFQYNGKKELPDNIFHGHKELATKIRLWH QQGEKCLYTGKNIPISDLIHNQYKYEIDHILPLSLSFDDSLSNKVLVLATANQEKGQRTPFQALDSMDDGWSYREFKSYVKESKLLGNKKKEYLLTEEDISKIEVKQKFIERNLVDTRYSSRVVLNALQDFYKAHKFDTTIS VVRGQFTSQLRRKWGLEKSRETYHHHAVDALIIAASSQLRLWKKQNNPLIAYKEGQFVDSETGEILSLSDDEYKELVFKAPYDHFVDTLRSKTFEDSILFSYQVDSKYNRKISDATIYATRKAKLDKDKSEETYVLGKIKDI YSQAGYDAFIKIYNKDKSKFLMYHKDPQTFEKVIEEILRTYPSKELNDKNKEIPCNPFEKYRQENGPIRKYSKKGNGPEIKCLKYYDNKLGNYIDITPDGSDNQVVLQSIAPWRTDVYYNHKTGKYEFLGLKYSDLYFEKGT GKYKISKEKYDNIKKIEGVVETSEFKFTLYKNDLILIKDVEKGQEQLFRFLSRNNKGKHQVQLKPMNKSDFEKGEKLIDIFGTVPNSTTQCVKGLNKSNISIFKVKTDVLGKKHIIKKEGDEPKLKFKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO: 36).

26. The protein of claim 17, wherein the oral Streptococcus SK313 Cas9 has an amino acid sequence that is at least 80% identical to MPAAKRVKLD GSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILA DECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSG PKKKRKV GT MNGLVLGL A IGIASVGVGILEKNSGKIIHANSRIFPAATADNNVERRKNRQARRLHRRKKHRGVRLQDIFEDYGLLTDFSKVSINLNPYRLRVDGLDQQLTNEELFIALKNIVKRRGISYLDDASEDGGTVSSDYGKAVEENRKLLAEKT PGQIQLERFEKYGQVRGDFTVVENGEKRRLINVFSTSAYRKEAERILRKQQEFNSKITDEFIEDCLKILTGKRKYYHGPGNEKSRTDYGRFRTDGTTLDNIFGILIGKCTFYPNEYRASKASHTAQEFNLLNDLNNLTVPT ETKKLSEEQKKVIVEYAKEAKTLGASTLLKYIAKMIDASVDQISGYRVDVNNKPEMHTFEVYRKMQSLETISVGELSRNVLDELAHILTLNTEREGIEEAINTKLKDSFSQDQVLELVQFRKNNSSLFSRGWHNFSLKLMM ELIPELYETSEEQMTILTRLGKQKSKETSKRTKYIDEKELTEEIYNPVVAKSVRQAIKIINEATKKYGIFDNIVIEMARENNEEDAKKDYIKRQKANQDEKNAAMEKAAFQYNGKKELPDNIFHGHKELATKIRLWHQQGE KCLYTGKNIPISDLIHNQYKYEIDHILPLSLSFDDSLSNKVLVLATANQEKGQRTPFQALDSMDDGWSYREFKSYVKESKLLGNKKKEYLLTEEDISKIEVKQKFIERNLVDTRYSSRVVLNALQDFYKAHKFDTTISVVR GQFTSQLRRKWGLEKSRETYHHHAVDALIIAASSQLRLWKKQNNPLIAYKEGQFVDSETGEILSLSDDEYKELVFKAPYDHFVDTLRSKTFEDSILFSYQVDSKYNRKISDATIYATRKAKLDKDKSEETYVLGKIKDIYS QAGYDAFIKIYNKDKSKFLMYHKDPQTFEKVIEEILRTYPSKELNDKNKEIPCNPFEKYRQENGPIRKYSKKGNGPEIKCLKYYDNKLGNYIDITPDGSDNQVVLQSIAPWRTDVYYNHKTGKYEFLGLKYSDLYFEKGTG KYKISKEKYDNIKKIEGVVETSEFKFTLYKNDLILIKDVEKGQEQLFRFLSRNNKGKHQVQLKPMNKSDFEKGEKLIDIFGTVPNSTTQCVKGLNKSNISIFKVKTDVLGKKHIIKKEGDEPKLKFKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO: 37).

27. The protein of claim 17, wherein the Staphylococcus wouleri strain 691 Cas9 has an amino acid sequence that is at least 80% identical to MPAAKRVKLD GSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILA DECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSG PKKKRKV GT MKEKYILGL A LGITSVGYGIINFETKKIIDAGVRLFPEANVDNNEGRRSKRGSRRLKRRRIHRLDRVKSLLTEYNLINREQIPTSNNPYQIRVKGLSEILSKDELAIALLHLAKRRGIHNINVSSEDEDASNELSTKEQINR NNKLLKNKYVCEVQLQRLKEGQIRGEKNRFKTTDILKEIDQLLKVQKDYHNLDIDFINQYKEIVETRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELRSVKYAYSADLFNALNDLNNLIIQRNDSE KLEYHEKYHIIENVFKQKKKPTLKQIAKEIGVNPEDIKGYRITKSGTPQFTEFKLYHDLKSIVFDKSILENEAILDQIAEILTIYQDEESIKEELNKLPEILNEQDKAEIAKLTGYNVTHRLSLKCIHLINE ELWQTSRNQMEIFNYLNIKPNKVDLSEQNKIPKDLVDEFILSPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQKKNEATRKRINEIIGQTGNQNGKRIVEKIRLHDQQEGKCLYSL ESIPLMDLLNNPQNYEVDHIIPRSVAFDNSIHNKVLVKQIENSKKGNRTPYQYLNSSDANLSYNQFKQHILNLSKSKDRISKKKKDYLLEERDINKFEVQKEFINRNLVDTRYATRELTSYLKAYFSANNMD VKVKTINGSFTNHLRKVWRFDKYRNHSYKHHAEDALIIANADFLFKENKKLQNANKILEKPTIENDTQKVTVEKEEDYNNMFETPKLVEDIKQYRDYKFSHRVDKKPNRQLIKDTLYSTRMKDEHNYIVQTIT DIYGKDNTNLKKQFNKNPEKFLMYQNDPKTFEKLSIIMKQYSDEKNPLAKYYEETGEYLTKYSKKNNGPIVKKIKLLGNKVGNHLDVTNKYENSTKKLVKLSIKNYRFDVYLTEKGYKFVTIAYLNVFKKDN YYYIPKDLYQELKAKKKIKDTDQFIASFYKNDLIKLNGDLYKIIGVNSDDRNIIELDYYDIKYKDYCEINNIKGEPRIKKTIGKKTESIEKLTTDVLGNLYLHTTEKAPQLIFKRGLKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO: 38).

28. The protein of claim 17, wherein the Staphylococcus squiraris strain SNUC 2430Cas9 has an amino acid sequence that is at least 80% identical to MPAAKRVKLD GSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILA DECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSG PKKKRKV GT MKRNYILGL A IGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRLQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEVEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNS KALEEKYVAELQLERLKTDGEVRGPNNRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVIARDENEK LEYYEKFQIIENVFKQKKKPTLKQIAKELLVNEEDIKGYRVTSIGKPEFTNFKIYHDIKGITERKEVLENAELLDQIAEILTIYQSSEDVQEELANLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDE LWHTNDNQMAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQMNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLE SIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRELMNLLRSYFRVNNLDV KVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQTVEEKQAESMPEIETEQEYKEIFITPHQIQHIKGFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNN LNGLYDKDNDKLKKLMNKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGKNLKAHLDITDDYPNSRNKVVKLSVKPYRFDVYLDNDIYKFVTVKNLDVIKK EDYYEVNSKCYKEAKKLKKISDQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMINITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKQKPQMIMKGKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO: 39).

29. The protein of claim 17, wherein the Streptococcus gallolyticum strain AM24-4 Cas9 has an amino acid sequence that is at least 80% identical to MPAAKRVKLD GSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILA DECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSG PKKKRKV GT MTNGKILGLAIGIASVGVGIIEAKTGKVVHANSRLFSAANAENNTERRGFRGSRRLNRRRKKHRVKRVRDLFEKHEIVTDFRNLNLSPYELRVKGLTEQLTNEELFAALRTISKRRGISYLDDAEDDSTGSTDYAKSIDENRRL LKNKTPGQIQLERLEKYGQLRGNFTVYDENGEAHRLINVFSTSDYEKEARKILETQADYNKKITAEFIDDYVEILTQKRKYYHGPGNEKSRTDYGRFRTDGTTLENIFGILIGKCNFYPDEYRASKASYTAQEYNFLNDLNNL KVPTETGKLPTEQKESLVEFAKNTATLGPSKLLKEIAKILDCKVDEIKGYREDDKGKPDLHTFEPYRKLKFNLESINIDDLSREVIDKLADILTLNTEREGIEDAIKRNLPNQFTEEQISEIIKVRKSQSTAFNKGWHSFSAK LMNELIPELYATSDEQMTILTRLEKFKVNKKSSKNTKTIDEKEVTDEIYNPVVAKSVRQTIKIINAAVKKYGDFDKIVIEMPRDKNADDEKKFIDKRNKENKKEKDDALKRAAYLYNSSDKLPDEVFHGNKQLETKIRLWYQQG ERCLYSGKPIPIHDLVHNSNNFEIDHILPLSLSFDDSLANKVLVYDWANQEKGQKTPYQVIDSMDAAWSFREMKDYVLKQKGLGKKKRDYLLTTENIDKIEVKKKFIERNLIDTRYASRVVLNSLQSALRELCKDTKVSVIRG QFTSQLRRKWKIDKSRETYHHHAVDALIIAASSQLKLWEKQDNLMFIDYGNNQVVDKETGEILSVSDDEYKELVFQPPYQGFVNTISSKGFEDEILFSYQVDSKYNRKVSDATIYSTRKAKIGKDKKEETYVLGKIKDIYSQN GFDTFIKKYNKDKTQFLMYQKDPLTWENVIEVILRDYPTTKKSEDGKNDVKCNPFEEYRRENGLICKYSKKGKGTPIKSLKYYDKKLGNCIDITPEESRNKVILQSINPWRADVYFNPETLKYELMGLKYSDLSFEKGTGKYH ISQEKYDAIKEKEGIGKKSEFKFTLYRNDLILIKDTASGEQEIYRFLSRTMPNVNHYAELKPYDKEKFDGGQELMEVFGKVANGGQCLKSLNKSNISIYKVRTDVLGNKYFVKKEGDKPKLNFKNNKKKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO: 40).

