The evolved Cas9 protein for gene editing

Abstract

Several aspects of this disclosure provide strategies, systems, reagents, methods, and kits useful for manipulating Cas9 variants that have increased activity against target sequences that do not contain Cas9 or classical PAM sequences. [Solution] In some embodiments, a fusion protein is provided that includes such a Cas9 variant and a nucleic acid editing domain, such as a deaminase domain.

Description

[Technical Field]

[0001] Related applications This application is related to USSN 62 / 245,828 filed on October 23, 2015, USSN 62 / 279,346 filed on January 15, 2016, USSN 62 / 311,763 filed on March 22, 2016, USSN 62 / 322,178 filed on April 13, 2016, USSN 62 / 357,352 filed on June 30, 2016, USSN 62 / 370,700 filed on August 3, 2016, USSN 62 / 398,490 filed on September 22, 2016, USSN 62 / 408,686 filed on October 14, 2016, and USSN 62 / 357,352 filed on June 30, 2016. Priority is claimed under §119(e) of the U.S. Patent Act to U.S. Provisional Patent Application 62 / 357,332; each of these is incorporated herein by reference. [Background technology]

[0002] Target-specific editing of nucleic acid sequences, such as target-specific cleavage or targeted introduction of specific modifications into genomic DNA, is a highly promising approach for studying gene function and also has the potential to provide new therapies for human genetic diseases. (1) An ideal nucleic acid editing technology possesses three characteristics: (1) high efficiency in implementing the desired modification; (2) minimal off-target activity; and (3) the ability to be programmed to precisely edit any site in a given nucleic acid, for example, any site in the human genome. (2) Current genome manipulation tools use manipulated zinc finger nucleases (ZFNs). (3) , transcription activator-like effector nucleases (TALENs) (4) , and most recently, the RNA-induced DNA endonuclease Cas9 (5)This process involves the inclusion of sequence-specific DNA breaks within the genome. These programmable breaks can result in DNA mutations at the break site via non-homologous end joining (NHEJ), or replacement of surrounding DNA via homologous recombination repair (HDR). (6、7) .

[0003] One drawback of current technologies is that both NHEJ and HDR are stochastic processes, which typically result in modest gene editing efficiency and unwanted gene modifications that may compete with the desired modifications. (8) In principle, many genetic disorders can be treated by causing specific nucleotide changes at specific locations in the genome (for example, a C-to-T change at a specific codon in a disease-related gene). (9) The development of programmable methods to achieve such high-precision gene editing would represent a powerful new research tool and potentially a new approach to gene-editing-based human therapy.

[0004] Another drawback of current genome engineering tools is that they are limited to the DNA sequences they can target. When using ZNFs or TALENS, a novel protein must be generated for each individual target sequence. While Cas9 can target virtually any target sequence by providing a suitable guide RNA, the strict requirement of a protospacer flanking motif (PAM), typically the nucleotide sequence 5'-NGG-3', located adjacent to the 3' end of the target DNA sequence to which the Cas9 protein binds and acts on the target sequence, still limits the range of sequences that can be targeted. Thus, the PAM requirement limits the sequences that can be efficiently targeted by the Cas9 protein. [Overview of the Initiative]

[0005] Importantly, 80–90 percent of protein mutations involved in human disease result from single nucleotide substitutions, deletions, or insertions.(6) 。Most current strategies for single - nucleotide gene editing involve engineered nucleases (creation of double - strand breaks (DSBs), followed by stochastic, inefficient homology - directed repair (HDR)) and DNA - RNA chimeric oligonucleotides. (22) 。The latter strategy involves design of the RNA / DNA base sequence for base pairs in genomic DNA that have specific sequences other than the nucleotide to be edited. The resulting inappropriate combinations are recognized and repaired by the endogenous repair systems in the cell, leading to changes in chimeric or genomic sequences. All of these strategies have the drawbacks of low gene - editing efficiency and unwanted gene changes, which are due to both the stochastic dependence of HDR and to both HDR and non - homologous end - joining (NHEJ). (23~25) 。The HDR efficiency varies depending on the position of the target gene within the genome (26) 、the state of the cell cycle (27) 、as well as the cell / tissue type. (28) 。The development of a direct, programmable, and non - stochastic method for introducing specific types of base modifications at precise sites within genomic DNA with enzymatic - like efficiency would be a powerful and novel approach for gene - editing - based research tools and human therapy.

[0006] The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system is a recently discovered prokaryotic adaptive immune system (10) that has been modified to enable robust and general genome engineering in a variety of organisms and cell lines. (11) 。The CRISPR - Cas (CRISPR - associated) system is a protein - RNA complex that uses an RNA molecule (sgRNA) as a guide to localize the complex to the target DNA sequence by base pairing. (12) 。Then, in the native system, the Cas protein functions as an endonuclease to cleave the target DNA sequence. (13)For the system to function, the target DNA sequence must be complementary to the sgRNA and also contain a "protospacer adjacent motif" (PAM) at the 3' end of the complementary region. (14) .