30. The protein of claim 17, wherein the Lactobacillus kurabacillus strain Biut2 Cas9 has an amino acid sequence that is at least 80% identical to MPAAKRVKLD GSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILA DECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSG PKKKRKV GT MKRVNEDYILGL A IGTNSCGAVTDKKNNLLKLRGKTAIGSHLFEEGHTAADRRGFRTTRRRLKRRKWRLRLLEEIFAEPMAKVDPGFFVRLHQSWVSPLDKDRKKYNAIVFPTAKEDQAFYKHYATIYHLRDELMTQDRQFDLREIFLAIHHIVKYRGNFLQDTPVKDFEASKIEVGPILSHINNA FAEKIVEDQDPIELNVANAADIEDVIRGKDAEKTVYKLDKVKKIAKLLTDSTAKEEKNVAKQIANAIMGYKTQFETILDKEIDKSDKAQWEFKLSDADADDKLDALLPDLDETDQTVVAEIEKLFSAITLSTIVDENKSLSQSMVEKYKKHKKDYKKLKKYINTLQDQTKAKKLL LAYDLYVNNRHGRLLEAKKTFEDKKKKKALTKDEFYKIVKDNLDDSDLAHEIQQEIAADNFMPKQRTNSNGVIPFQLHQIELDKIIANQGKYYPFLAAENPVEDHRKQAPYKLDELVRFRVPYYVGPMITADEQEKTSGKSFAWMVRKEDGQITPWNFEQKVDRQESANKFIKRM TIKDTYLLSEDVLPANSLLYQRFEVLNELNNIRINGSRISVDLKQQIFNDLFEEKKTVTEKSLTSYLKQNLHLPTVEIKGLADPTKFNSSLASYYHLKSLHVFDKELADPQYQKDFEKIIEYSSIFEDKKIFQDKLHAEFKWLTPEQFKAISTWRLQGWGRLSRKLLVELHDTNG QNIMEQLWDSQKNFMQIVTEPDFKDAIAKENQNVTRANGVEEILADAYTSPANKKAIRQVVKVVADVVKAAGGKKPAQFAIEFTRDPDKNPQLSHIRGTKLLKAYQETAGELVDQKLTDSLKEAMTSRKLLKDKYFLYFMQAGRDAYTGQKINIDEVSTNYQIDHILPQSFIKDD SFDNRVLTATPLNAEKSDDVPYKRFANNYVSDMKMTVGEMWKHWQKAGIINKHKLGNLLLDPDRLNKFQKSGFINRQLVETSQIIKLVSVILQNKYPDAEIITVKAGDNSALRQRLNLYKSRDVNDYHHAIDAYLSIICGNFLYQVYPKYRPYFVYGKYKKFSQDPDLQKEVIKH FKGFTFMWPLLQKDNSERKAPEKIKENNSDRIVFYKHPDIFDKLRKAYNYKYMLVSRETTTENSGLFDVTIYPRGERDLAKTRKLIPKSNGLDPKIYGGYSGNTDAYMVIVKIDKGKESIYKVIGVPMRALASLNRAKKQGNYKEELHQVLEPQIMFDKNGKPKRSVKGFRIIKD HVPFKQVVLDGDKKFMLNSSTYEINAKQLTLTPETMRIVTDNLKKGEDQDQLLVKAYDEILQKVDQYLPLFDVNKFRNSLHLGRAKFLDLAVNDKKITLTNILNGLHDNLVTPDLKNIGIKTPLGKLQVPSGIVLSSEAILIFQSPTGLFEKRVRIADLKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO: 41).

31. The protein of claim 17, wherein the Streptococcus suis strain LSS83 Cas9 has an amino acid sequence that is at least 80% identical to MPAAKRVKLD GSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILA DECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSG PKKKRKV GT MSNGKILGL A IGIASVGVGVIDAQTGEIIHASSRIFPSANAANNAERRTFRGSRRLIRRKKHRIKRLDDLFNDFHINLDGEMSTDNPYVLRVKGLSQKLTVEELYISIKNIMKRRGISYLDDAESDNEAGRSDYAKAIERNRQLLTSKTPG EIQLERLEKYGQLRGNFTIIDEEGQSQQIINVFSTSDYVKEVEKILDCQKMYHKFISDEFCDKLIELLREKRKYYVGPGNEKSRTDYGIYRTDGTTLENLFGILIGKCTFYPDQYRSSRASYTAQEFNFLNDLNNLTVPTE TKKLSQEQKEFLVNYAKETSVLGAGKILQQIAKLADCKVEDIRGYRLDNKDKPELHTFETYRAMKGLVPLVDIGVLSREQLDILADILTLNTDFEGIREALKKQLPNVFDEKQVKGLASFRKSKSQLFAKGWHNLSQKIML EVIPELYATSDEQMTILTRLGKFEKSSVAEYPSSINVDEITDEIYNPVVAKSIRQTIKIINASIKKWDEFDQIVIEMPRDRNEDEEKKRIADGQKANAKEKADSILRAAELYCAGKVLPDYVYNGHNQLATKIRLWYQQGE RCIYTGQPISIHDLIHNQNQYEIDHILPLSLTFDDSLSNKVLVLATANQEKAQRTPYNYLKSATSAWSYREFKDYVTKRKGIGKKKCEYLTFEEDINGFEVRSKFIQRNLVDTRYASKVILNALQDYFKISGIQTKVSVVR GQFTSQLRHKWGIEKTRETYHHHAVDALIIAASSQLRLWKKQESPLVVDYQEGRQVDLETGEILELTDEQYKELVYQPPYQGFVNTISSSAFDNEILFSYQVDSKVNRKISDATIYATRNAQLGKDKTEGIYVLGKIKDIY TQAGYEAFLKRYTKDKTSFLMYHKDLDTWEKVIEIILRDYREYDEKGKEIGNPFERYRRENGYVKKYSRKGNGTAIKSLKYYDNKLGNHIDITPENSRNAVVLQSLKPWRTDVYFNKETGKYEFLGIKYSDLSFEKGTGEY GISQEKYDSIKIAEGVAKKSIFKFTLYKQDLLFIKDIENNFGKLLRFTSKNDTSKHYVELKPYDKNKFGTEEPLLPVLGNVAKSGQCIKGLNKSNISIYKVRTDILGYRHFIKQEGEHPQLKFKKKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO: 42).

32. The protein of any one of the preceding claims, wherein the amino acid sequence of the Cas9 protein comprises SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least 10 mutations of , 13 or 14.

33. The protein of claim 32, wherein the mutation is an amino acid substitution.

34. The protein of any one of the preceding claims, wherein the Cas9 protein has nickase activity.

35. The protein of claim 32, wherein said at least one mutation results in inactive Cas9 (dCas9).

36. The protein of any one of the preceding claims, wherein the Cas9 protein comprises at least one amino acid mutation in the PAM interaction, HNH and / or RuvC domains.

37. An engineered, non-naturally occurring Cas9 fusion protein comprising SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 A Cas9 protein having at least 80% identity, and wherein the Cas9 protein is fused to a histone demethylase, transcriptional activator or deaminase.

38. The Cas9 protein of claim 37, wherein the Cas9 protein is fused to cytosine deaminase or to adenosine deaminase.

39. The Cas9 protein of claim 38, wherein the Cas9 protein is fused to adenosine deaminase and has an amino acid sequence that is at least 80% identical to (a) MPAAKRVKLD GSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILA DECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSG PKKKRKVGTMEKSYSIGLAIGTNSVGWSVITDDYKVPAKKMRVLGNTDKKYIKKNLLGALLFDSGETAEATRLKRTARRRYTRRKNRLRYLQEIFAKEMAKVDESFFQRLDESFLTDDDKTFDSHPIFGNKAEEDAYHQKFPTIYHLRKHLADSQEKADLRLIYLALAHMIKFRGHF LYDNFNDDNFDWRNIDIQKRYEEFIETYDSTLGESYLADISVDAASILEEKVSKTERLENLLKYYPTEKKTTFFGNLIKLILGQQAKFKAIFNLEDEISLQFATPTYDEDLEELLGKIDNGDSYSELFVAAQNLYNTILLASFLKTDNKSAKAPLSTSMIERYENHKKDLAKLKDFVK KNCPDQYHDIFRDKSKNGYAGYIDNGVSQNDFYTFIGKCLEESLKKDKGAQYFLDKIDRDDFLRKQRTFDNGAFPYQIHLQEMHAILRRQGDYYPFLKENQDKIEKILTFRIPYYVGPLARKDSRFAWANYRSDEAITPWNFDEVVDKEKSAEKFITRMTLNDLYLPEEKVLPKHSLI YETFTVYNELTNIKYVNDQGNAIHFDSELKEKIFNQLFKENRKVSKKTLIDFLNNSEGIYTDKLVGIDEEVKYLNASLGTYHDLKKILESFMDDEINEKIIEDIIQTLTLFEDIEMKRQRLQKYDDIFTPKQLKELARRNYTGWGRLSYKLINGIRNKENNKTILDYLKNGNRNFMQL INDDRLSFKQIIIDARKIEKLDNIESVVYNLPGSPAIKKGILQSIKIVDELVKVMGHNPDNIVIEMARENQTTNQGKNRSQQRLKRLQDSMSNFKDSSISLKDVDNSDLQNDRLFLYYIQNGKDMYTGEELDIDHLSDYDIDHIIPQSFIKDNSIDNRVLTSSAKNRGKSDDVPGRDV VLKMKPFWKKLYDVKLISKRKFDNLTKSEHGGLTESDKAGFIKRQLVETRQITKYVAQILDGRFNTKRDDNNKVIRDVKVITLKSSLVSQFRKDFGFYKVREINDYHHAHDAYLNAVVGTAILKKYPKLAPEFVYGEYKKCDVRKLIAKSGDKSEIGKATAKYFFYSNLMNFFKRVIR YSNGMIVVRPVIEYSKDTGEIAWDKEKDFKTVCKVLSCPQVNIVKKVEKQSHGLDRGKPKGFYNANPSPKPKKGSKVNLVPIKANLNPKNYGGYAGISNSYAVLVDATIEKGAKKKLTRIQEFQGISIIDREKYEKNKVEFLKGLGYKEIYSIITLPKYSLFELADGSRRMLASILST NNKRGEIHKGNELVLPAKYIPLLYHANRIHNTFETGHREYVEKHIAEFKEIAEIILEFNNKYVNAKKNSSIIEKALESFDSFSLDEICDSFVGKLKKNNTKKNSGLFELVSLGSASDFEFLETKVPRYRDYTPSSLLNATLIHQSITGLYETRIDLSKLGEEKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO: 29). (b) MPAAKRVKLD GSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILA DECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSG PKKKRKV GT MTKDYTIGL A IGTNSVGWAVLTDDYQLMKRKMSVHGNTEKKKIKKNFWGARLFDEGQTAEFRRTKRTNRRRLARRKYRLSKLQDLFAEELCKQDDCFFVRLEESFLVPEEKQYKPASIFPTLEEEKEYYQKYPTIYHLRQKLVDSTEKEDLRLVYLALAHLLKYRGHFLFEGDLDTE NTSIEESFRVFLEQYSKQSDQPLIVHQPVLTILTDKLSKTKKVEEILKYYPTEKINSFFAQCLKLIVGNQANFKRIFDLEAEVKLQFSKETYEEDLESLLEKIGDEYLDIFLQAKKVHDAILLSEIISSTVKHTKAKLSSGMVERYERHKADLAKFKQFVKENVPQKS TAFFKDTTKNGYAGYIKGKTTQEEFYKFVKKELSGVVGSEPFLEKIDQETFLLKQRTYTNGVIPHQVHLIELKAIIDQQKQHYPFLEEAGPKIIALFKFRIPYYVGPLAKEQEASSFAWIERKTAEKIHPWNFSEVVDIEKSAMRFIQRMTKQDTYLPTEKVLPKNSL LYQKYMIFNELTKVSYKDERGVKQYFSGDEKQQIFKQLFQKERGKITVKKLQNFLYTHYHIENAQIFGIEKAFNASYSTYHDFMKLAKTNQKAMQEWLEQPEMEPIFEDIVKILTIFEDRQMIKHQLSKYQEVFGEKLLKEFARKHYTGWGRFSAKLIHGIRDRKTNK TILDYLINDDDVPANRNRNLMQLINDEHLSFKEEIAKATVFSKHKSLVDVIQDLPGSPAIKKGIWQSLKIVEELIAIIGYKPKNIVIEMARENQKTHRTKPRLKALENGLKQIGSTLLKEQPTDNKALQKERLYLYYLQNGRDMYTGEPLEIENLHQYEVDHIIPRS FIVDNSIDNKVLVARKQNQKKRDDVPKKQIVNEQRIFWNQLKEAKLISPKKYAYLTKIELTPEDKARFIQRQLVETRQITKHVANILHQSFHQEEEGTDCDGVQIITLKATLTSQFRQTFGLYKVREINPHHHAHDAYLNGFIANVLLKRYPKLAPEFVYGKYVKYSL ARENKATAKKEFYSNILKFFESDEPFCDENGEIYWEKSHHLPRIKKVLSSHQVNVVKKVEQQKGGFYKETVNSKEKPDKLIERKNNWDVTKYGGFGSPVIAYAIAFVYAKGKTQKKTRAIEGITIMEQAAFEKDPTTFLKEKGFPQVTEFIKLPKYTLFEFDNGRRRF LASHKESQKGNPFILSDQLVTLLYHAQHYDKITYQESFDYVNTHLSDFSAILTEVLAFAEKYTLADKNIERIQELYEENKYGEISMIAQSFLQLLQFNAIGAPADFKFFGVTIPRKRYTSLTEIWDATIIYQSVTGLYETRIRMGDLWAGEQKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA(SEQID NO:30) (c) MPAAKRVKLD GSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILA DECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSG PKKKRKV GT MTNGKILGLAIGVASVGVGIIEAKTGKVIHANSRLFSAANAENNAERRGFRGARRLTRRKKHRVKRVRDLFEKYDISTDFRNLNLNPYELRVKGLSEQLTNEELFAALRTIAKRRGISYLDDAEDDSTGSSDYAKSIDENRRL LKSMTPGQIQLERLEKYGQLRGNFTVYDENGEAHRLINVFSTSDYKNEARKILETQSNYNKQITDEFIEDYIEILTQKRKYYHGPGNEKSRTDYGRFRTDGTTLENIFGILIGKCSFYPEEYRASKASYTAQEFNFLNDLNNL KVPTETGKLSTEQKEYLVDFAKNSKTLGASKLLKEIAKLVDGDVKDISGYREDSKGKPDLHTFEPYRKLKFNLTTVDIDNLSRDILDKLANILTLNTEREGIEDAINRNLPEQFTKEQISEIVQIRKSQSSAFNKGWHSFSAK LMNELIPELYATSEEQMTILTRLEKFKATKKSSKNTKTIDEKEITDEIYNPVVAKSVRQTIKIINAAVKKYGDFDKIVIEMPRDKNADDEKKFIDKKNKENKKEKDDSLKRAAYLYNGTDKLPDDVFHGNKQLATKIRLWYQQG ERCLYSGKPILIQDLVHNSNNFEIDHILPLSLSFDDSLANKVLVYAWTNQEKGQKTPYQVIDSMDAAWSFREMKDYVLKQKGIGKKKREYLLTTENIDKIEVKKKFIERNLVDTRYASRVVLNSLQTALKELGKDTKVSVVRG QFTSQLRRKWKIDKSRETYHHHAVDALIIAASSQLKLWQKHENLMFENYGENQVVNKETGEILSISDDEYKELVFQPPYQGFVNTISSKAFEDEILFSYQVDSKFNRKVSDATIYSTRKAKLGKDKKEETYVLGKIKDIYSQD GFDTFIKRYKKDKTQFLMYQKDPLTWENVIEVILRDYPTTKKSEDGKNDVKCNPFEEYRRENGLICKYSKKGKGTPIKSLKYYDKKLGNHISITPKESKNDVVLQSLNPWRADLYFNPDTLKYELMGLKYSDLSFEKGTGKYH ISQEKYDEIKEKEGIGQNSEFKFTLYRNDLILIKDTESGEQEIYRFLSRTMPNVKHYVELKPYDKEKFNGGQELIKSLGEADKVGRCLKGLSKPGISIYKVRTDVLGNKFFVKKEGDKPKLDFKNNKKKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO: 31). (d) MPAAKRVKLD GSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILA DECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSG PKKKRKV GT MNGLVLGL A