[0007] In the present invention, a novel Cas9 variant is provided that exhibits activity against target sequences that do not contain a classical PAM sequence (5'-NGG-3', where N is any nucleotide) at the 3'-end. Such Cas9 variants are not limited to target sequences containing a classical PAM sequence at the 3'-end.

[0008] Among known Cas proteins, Streptococcus pyogenes Cas9 has been mainly widely used as a tool for genome engineering. (15) . This Cas9 protein is a large multi-domain protein containing two separate nuclease domains. Point mutations can be introduced into Cas9 to abolish nuclease activity, resulting in an inactive Cas9 (dCas9) that still retains its ability to bind to DNA in a manner programmed by sgRNA. (16) . In principle, such Cas9 variants can target the protein to substantially any DNA sequence simply by co-expression with an appropriate sgRNA when fused to another protein or domain. Thus, the disclosure of the present invention also includes such Cas9 variants and fusion proteins comprising a DNA repair domain (e.g., deaminase, nuclease, nickase, recombinase, methyltransferase, methylase, acetylase, acetyltransferase, transcription activator or transcription repressor domain), and the use of such fusion proteins to correct mutations associated with diseases within the genome (e.g., the genome of a human subject), or to generate mutations within the genome (e.g., the human genome) to reduce or prevent gene expression.

[0009] In some embodiments, any of the Cas9 proteins provided in the present invention can be fused with a protein having enzymatic activity. In some embodiments, the enzymatic activity modifies target DNA. In some embodiments, the enzymatic activity is nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurine activity, oxidation activity, pyrimidine dimerization activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, or glycosylase activity. In some embodiments, the enzymatic activity is nuclease activity. In some embodiments, the nuclease activity cleaves the double strand of target DNA. In some embodiments, the enzymatic activity modifies target polypeptide associated with target DNA. In some embodiments, the enzyme activity is methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity. In some embodiments, the target polypeptide is a histone, and the enzyme activity is methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, or deubiquitination activity.

[0010] In some embodiments, any of the Cas9 proteins provided in the present invention can be fused with a protein having enzymatic activity. In some embodiments, the enzymatic activity modifies polypeptides related to DNA (e.g., histones). In some embodiments, the enzymatic activity is methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity (i.e., ubiquitinating activity), deubiquitinating activity, adenylating activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, non-myristoylation activity, glycosylation activity (e.g., from O-GlcNAc transferase), or deglycosylation activity. The enzymatic activities listed herein catalyze covalent modifications to proteins. Such modifications to alter the stability or activity of target proteins are known techniques (e.g., phosphorylation by kinase activity can stimulate or quench protein activity depending on the target protein). Histones are particularly well-known as target proteins. Histone proteins are known in the prior art as binding to DNA and forming complexes known as nucleosomes. Modifying histones (e.g., by methylation, acetylation, ubiquitination, or phosphorylation) alters the structure of the surrounding DNA, thereby controlling the accessibility of potentially large portions of DNA to interacting factors (e.g., transcription factors, polymerases, etc.). A single histone can be modified in various ways and in various combinations (e.g., trimethylation of lysine 27 (H3K27) of histone 3 is involved in transcriptional repression of a DNA region, and trimethylation of lysine 4 (H3K4) of histone 3 is involved in active transcription of a DNA region). Thus, site-directed modification polypeptides with histone modification activity offer use for site-directed control of DNA structure and can be used to alter the histone modification pattern of selected regions of target DNA. Such methods can be used for research and clinical applications.

[0011] In some embodiments, the deaminase domain catalyzes the removal of amine groups within a molecule. In further embodiments, the cytidine deaminase domain deaminates cytosine to produce uracil. In other embodiments, the nuclease domain has enzymatic activity and can cleave phosphodiester bonds between nucleotide subunits of nucleic acids. In some embodiments, recombinase domains that rejoin specific sequences of DNA can be used to manipulate genomic structures and control gene expression. In further embodiments, methylase domains can be used for methylation of their respective substrates, while acetylase domains can be used for acetylation of their respective substrates. In other embodiments, acetyltransferase domains can be used for the transfer of acetyl groups. Examples of acetyltransferase molecules include, but are not limited to, histone acetyltransferases (e.g., CBP histone acetyltransferase), choline acetyltransferase, chloramphenicol acetyltransferase, serotonin N-acetyltransferase, NatA acetyltransferase, and NatB acetyltransferase. The disclosure of the present invention also encompasses transcriptional activator and transcriptional repressor domains. A transcriptional activator domain is a region of a transcription factor that can activate transcription from a promoter through one or more interactions with a DNA-binding domain, a common transcription factor, and RNA polymerase. A transcriptional repressor domain is a region of a transcription factor that can repress transcription from a promoter through one or more interactions with a DNA-binding domain, a common transcription factor, and RNA polymerase.