IGIASVGVGILNKETGEIIHVNSRIFPAATADSNVERRGFRQGRRLGRRKKHRSARLNDLFEEFGFITDFSAVPLNLNPYALRVKGLSEELTNEELFIALKNIIKRRGISYLDDASEDGETASNEYGKAVEENRKLLADKT PGQIQLERFEKYGQVRGDFTVVENGENHRLINVFSTSAYKKEAERILRRQQEFNVRISDEFIEAYLTILTGKRKYYHGPGNEKSRTDYGRFRTDGTTLDNIFGILIGKCTFYPDEYRAAKASYTAQEFNLLNDLNNLTVPTE TKKLRPEQKRQIVEYARTAKTLGTPTLLKYIAKLVDGSIDDIKGYRIDKSDKPEMHTFDAYRKMRTLELVDVDILSRETLDDLAYILTLNTESEGILEALNSKMPGTFTKEQIDELIQFRKKNSAVFGKGWHNFSLKLMNE LISELYETSEEQMTILTRLGKQRSREISKRTKYIDEKELTDEIYNPVVAKSVRQAIKIINLATKKYGIFDNIVIEMARESNEDDEKKAIQNVQKANEDEKKAAMEKAADLYNGKKELPDSIFHGHKELATKIRLWHQQGERC LYTGKNISIHDLIHNPHQYEIDHILPLSLSFDDGLANKVLVLATANQEKGQRTPFQALDSMDDAWSYIEFKQYVRNSKSLSNKKKDYLLTEEDISKIEVKQKFIERNLVDTRYSSRVVLNTLQEFYKTNDFDTKISVVRGQ FTSQLRRKWKIEKSRDTYHHHAVDALIIAASSQLRLWKKQNNPLISYREGQLVDPETGEILSLLTDDEYKELVFRPPYDYFVDTLKSKSFEDSILFSYQVDSKYNRKISDATIYGTRKAQLGKDKQEETYVLGKIKDIYSQKG YEDFIKRYKKDTTQFLMYHKDPQTFAKVIEEILKTYPDKELNEKGKEIPCNPFEKYRQENGPIRKYSKKGKGPEIKSLKYYDNKLGNHIDITPVNSQNQVVLQSLKPWRTDVYFNPQTSKYELMGLKYSDLRFEKGSGSYG ISPEKYNKVKAKEGVDEDSEFKFTLYKNDLILIKDTETGEQQLFRYGSRNDTSKHYVELKPYEKAKFEGNQQLMNLLGTVAKGGQCLKGINKPNLSIYKVKTDVLGNKHFIKKEGDQPQLNFKKKIKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO: 32). (e) MPAAKRVKLDGS EVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILA DECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSG PKKKRKV GT MNNSYILGLAIGITSVGYGIIEYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRLQRVKKMLFDYKLLNEDSEISGINPYEARVKGLSEKLSDEEFSAALLHLAKRRGVHNVSDVEEDTGNELS TKEQIARNNKALEDKYVAELQLERLKEQGEVRGAANRFKTSDYIKEAKQLLKTQSDYHKIDETFIETYISLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVI ARDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNFKIYHDIKGITSRKEILENADLLNQIAEILTIYQSAEDIQEELAELEPKLTQEEIEQISNLTGYTGTHRLSLKAI NLILDELWNTSDNQMTIFNRLKLVPKKVDLSQQKEIPTSLVDDFILSPVVKRSFIQSIKVINAIIKKFGLPKDIIIELAREKNSKEAQKFINEMQKRNRQTNERIEKIIKETGKEKAKFLIEKIKLHDMQEGKC LYSLESIPLEDLLNNPYHYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDAKISYETFKKHILNLSKGKGRVSKKKKEYLLEERDINRFSVQKDFINRNLVDTRYATRELMNLLRSYFRVN DLDVKVKSINGGFTSFLRTKWKFKKERNQGYKHHAEDALVIANADFIFKEWKKLDTTNKVMENQTVEEQQAENMPGIETDDEYKEIFVIPRQIQSIKDFKDYKYSHRVDKKPNRELVNDTLYSTRKDDKGNTLI INNIKGLYDKDNDKLKNLIKKSPEKLLMYHHDPQTYQKLKTIMEQYSNEKNPLYKYHEETGNYLTKYSKKDNGPIIKKVKYYGKKLNAHLDITNDYSNSQNKIVKLSLKPYRFDVYLDNGGYKFVTVKNLDVIK KEGFFKIDSNAYEKAKSEKKIDENAVFIASFYNNDLIKIDGELYRIVGVNNDTRNVVELNMIPITYKEYLENINDKRTPRILKTISQKTYSIEKYSTDILGNLYKVKSKKKPQMIMKGKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO: 33). (f) MPAAKRVKLD GSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILA DECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSG PKKKRKV GT MENKNYSIGLAIGTNSVGWAVITDDYKVPSKKMKVFGNTDKHFIKKNLIGALLFDEGATAEDRRLKRTARRRYTRRKNRLRYLQEIFSEEISKLDSSFFHRLDDSFLVPKDKRGSKYPIFATLEEEKEYHKKFPTIYHLRKHLADSKEKTDLRLIYLALAHMIKYRGHFLYEESFD IKNNDIQKIFNEFISIYDNTFEGSSLSGQNAQVEAIFTDKISKSAKRERVLKLFSDEKSTSLFSEFLKLIVGNQADFKKHFDLEEKAPLQFSKDTYDEDLENLLGQIGDGFTDLFLVAKKLYDAILLSGILTVTDPSTKAPLSASMIERYESHQKDLAALKQFIKNNLPKRYNEVF SDQSKDGYAGYIDGKTTQEAFYKYIKNLLSKFEGADYFLDKIEREDFLRKQRTFDNGSIPHQIHLQEMNAILRRQGEHYPFLKENREKIEKILTFRIPYYVGPLARGNRDFAWLTRNSDQAIRPWNFEEIVDKASSAEEFINKMTNYDLYLPEEKVLPKHSLLYETFAVYNELTKV KFIAEGLRDYQFLDSGQKKQIVNQLFKEKRKVTEKDIIHYLHNVDGYDGIELKGIEKQFNASLSTYHDLLKIIKDKEFMDDPKNEEILENIVHTLTIFEDREMIKQRLAQYDSIFDEKVIKALTRRHYTGWGKLSAKLINGICDKQTGDTILDYLIDDGKINRNFMQLINDDGLSF KEIIQKAQVVGKTDDVKQVVQELPGSPAIKKGILQSIKIVDELVKVMGYAPESIVIEMARENQTTARGKKNSQQRYKRIEDSLKNLAPGLDSNILKENPTDNIQLQNDRLFLYYLQNGKDMYTGKPLDIDQLSSYDIDHIIPQAFIKDDSIDNRVLTSSKDNRGKSDNVPSLEVVQ KRKAFWQQLLDSKLISERKFNNLTKAERGGLDERDKVGFIRRQLVETRQITKHVAQILDASFNTEVNEKNQKIRTVKIITLKSNLVSNFRKEFELYKVREINDYHHAHDAYLNAVVAKAILKKYPKLEPEFVYGDYQKYDLKRYISRFKPSKEIEKATEKYFFYSNLLNFFKEEVH YADGIIVKRENIEYSKDTGEIAWNKEKDFATIKKVLSYPQVNIVKKTEIQTHGLDRGKPKGLFNSNPSPKPSEDSKENLVPIKQGLDPRKYGGYAGISNSYAVLVKAIVEKGAKKQQKTILEFQGISILDKINFENNKENYLLKKRYIEILSTITLPKYSLFEFPDGTRRRLASIL STNNKRGEIHKGNELVLPGKYTTLLYHAKNINKKLEPEHLEYVEKHRNDFAKLLECVLNFNDKYVGALKNGERIRQAFTDWETVDIEKLCFSFIGPENSKNAGLFELTSQGSASDFEFLGVKIPRYRDYAPSSLLKATLIHQSITGLYETRIDLSKLGEDKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO: 34). (g) MPAAKRVKLD GSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILA DECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSG PKKKRKV GT MSDLVLGL A IGIGSVGVGILNKVTGEIIHKNSRIFPAAQAENNLERRTNRQGRRLTRRKKHRRVRLNHLFEESGLITDFTKVSINLNPYQLRVKGLTAELSNEELFIALKNMVKHRGISYLDDASDDGNSSVGDYAQIVKENSKQLETKTPG QIQLERYQKYGQLRGDFTVEEDGRKHRLINVFPTSAYHAEALRILQTQQEFNPQITDEFINSYLEILTGKRKYYHGPGNEKSRTDYGKYTTKKDAQGQYITLNNIFGILIGKCTFYPEEYRAAKASYTAQEFNLLNDLNNLTV PTETKKLSEEQKYQIITYVKNEKAMGPAKLFKYIAKLLSCDVADIKGYRIDKSDKAEIHTFEAYRKMKTLETLDVKKMAREELDKLAYVLTLNTEREGIQEALDHEFADGTFSQEQVDELVQFRKANSSIFGKGWHSFSVKLM MELIPELYATSEEQMTILTRLGKQKTTSSSNKTKYIDEKQLTEEIYNPVVAKSVRQAIKIVNAAIKKYGDFDNIVIEMARETNEDDEKKAIQKIQKANKAEKDAAMRKAANQYNGKAELPHSVFHGHKQLATKIRLWHQQGERC LYTGKTISIHDLINNPNQFEIDHILPLSITFDDSLANKVLVYATANQEKGQRTPYQALDSMDDAWSFRELKAFVRESKALSNKKKEYLLTEEDISKFDVRKKFIERNLVDTRYASRVVLNALQEHFRTHKIDTKVSVVRGQFT SQLRRHWGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKNTLVSYSEDQLLDIETGELISDDEYKESVFKAPYQHFVDTLKSKEFEDSILFSYQVDSKFNRKISDATIYATRKAKLDKEKKEYTYTLGKIKDIYALGTKTPSK TGFYKFLDLYKKDKSQFLMYQKDRRTWDEVIEKILEQYRPFKEKDKNGKEVDFNPFEKYRIENGPIRKYSRKGNGPEIKSLKYYDNLLGRFVDITPSESKNPVALLSLNPWRTDVYYNTETRKYEFLGLKYADLCFEKGGSYG ISKVKYNKIREKEGIGKNSEFKFTLYKNDLILIKDTETNRQQIFRFWSRTGKDNPKSFEKHKLELKPYEKTRFEKGEELKVLGKVPPSSNRLQKNMQIENLSIYKVRTDVLGNQHIIKNEGDKPKLDFKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO: 35). (h) MPAAKRVKLD GSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILA DECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSG PKKKRKV GT MNGLVLGLAIGIASVGVGILEKNSGKIIHANSRIFPAATADNNVERRKNRQARRLHRRKKHRGVRLQDIFEDYGLLTDFSKVSINLNPYRLRVDGLDQQLTNEELFIALKNIVKRRGISYLDDASEDGGTVSSDYGKAVEEN RKLLAEKTPGQIQLERFEKYGQVRGDFTVVENGEKRRLINVFSTSAYRKEAERILRKQQEFNSKITDEFIEDCLKILTGKRKYYHGPGNEKSRTDYGRFRTDGTTLDNIFGILIGKCTFYPNEYRASKASHTAQEFNLLNDL NNLTVPTETKKLSEEQKKVIVEYAKEAKTLGASTLLKYIAKMIDASVDQISGYRVDVNNKPEMHTFEVYRKMQSLETISVGELSRNVLDELAHILTLNTEREGIEEAINTKLKDSFSQDQVLELVQFRKNNSSLFSRGWHNF SLKLMMELIPELYETSEEQMTILTRLGKQKSKETSKRTKYIDEKELTEEIYNPVVAKSVRQAIKIINEATKKYGIFDNIVIEMARENNEEDAKKDYIKRQKANQDEKNAAMEKAAFQYNGKKELPDNIFHGHKELATKIRLWH QQGEKCLYTGKNIPISDLIHNQYKYEIDHILPLSLSFDDSLSNKVLVLATANQEKGQRTPFQALDSMDDGWSYREFKSYVKESKLLGNKKKEYLLTEEDISKIEVKQKFIERNLVDTRYSSRVVLNALQDFYKAHKFDTTIS VVRGQFTSQLRRKWGLEKSRETYHHHAVDALIIAASSQLRLWKKQNNPLIAYKEGQFVDSETGEILSLSDDEYKELVFKAPYDHFVDTLRSKTFEDSILFSYQVDSKYNRKISDATIYATRKAKLDKDKSEETYVLGKIKDI YSQAGYDAFIKIYNKDKSKFLMYHKDPQTFEKVIEEILRTYPSKELNDKNKEIPCNPFEKYRQENGPIRKYSKKGNGPEIKCLKYYDNKLGNYIDITPDGSDNQVVLQSIAPWRTDVYYNHKTGKYEFLGLKYSDLYFEKGT GKYKISKEKYDNIKKIEGVVETSEFKFTLYKNDLILIKDVEKGQEQLFRFLSRNNKGKHQVQLKPMNKSDFEKGEKLIDIFGTVPNSTTQCVKGLNKSNISIFKVKTDVLGKKHIIKKEGDEPKLKFKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO: 36). (i) MPAAKRVKLD GSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILA DECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSG PKKKRKV GT MNGLVLGL A IGIASVGVGILEKNSGKIIHANSRIFPAATADNNVERRKNRQARRLHRRKKHRGVRLQDIFEDYGLLTDFSKVSINLNPYRLRVDGLDQQLTNEELFIALKNIVKRRGISYLDDASEDGGTVSSDYGKAVEENRKLLAEKT PGQIQLERFEKYGQVRGDFTVVENGEKRRLINVFSTSAYRKEAERILRKQQEFNSKITDEFIEDCLKILTGKRKYYHGPGNEKSRTDYGRFRTDGTTLDNIFGILIGKCTFYPNEYRASKASHTAQEFNLLNDLNNLTVPT ETKKLSEEQKKVIVEYAKEAKTLGASTLLKYIAKMIDASVDQISGYRVDVNNKPEMHTFEVYRKMQSLETISVGELSRNVLDELAHILTLNTEREGIEEAINTKLKDSFSQDQVLELVQFRKNNSSLFSRGWHNFSLKLMM ELIPELYETSEEQMTILTRLGKQKSKETSKRTKYIDEKELTEEIYNPVVAKSVRQAIKIINEATKKYGIFDNIVIEMARENNEEDAKKDYIKRQKANQDEKNAAMEKAAFQYNGKKELPDNIFHGHKELATKIRLWHQQGE KCLYTGKNIPISDLIHNQYKYEIDHILPLSLSFDDSLSNKVLVLATANQEKGQRTPFQALDSMDDGWSYREFKSYVKESKLLGNKKKEYLLTEEDISKIEVKQKFIERNLVDTRYSSRVVLNALQDFYKAHKFDTTISVVR GQFTSQLRRKWGLEKSRETYHHHAVDALIIAASSQLRLWKKQNNPLIAYKEGQFVDSETGEILSLSDDEYKELVFKAPYDHFVDTLRSKTFEDSILFSYQVDSKYNRKISDATIYATRKAKLDKDKSEETYVLGKIKDIYS QAGYDAFIKIYNKDKSKFLMYHKDPQTFEKVIEEILRTYPSKELNDKNKEIPCNPFEKYRQENGPIRKYSKKGNGPEIKCLKYYDNKLGNYIDITPDGSDNQVVLQSIAPWRTDVYYNHKTGKYEFLGLKYSDLYFEKGTG KYKISKEKYDNIKKIEGVVETSEFKFTLYKNDLILIKDVEKGQEQLFRFLSRNNKGKHQVQLKPMNKSDFEKGEKLIDIFGTVPNSTTQCVKGLNKSNISIFKVKTDVLGKKHIIKKEGDEPKLKFKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO: 37). (j) MPAAKRVKLD GSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILA DECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSG PKKKRKV GT MKEKYILGL A LGITSVGYGIINFETKKIIDAGVRLFPEANVDNNEGRRSKRGSRRLKRRRIHRLDRVKSLLTEYNLINREQIPTSNNPYQIRVKGLSEILSKDELAIALLHLAKRRGIHNINVSSEDEDASNELSTKEQINR NNKLLKNKYVCEVQLQRLKEGQIRGEKNRFKTTDILKEIDQLLKVQKDYHNLDIDFINQYKEIVETRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELRSVKYAYSADLFNALNDLNNLIIQRNDSE KLEYHEKYHIIENVFKQKKKPTLKQIAKEIGVNPEDIKGYRITKSGTPQFTEFKLYHDLKSIVFDKSILENEAILDQIAEILTIYQDEESIKEELNKLPEILNEQDKAEIAKLTGYNVTHRLSLKCIHLINE ELWQTSRNQMEIFNYLNIKPNKVDLSEQNKIPKDLVDEFILSPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQKKNEATRKRINEIIGQTGNQNGKRIVEKIRLHDQQEGKCLYSL ESIPLMDLLNNPQNYEVDHIIPRSVAFDNSIHNKVLVKQIENSKKGNRTPYQYLNSSDANLSYNQFKQHILNLSKSKDRISKKKKDYLLEERDINKFEVQKEFINRNLVDTRYATRELTSYLKAYFSANNMD VKVKTINGSFTNHLRKVWRFDKYRNHSYKHHAEDALIIANADFLFKENKKLQNANKILEKPTIENDTQKVTVEKEEDYNNMFETPKLVEDIKQYRDYKFSHRVDKKPNRQLIKDTLYSTRMKDEHNYIVQTIT DIYGKDNTNLKKQFNKNPEKFLMYQNDPKTFEKLSIIMKQYSDEKNPLAKYYEETGEYLTKYSKKNNGPIVKKIKLLGNKVGNHLDVTNKYENSTKKLVKLSIKNYRFDVYLTEKGYKFVTIAYLNVFKKDN YYYIPKDLYQELKAKKKIKDTDQFIASFYKNDLIKLNGDLYKIIGVNSDDRNIIELDYYDIKYKDYCEINNIKGEPRIKKTIGKKTESIEKLTTDVLGNLYLHTTEKAPQLIFKRGLKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO: 38). (k) MPAAKRVKLD GSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILA DECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSG PKKKRKV GT MKRNYILGL A IGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRLQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEVEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNS KALEEKYVAELQLERLKTDGEVRGPNNRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVIARDENEK LEYYEKFQIIENVFKQKKKPTLKQIAKELLVNEEDIKGYRVTSIGKPEFTNFKIYHDIKGITERKEVLENAELLDQIAEILTIYQSSEDVQEELANLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDE LWHTNDNQMAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQMNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLE SIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRELMNLLRSYFRVNNLDV KVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQTVEEKQAESMPEIETEQEYKEIFITPHQIQHIKGFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNN LNGLYDKDNDKLKKLMNKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGKNLKAHLDITDDYPNSRNKVVKLSVKPYRFDVYLDNDIYKFVTVKNLDVIKK EDYYEVNSKCYKEAKKLKKISDQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMINITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKQKPQMIMKGKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO: 39). (l) MPAAKRVKLD GSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILA DECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSG PKKKRKV GT MTNGKILGLAIGIASVGVGIIEAKTGKVVHANSRLFSAANAENNTERRGFRGSRRLNRRRKKHRVKRVRDLFEKHEIVTDFRNLNLSPYELRVKGLTEQLTNEELFAALRTISKRRGISYLDDAEDDSTGSTDYAKSIDENRRL LKNKTPGQIQLERLEKYGQLRGNFTVYDENGEAHRLINVFSTSDYEKEARKILETQADYNKKITAEFIDDYVEILTQKRKYYHGPGNEKSRTDYGRFRTDGTTLENIFGILIGKCNFYPDEYRASKASYTAQEYNFLNDLNNL KVPTETGKLPTEQKESLVEFAKNTATLGPSKLLKEIAKILDCKVDEIKGYREDDKGKPDLHTFEPYRKLKFNLESINIDDLSREVIDKLADILTLNTEREGIEDAIKRNLPNQFTEEQISEIIKVRKSQSTAFNKGWHSFSAK LMNELIPELYATSDEQMTILTRLEKFKVNKKSSKNTKTIDEKEVTDEIYNPVVAKSVRQTIKIINAAVKKYGDFDKIVIEMPRDKNADDEKKFIDKRNKENKKEKDDALKRAAYLYNSSDKLPDEVFHGNKQLETKIRLWYQQG ERCLYSGKPIPIHDLVHNSNNFEIDHILPLSLSFDDSLANKVLVYDWANQEKGQKTPYQVIDSMDAAWSFREMKDYVLKQKGLGKKKRDYLLTTENIDKIEVKKKFIERNLIDTRYASRVVLNSLQSALRELCKDTKVSVIRG QFTSQLRRKWKIDKSRETYHHHAVDALIIAASSQLKLWEKQDNLMFIDYGNNQVVDKETGEILSVSDDEYKELVFQPPYQGFVNTISSKGFEDEILFSYQVDSKYNRKVSDATIYSTRKAKIGKDKKEETYVLGKIKDIYSQN GFDTFIKKYNKDKTQFLMYQKDPLTWENVIEVILRDYPTTKKSEDGKNDVKCNPFEEYRRENGLICKYSKKGKGTPIKSLKYYDKKLGNCIDITPEESRNKVILQSINPWRADVYFNPETLKYELMGLKYSDLSFEKGTGKYH ISQEKYDAIKEKEGIGKKSEFKFTLYRNDLILIKDTASGEQEIYRFLSRTMPNVNHYAELKPYDKEKFDGGQELMEVFGKVANGGQCLKSLNKSNISIYKVRTDVLGNKYFVKKEGDKPKLNFKNNKKKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO: 40). (m) MPAAKRVKLD GSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILA DECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSG PKKKRKV GT MKRVNEDYILGL A IGTNSCGAVTDKKNNLLKLRGKTAIGSHLFEEGHTAADRRGFRTTRRRLKRRKWRLRLLEEIFAEPMAKVDPGFFVRLHQSWVSPLDKDRKKYNAIVFPTAKEDQAFYKHYATIYHLRDELMTQDRQFDLREIFLAIHHIVKYRGNFLQDTPVKDFEASKIEVGPILSHINNA FAEKIVEDQDPIELNVANAADIEDVIRGKDAEKTVYKLDKVKKIAKLLTDSTAKEEKNVAKQIANAIMGYKTQFETILDKEIDKSDKAQWEFKLSDADADDKLDALLPDLDETDQTVVAEIEKLFSAITLSTIVDENKSLSQSMVEKYKKHKKDYKKLKKYINTLQDQTKAKKLL LAYDLYVNNRHGRLLEAKKTFEDKKKKKALTKDEFYKIVKDNLDDSDLAHEIQQEIAADNFMPKQRTNSNGVIPFQLHQIELDKIIANQGKYYPFLAAENPVEDHRKQAPYKLDELVRFRVPYYVGPMITADEQEKTSGKSFAWMVRKEDGQITPWNFEQKVDRQESANKFIKRM TIKDTYLLSEDVLPANSLLYQRFEVLNELNNIRINGSRISVDLKQQIFNDLFEEKKTVTEKSLTSYLKQNLHLPTVEIKGLADPTKFNSSLASYYHLKSLHVFDKELADPQYQKDFEKIIEYSSIFEDKKIFQDKLHAEFKWLTPEQFKAISTWRLQGWGRLSRKLLVELHDTNG QNIMEQLWDSQKNFMQIVTEPDFKDAIAKENQNVTRANGVEEILADAYTSPANKKAIRQVVKVVADVVKAAGGKKPAQFAIEFTRDPDKNPQLSHIRGTKLLKAYQETAGELVDQKLTDSLKEAMTSRKLLKDKYFLYFMQAGRDAYTGQKINIDEVSTNYQIDHILPQSFIKDD SFDNRVLTATPLNAEKSDDVPYKRFANNYVSDMKMTVGEMWKHWQKAGIINKHKLGNLLLDPDRLNKFQKSGFINRQLVETSQIIKLVSVILQNKYPDAEIITVKAGDNSALRQRLNLYKSRDVNDYHHAIDAYLSIICGNFLYQVYPKYRPYFVYGKYKKFSQDPDLQKEVIKH FKGFTFMWPLLQKDNSERKAPEKIKENNSDRIVFYKHPDIFDKLRKAYNYKYMLVSRETTTENSGLFDVTIYPRGERDLAKTRKLIPKSNGLDPKIYGGYSGNTDAYMVIVKIDKGKESIYKVIGVPMRALASLNRAKKQGNYKEELHQVLEPQIMFDKNGKPKRSVKGFRIIKD HVPFKQVVLDGDKKFMLNSSTYEINAKQLTLTPETMRIVTDNLKKGEDQDQLLVKAYDEILQKVDQYLPLFDVNKFRNSLHLGRAKFLDLAVNDKKITLTNILNGLHDNLVTPDLKNIGIKTPLGKLQVPSGIVLSSEAILIFQSPTGLFEKRVRIADLKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO: 41). (n) MPAAKRVKLD GSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLYDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPGMNHRVEITEGILA DECAALLCRFFRMPRRVFNAQKKAQSSTDGSSGSETPGTSESATPESSG PKKKRKV GT MSNGKILGL A IGIASVGVGVIDAQTGEIIHASSRIFPSANAANNAERRTFRGSRRLIRRKKHRIKRLDDLFNDFHINLDGEMSTDNPYVLRVKGLSQKLTVEELYISIKNIMKRRGISYLDDAESDNEAGRSDYAKAIERNRQLLTSKTPG EIQLERLEKYGQLRGNFTIIDEEGQSQQIINVFSTSDYVKEVEKILDCQKMYHKFISDEFCDKLIELLREKRKYYVGPGNEKSRTDYGIYRTDGTTLENLFGILIGKCTFYPDQYRSSRASYTAQEFNFLNDLNNLTVPTE TKKLSQEQKEFLVNYAKETSVLGAGKILQQIAKLADCKVEDIRGYRLDNKDKPELHTFETYRAMKGLVPLVDIGVLSREQLDILADILTLNTDFEGIREALKKQLPNVFDEKQVKGLASFRKSKSQLFAKGWHNLSQKIML EVIPELYATSDEQMTILTRLGKFEKSSVAEYPSSINVDEITDEIYNPVVAKSIRQTIKIINASIKKWDEFDQIVIEMPRDRNEDEEKKRIADGQKANAKEKADSILRAAELYCAGKVLPDYVYNGHNQLATKIRLWYQQGE RCIYTGQPISIHDLIHNQNQYEIDHILPLSLTFDDSLSNKVLVLATANQEKAQRTPYNYLKSATSAWSYREFKDYVTKRKGIGKKKCEYLTFEEDINGFEVRSKFIQRNLVDTRYASKVILNALQDYFKISGIQTKVSVVR GQFTSQLRHKWGIEKTRETYHHHAVDALIIAASSQLRLWKKQESPLVVDYQEGRQVDLETGEILELTDEQYKELVYQPPYQGFVNTISSSAFDNEILFSYQVDSKVNRKISDATIYATRNAQLGKDKTEGIYVLGKIKDIY TQAGYEAFLKRYTKDKTSFLMYHKDLDTWEKVIEIILRDYREYDEKGKEIGNPFERYRRENGYVKKYSRKGNGTAIKSLKYYDNKLGNHIDITPENSRNAVVLQSLKPWRTDVYFNKETGKYEFLGIKYSDLSFEKGTGEY GISQEKYDSIKIAEGVAKKSIFKFTLYKQDLLFIKDIENNFGKLLRFTSKNDTSKHYVELKPYDKNKFGTEEPLLPVLGNVAKSGQCIKGLNKSNISIYKVRTDILGYRHFIKQEGEHPQLKFKKKRPAATKKAGQAKKKK GS YPYDVPDYAYPYDVPDYAYPYDVPDYA (SEQ ID NO: 42).