[0012] The Cas9 system has immense potential for genome manipulation. Its unique ability to deliver proteins to specific sites in the genome programmed by sgRNAs can be developed into a wide range of site-specific genome manipulation tools beyond nucleases, including transcription activators, transcriptional repressors, histone-modifying proteins, integrases, deaminases, and recombinases.(11) Some of their potential applications have recently been implemented by dCas9 fusions with transcription activators, and RNA-induced transcription activators. (17、18) , transfer repressor (16、19、20) , and provided chromatin modifying enzymes. (21) Simple co-expression of these fusions with various sgRNAs results in the specific expression of target genes. These pioneering studies paved the way for the design and construction of easily programmable sequence-specific effectors for precise genome manipulation.

[0013] Some aspects of the disclosure of the present invention provide strategies, systems, proteins, nucleic acids, compositions, cells, reagents, methods, and kits useful for target-specific binding, editing, and / or cleavage of nucleic acids, such as editing a single site within a subject's genome (e.g., the genome of a human subject). In some aspects, recombinant Cas9 proteins, compared to naturally occurring Cas9 proteins, contain 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 ten mutations and exhibit activity against target sequences that do not contain the 3'-terminal classical PAM (5'-NGG-3', where N is any nucleotide). Examples of such Cas9 protein mutations are shown in Tables 3, 5, 8, and 9. In some aspects, fusion proteins of Cas9 with nucleic acid editing enzymes or enzymatic domains (e.g., deaminase domains) are provided. In some aspects, methods for binding, editing, and / or cleavage of target-specific nucleic acids are provided. In some embodiments, reagents and kits are provided for generating target-specific nucleic acid binding, editing, and / or cleavage proteins (e.g., fusion proteins of Cas9 variants with nucleic acid editing enzymes or domains).

[0014] Some aspects of the disclosure of the present invention provide recombinant Cas9 proteins comprising an amino acid sequence that is at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent or at least 99.5 percent identical to the amino acid sequence of Cas9 represented by any of SEQ ID NOs: 9 to 262, wherein 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 or at least seven mutations of amino acid residues selected from the group consisting of amino acid residues 122, 137, 182, 262, 294, 409, 480, 543, 660, 694, 1219 and 1329 of Cas9 of S. pyogenes having the amino acid sequence shown in SEQ ID NOs: 9, or the corresponding amino acid residues of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262. In some embodiments, the recombinant Cas9 protein includes RuvC and HNH domains. In some embodiments, the amino acid sequence of the recombinant Cas9 protein is not identical to the amino acid sequence of the naturally occurring Cas9 protein. In some embodiments, the amino acid sequence of the Cas9 protein includes at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations selected from the group consisting of X262T, X294R, X409I, X480K, X543D, X694I, and X1219V of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10-262, where X represents any of the amino acids. In some embodiments, the amino acid sequence of the Cas9 protein includes at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations selected from the group consisting of A262T, K294R, S409I, E480K, E543D, M694I, and E1219V of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

[0015] Another aspect of the disclosure of the present invention provides a recombinant Cas9 protein comprising an amino acid sequence that is at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent or at least 99.5 percent identical to the amino acid sequence of Cas9 represented by any of SEQ ID NOs. 10 to 262, wherein 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 or at least seven mutations of amino acid residues selected from the group consisting of amino acid residues 262, 267, 294, 405, 409, 480, 543, 694, 1219, 1224, 1256 and 1362 of the amino acid sequence represented by SEQ ID NOs. 9, or the corresponding amino acid residues of any of the amino acid sequences represented by SEQ ID NOs. 10 to 262, and the amino acid sequence of the recombinant Cas9 protein is not identical to the amino acid sequence of the naturally occurring Cas9 protein. In some embodiments, the Cas9 protein includes RuvC and HNH domains. In some embodiments, the amino acid sequence of the Cas9 protein includes at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations selected from the group consisting of X262T, X267G, X294R, X405I, X409I, X480K, X543D, X694I, X1219V, X1224K, and X1256K of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids. In some embodiments, the amino acid sequence of the Cas9 protein includes at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations selected from the group consisting of A262T, S267G, K294R, F405I, S409I, E480K, E543D, M694I, E1219V, N1224K, and Q1256K of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