40. The Cas9 protein of claim 38, wherein the Cas9 protein is fused to cytosine deaminase.

41. The Cas9 protein of claim 2, wherein the Cas9 protein recognizes a PAM sequence comprising 5'-NRGNR-3'.

42. The Cas9 protein of claim 3, wherein the Cas9 protein recognizes a PAM sequence comprising 5'-NRG-3', wherein H is adenine, cytosine, or thymine.

43. The Cas9 protein of claim 4, 11 or 12, wherein the Cas9 protein recognizes a PAM sequence comprising 5'-NNGR-3', wherein H is adenine, cytosine or thymine, and R is adenine. Purine or guanine.

44. The Cas9 protein of claim 5, wherein the Cas9 protein recognizes a PAM sequence comprising 5'-NNGRRT-3'.

45. The Cas9 protein of claim 6, wherein the Cas9 protein recognizes a PAM sequence comprising 5'-NNAAAA-3'.

46. ​​The Cas9 protein of claim 7, wherein the Cas9 protein recognizes a PAM sequence comprising 5'-NGGNG-3'.

47. The Cas9 protein of claim 8, wherein the Cas9 protein recognizes a PAM sequence comprising 5'-NNGNRG-3'.

48. The Cas9 protein of claim 9, wherein the Cas9 protein recognizes a PAM sequence comprising 5'-NNAAAC-3'.

49. The Cas9 protein of claim 10, wherein the Cas9 protein recognizes a PAM sequence comprising 5'-NNRAAG-3'.

50. The Cas9 protein of claim 13, wherein the Cas9 protein recognizes a PAM sequence comprising 5'-NNAYAA-3'.

51. The Cas9 protein of claim 13, wherein the Cas9 protein recognizes a PAM sequence comprising 5'-NNGAAA-3'.

52. The Cas9 protein of claim 13, wherein the Cas9 protein recognizes a PAM sequence comprising 5'-NNAAA-3' (H=A, C or T; R=A or G).

53. A nucleic acid encoding the Cas9 protein according to any one of the preceding claims.

54. The nucleic acid of claim 53, wherein the nucleic acid is codon optimized for expression in mammalian cells.

55. The nucleic acid of claim 53, wherein the nucleic acid is codon optimized for expression in human cells.

56. A eukaryotic cell comprising the Cas9 protein according to any one of claims 1 to 52.

57. The eukaryotic cell of claim 56, wherein said cell is a human cell.

58. A method for cutting target nucleic acids in eukaryotic cells, which include: Contacting the cell with Cas9 and an RNA guide according to any one of claims 1 to 52 or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a positive sequence repeat capable of hybridizing to the target nucleic acid and spacer sequence, and Wherein the Cas9 protein is capable of binding to the RNA guide and causing a break in the target nucleic acid sequence complementary to the RNA guide.

59. A method of changing the expression of a target nucleic acid in a eukaryotic cell, which include: Contacting the cell with Cas9 and an RNA guide according to any one of claims 1 to 52 or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a positive sequence repeat capable of hybridizing to the target nucleic acid and spacer sequence, and Wherein the Cas9 protein is capable of binding to the RNA guide and causing a break in the target nucleic acid sequence complementary to the RNA guide.

60. A method of changing the expression of a target nucleic acid in a eukaryotic cell, which include: Contacting the cell with Cas9 and an RNA guide according to any one of claims 1 to 52 or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a positive sequence repeat capable of hybridizing to the target nucleic acid and spacer sequence, and Wherein the Cas9 protein is capable of binding to the RNA guide and editing the target nucleic acid sequence complementary to the RNA guide.

61. A method for modifying target nucleic acids in eukaryotic cells, which include: Contacting the cell with Cas9 and an RNA guide according to any one of claims 1 to 52 or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a positive sequence repeat capable of hybridizing to the target nucleic acid and spacer sequence, and Wherein the Cas9 protein is capable of binding to the RNA guide and editing the target nucleic acid sequence complementary to the RNA guide.

62. The method of claim 60 or 61, wherein the Cas9 protein is inactivated Cas9 (dCas9).

63. The method of claim 62, wherein said dCas9 is fused to a deaminase.

64. The method of any one of claims 59 to 63, wherein the RNA guide comprises crRNA and tracrRNA.

65. The method of any one of claims 59 to 64, wherein the RNA guide comprises sgRNA.

66. The method of claim 65, wherein the sgRNA for use with Seq2Cas9 comprises a scaffold comprising at least about 80% identity to 5'-GUUUUAGUACUCUGUAAUUUUGAAAAAAUACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGACGAGAUUUUUU-3' (SEQ ID NO: 43) sequence.

67. The method of claim 65, wherein the sgRNA for use with EhiCas9 comprises a scaffold comprising at least about 80% identity to 5'-GUUUUAGAGCUAUGUUGGAAACAACAUAGCGAGUUAAAAUAAGGCAUUGUCCGUUAUCAGCUUUUAAAGCAAGCACUGUCUCGGUGCUUUUUU-3' (SEQ ID NO: 44) sequence.

68. The method of claim 65, wherein the sgRNA for use with SeqCas9 comprises a scaffold comprising at least about 80% identity to 5'-GUUUUUGUACUCUCAAGAUUUGAAAAAAUCUUGCAGAGCCUACAAAGAUAAGGCUUCAUGCCGAAUUCAAGCACCCAUGUUUACAUGGGGUGCUUUU-3' (SEQ ID NO: 45) sequence.

69. The method of claim 65, wherein the sgRNA for use with SsiCas9 comprises a scaffold comprising at least about 80% identity to 5'-GUUUUAGUACUCUGUAAUUUUGAAAAAAUACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUUGGCGAGAUUUUUU-3' (SEQ ID NO: 46) sequence.

70. The method of claim 65, wherein the sgRNA for use with SinCas9 comprises a scaffold comprising at least about 80% identity to 5'-GUUUUUGUACUCUCAAGAUUUGAAAAAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUCAACCCUGUCUAUGACGGGGUGUUUU-3' (SEQ ID NO: 47) sequence.

71. The method of claim 65, wherein the sgRNA for use with SsaCas9 comprises a scaffold comprising at least about 80% identity to 5'-GUUUUAGAGCUGUGUUGUGAAAACACACAGCAAGUUAAAAUAAGGCUUUGUCCGUACACAACUUGAAAAAGUGCGCACCGAUUCGGUGCUUUUUU-3' (SEQ ID NO: 48) sequence.

72. The method of claim 65, wherein the sgRNA for use with Ssc2Cas9 comprises a scaffold comprising at least about 80% identity to 5'-GUUUUUGUACUCUCAAGAUUUGAAAAAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUCAACACCCUGUCAUUUUAUGGCAGGGUGUUU-3' (SEQ ID NO: 49) sequence.

73. The method of claim 65, wherein the sgRNA for use with Sor2Cas9 comprises a scaffold comprising at least about 80% identity to 5'-GUUUUUGUACUCUCAAGAUUUGAAAAAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAUUCAACACCCUGUCAUUUAUGGCGGGGUUUU-3' (SEQ ID NO: 50) sequence.

74. The method of claim 65, wherein the sgRNA for use with SorCas9 comprises a scaffold comprising at least about 80% identity to 5'-GUUUUUGUACUCUCAAGAUUUGAAAAAAUCUUGUAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAUUCAACACCCUGUCAUUUAUGGCGGGGGUUUCGUUU-3' (SEQ ID NO: 51) sequence.

75. The method of claim 65, wherein the sgRNA for use with SwaCas9 comprises a scaffold comprising a 5'- GUUUUAGUACUCUGUAAUUUUGAAAAAAAUUACAGAAUCUACUGAAACAAGACUAUAUGUCGUGUUUAUCCCACUAAUUUAUUAGUGGGAUUUUUU-3' (SEQ ID NO: 52) A sequence having at least about 80% identity.

76. The method of claim 65, wherein the sgRNA for use with SscCas9 comprises a scaffold comprising at least about 80% identity to 5'-GUUUUAGUACUCUGUAAUUUUGAAAAAAAUUACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGACGAGAUUUUUU-3' (SEQ ID NO: 53) sequence.

77. The method of claim 65, wherein the sgRNA for use with SgaCas9 comprises a scaffold comprising at least about 80% identity to 5'-GUUUUUGUACUCUCAAGAUUUGAAAAAAUCUUGCUGAGCCUACAAAGAUAAGGCUUUAUGCCGAAUUCAAGCACCCAUGUUUUGACAUGGGGUGCUUUU-3' (SEQ ID NO: 54) sequence.

78. The method of claim 65, wherein the sgRNA for use with LkuCas9 comprises a scaffold comprising 5'-GUUUUAGAUGAUUGUUAGAUCGAAAGAUCUAACAACCAGAUUUUAAAAUCAAACAAUGUAUCUUUGAUACUAAGUUUCAACGCGGUAUUAUUACCGUCCUGCCUCAGCUCUAUAGCGGAGGUUUUU-3' (SEQ IDNO:55) has a sequence of at least about 80% identity.

79. The method of claim 65, wherein the sgRNA for use with SsuCas9 comprises a scaffold comprising at least about 80% identity to 5'-GUUUUUGUACUCUCAAGAUUUGAAAAAAUCUUGCAGAGCCUACAAAGAUAAGGCUUUAUGCCGAAAUCAAGCACCCGUUUCGUACGGGGUGCUUUU-3' (SEQ ID NO: 56) sequence.

80. The method of claim 64, wherein the crRNA comprises a guide sequence between about 16 nucleotides long and 26 nucleotides long.

81. The method of claim 64, wherein the crRNA comprises a guide sequence between 18 nucleotides long and 24 nucleotides long.

82. The method of claim 58 or 59, wherein the break in the target nucleic acid is a single-stranded or double-stranded break.

83. The method of claim 82, wherein the break in the target nucleic acid is a single-strand break.

84. The method of claim 60 or 61, wherein the Cas9 protein is a nuclease that cleaves both strands of the target nucleic acid sequence, or a nickase that cleaves one strand of the target nucleic acid sequence.

85. The method of any one of claims 58 to 84, wherein the target nucleic acid is 5' to a protospacer adjacent motif (PAM) sequence.

86. The method of any one of claims 58 to 85, wherein the Cas9 is operably linked to a promoter sequence for expression in a eukaryotic cell, and wherein the guide RNA is operably linked to Promoter sequences for expression in eukaryotic cells.

87. The method of claim 86, wherein the eukaryotic cell is a human cell.

88. The method of claim 86, wherein the promoter sequence is a eukaryotic promoter or a viral promoter.

89. An engineered, non-naturally occurring CRISPR-Cas system which include: An RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes a positive sequence repeat sequence and a spacer sequence capable of hybridizing to a target nucleic acid; as well as CRISPR-associated (Cas) protein having at least 80% sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, and wherein The Cas protein is capable of binding to the RNA guide and causing a break in the target nucleic acid sequence that is complementary to the RNA guide.

90. An engineered, non-naturally occurring CRISPR-Cas system which include: An RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide includes a positive sequence repeat sequence and a spacer sequence capable of hybridizing to a target nucleic acid; as well as CRISPR-associated (Cas) protein having at least 80% sequence identity with SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14; wherein the Cas protein is fused to a deaminase, and wherein the Cas protein fusion is capable of binding to the RNA guide and editing the target nucleic acid sequence complementary to the RNA guide.

91. The system of claim 90, wherein the Cas9 protein is inactive Cas9 (dCas9).

92. The system of any one of claims 89 to 91, wherein the RNA guide comprises crRNA and tracrRNA.

93. The system of any one of claims 89 to 92, wherein the RNA guide comprises sgRNA.

94. The system of claim 93, wherein the sgRNA for use with Seq2Cas9 comprises a scaffold comprising at least about 80% identity to 5'-GUUUUAGUACUCUGUAAUUUUGAAAAAAUACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGACGAGAUUUUUU-3' (SEQ ID NO: 43) sequence.

95. The system of claim 93, the sgRNA for use with EhiCas9 comprising a scaffold comprising a sequence that is at least about 80% identical to 5'-GUUUUAGAGCUAUGUUGGAAACAACAUAGCGAGUUAAAAUAAGGCAUUGUCCGUUAUCAGCUUUUAAAGCAAGCACUGUCUCGGUGCUUUUU-3' (SEQ ID NO: 44) .

96. The system of claim 93, wherein the sgRNA for use with SeqCas9 comprises a scaffold comprising at least about 80% identity to 5'-GUUUUUGUACUCUCAAGAUUUGAAAAAAUCUUGCAGAGCCUACAAAGAUAAGGCUUCAUGCCGAAUUCAAGCACCCAUGUUUACAUGGGGUGCUUUU-3' (SEQ ID NO: 45) sequence.

97. The system of claim 93, wherein the sgRNA for use with SsiCas9 comprises a scaffold comprising at least about 80% identity to 5'-GUUUUAGUACUCUGUAAUUUUGAAAAAAAUUACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUUGGCGAGAUUUUUU-3' (SEQ ID NO: 46) sequence.

98. The system of claim 93, wherein the sgRNA for use with SinCas9 comprises a scaffold comprising at least about 80% identity to 5'-GUUUUUGUACUCUCAAGAUUUGAAAAAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUCAACCCUGUCUAUGACGGGGUGUUUU-3' (SEQ ID NO: 47) sequence.

99. The system of claim 93, wherein the sgRNA for use with SsaCas9 comprises a scaffold comprising at least about 80% identity to 5'-GUUUUAGAGCUGUGUUGUGAAAACACACAGCAAGUUAAAAUAAGGCUUUGUCCGUACACAACUUGAAAAAGUGCGCACCGAUUCGGUGCUUUUUU-3' (SEQ ID NO: 48) sequence.

100. The system of claim 93, wherein the sgRNA for use with Sor2Cas9 comprises a scaffold comprising at least about 80% identity to 5'-GUUUUUGUACUCUCAAGAUUUGAAAAAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUCAACACCCUGUCAUUUUAUGGCAGGGUGUUUU-3' (SEQ ID NO: 49) sequence.

101. The system of claim 93, wherein the sgRNA for use with Ssc2Cas9 comprises a scaffold comprising at least about 80% identity to 5'-GUUUUUGUACUCUCAAGAUUUGAAAAAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUCAACACCCUGUCAUUUUAUGGCAGGGUGUUUU-3' (SEQ ID NO: 50) sequence.

102. The system of claim 93, wherein the sgRNA for use with Sor2Cas9 comprises a scaffold comprising at least about 80% identity to 5'-GUUUUUGUACUCUCAAGAUUUGAAAAAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAUUCAACACCCUGUCAUUUAUGGCGGGGUUUU-3' (SEQ ID NO: 51) sequence.

103. The system of claim 93, wherein the sgRNA for use with SwaCas9 comprises a scaffold comprising at least about 80% identity to 5'-GUUUUAGUACUCUGUAAUUUUGAAAAAAAUACAGAAUCUACUGAAACAAGACUAUAUGUCGUGUUUAUCCCACUAAUUUAUUAGUGGGAUUUUU-3' (SEQ ID NO: 52) sequence.

104. The system of claim 93, wherein the sgRNA for use with SscCas9 comprises a scaffold comprising at least about 80% identity to 5'-GUUUUAGUACUCUGUAAUUUUGAAAAAAUACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGACGAGAUUUUUU-3' (SEQ ID NO: 53) sequence.

105. The system of claim 93, wherein the sgRNA for use with SgaCas9 comprises a scaffold comprising at least about 80% identity to 5'-GUUUUUGUACUCUCAAGAUUUGAAAAAAUCUUGCUGAGCCUACAAAGAUAAGGCUUUAUGCCGAAUUCAAGCACCCAUGUUUUGACAUGGGGUGCUUUU-3' (SEQ ID NO: 54) sequence.

106. The system of claim 93, wherein the sgRNA for use with LkuCas9 comprises a scaffold comprising 5'-GUUUUAGAUGAUUGUUAGAUCGAAAGAUCUAACAACCAGAUUUUAAAAUCAAACAAUGUAUCUUUGAUACUAAGUUUCAACGCGGUAUUAUUACCGUCCUGCCUCAGCUCUAUAGCGGAGGUUUUU-3' (SEQ IDNO:55) has a sequence of at least about 80% identity.

107. The system of claim 93, wherein the sgRNA for use with SsuCas9 comprises a scaffold comprising at least about 80% identity to 5'-GUUUUUGUACUCUCAAGAUUUGAAAAAAUCUUGCAGAGCCUACAAAGAUAAGGCUUUAUGCCGAAAUCAAGCACCCCGUUUCGUACGGGGUGCUUUU-3' (SEQ ID NO: 56) sequence.

108. The system of any one of claims 89 to 97, wherein the nucleic acid encoding the Cas protein is operably linked to a promoter sequence for expression in a eukaryotic cell, and wherein the guide RNA can Operably linked to a promoter sequence for expression in eukaryotic cells.

109. The system of claim 108, wherein the eukaryotic cells are human cells.

110. The system of claim 108, wherein the promoter sequence is a eukaryotic promoter sequence.

111. A nucleic acid encoding the system according to any one of claims 89 to 110.

112. A vector comprising a system according to any one of claims 89 to 110.

113. The vector of claim 112, wherein the vector is a plasmid vector or a viral vector.

114. The vector of claim 113, wherein the viral vector is an adeno-associated virus (AAV) vector or a lentiviral vector.

115. The vector of claim 114, wherein the viral vector is an AAV vector.

116. The vector of claim 115, wherein more than one AAV vector is used for packaging the system of claims 89 to 110.

117. A method of treating a disorder or disease in a subject in need thereof, said method comprising administering to said subject a system according to any one of claims 89 to 110. in, The guide RNA is complementary to at least 10 nucleotides of a target nucleic acid associated with a disorder or disease; wherein the Cas protein is associated with the guide RNA; wherein said guide RNA binds to said target nucleic acid; wherein said Cas protein causes a break in said target nucleic acid, optionally wherein said Cas9 is inactive Cas9 fused to a deaminase (dCas9) and results in editing of one or more bases in said target nucleic acid, To treat illness or disease.

118. The method of claim 117, wherein the guide RNA is complementary to about 18 to 24 nucleotides.

119. The method of claim 118, wherein the guide RNA is complementary to 20 nucleotides.

120. A base editor comprising the fusion protein according to any one of claims 37 to 40.

121. The base editor of claim 120, comprising an adenosine deaminase domain or a cytidine deaminase domain.

122. A method of editing a nucleobase of a polynucleotide, the method comprising contacting the polynucleotide with a base editor according to claim 120, the base editor being in contact with one or more a guide RNA complex, wherein the base editor comprises an adenosine deaminase domain, and wherein the one or more guide RNAs target the base editor to achieve A in the polynucleotide ·T to G·C changes.

123. A method of editing a nucleobase of a polynucleotide, the method comprising contacting the polynucleotide with a base editor according to claim 120, the base editor being in contact with one or more a guide RNA complex, wherein the base editor comprises a cytidine deaminase domain, and wherein the one or more guide RNAs target the base editor to achieve C in the polynucleotide ·G to T·A change.

124. The method of claim 122 or 123, wherein the editing results in the formation of less than 50% indels in the target polynucleotide sequence.

125. The method of any one of claims 122 to 124, wherein said editing creates a point mutation.

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