[0016] The amino acid mutations described herein, such as an amino acid mutation from a first amino acid residue (e.g., A) to a second amino acid residue (e.g., T) (e.g., A262T), should be understood to include (conservative) mutations from the first amino acid residue to an amino acid residue similar to the second amino acid residue. For example, a mutation of alanine to threonine (e.g., the A262T mutation) may also be a mutation from alanine to an amino acid (e.g., serine) that is similar to threonine in terms of size and chemical properties. Further similar amino acid pairs include, but are not limited to, phenylalanine and tyrosine, asparagine and glutamine, methionine and cysteine, aspartic acid and glutamic acid, and arginine and lysine. Those skilled in the art will recognize that such conservative amino acid substitutions likely have only minor effects on protein structure and do not result in loss of function. In some embodiments, any amino acid mutation from an amino acid to threonine provided in the present invention may also be an amino acid mutation to serine. In some embodiments, any amino acid mutation from an amino acid to arginine provided in the present invention may also be an amino acid mutation to lysine. In some embodiments, any amino acid mutation from an amino acid to isoleucine provided in the present invention may also be an amino acid mutation to alanine, valine, methionine, or leucine. In some embodiments, any amino acid mutation from an amino acid to lysine provided in the present invention may also be an amino acid mutation to arginine. In some embodiments, any amino acid mutation from an amino acid to aspartic acid provided in the present invention may also be an amino acid mutation to glutamic acid or asparagine. In some embodiments, any amino acid mutation from an amino acid to valine provided in the present invention may also be an amino acid mutation to alanine, isoleucine, methionine, or leucine. In some embodiments, any amino acid mutation from an amino acid to glycine provided in the present invention may also be an amino acid mutation to alanine.However, it should be recognized that further conserved amino acid residues are known to those skilled in the art, and that any amino acid mutation to other conserved amino acid residues falls within the scope of the disclosure of this invention.

[0017] In some embodiments, the Cas9 protein is the Cas9 domain of a fusion protein. In some embodiments, the amino acid sequence of the Cas9 protein includes the X1219V mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10-262, where X represents any of the amino acids. In some embodiments, the mutation is X1219A, X1219I, X1219M, or X1219L.

[0018] In some embodiments, the amino acid sequence of the Cas9 protein includes the E1219V mutation in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in one of the amino acid sequences shown in SEQ ID NOs: 10-262. In some embodiments, the mutation is E1219A, E1219I, E1219M, or E1219L.

[0019] In some embodiments, the amino acid sequence of the Cas9 protein includes the X480K mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10-262, where X represents any of the amino acids. In some embodiments, the mutation is X480R.

[0020] In some embodiments, the amino acid sequence of the Cas9 protein includes the E480K mutation in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation in either of the amino acid sequences shown in SEQ ID NOs: 10-262. In some embodiments, the mutation is E480R.

[0021] In some embodiments, the amino acid sequence of the Cas9 protein includes the X543D mutation in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10-262, where X represents any of the amino acids. In some embodiments, the mutation is X543N.

[0022] In some embodiments, the amino acid sequence of the Cas9 protein includes the E543D mutation in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation in either of the amino acid sequences shown in SEQ ID NOs: 10-262. In some embodiments, the mutation is E543N.

[0023] In some embodiments, the amino acid sequence of the Cas9 protein includes mutations X480K, X543D, and X1219V of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

[0024] In some embodiments, the amino acid sequence of the Cas9 protein comprises the X262T, X409I, X480K, X543D, X694I, and X1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10-262, where X represents any of the amino acids.

[0025] In some embodiments, the amino acid sequence of the Cas9 protein comprises the X294R, X480K, X543D, X1219V, X1256K, and X1362P mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10-262, where X represents any of the amino acids.

[0026] In some embodiments, the amino acid sequence of the Cas9 protein includes the X294R, X480K, X543D, X1219V, and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10-262, where X represents any of the amino acids.

[0027] In some embodiments, the amino acid sequence of the Cas9 protein includes the X267G, X294R, X480K, X543D, X1219V, X1224K, and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10-262, where X represents any of the amino acids.

[0028] In some embodiments, the amino acid sequence of the Cas9 protein comprises the X262T, X405I, X409I, X480K, X543D, X694I, and X1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10-262, where X represents any of the amino acids.

[0029] In some embodiments, the amino acid sequence of the Cas9 protein includes mutations E480K, E543D, and E1219V in the amino acid sequence shown in SEQ ID NO: 9, or corresponding mutations in any of the amino acid sequences shown in SEQ ID NOs: 10-262.

[0030] In some embodiments, the amino acid sequence of the Cas9 protein includes the A262T, S409I, E480K, E543D, M694I, and E1219V mutations in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations in any of the amino acid sequences shown in SEQ ID NOs: 10-262.

[0031] In some embodiments, the amino acid sequence of the Cas9 protein includes the K294R, E480K, E543D, E1219V, Q1256K, and L1362P mutations in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations in any of the amino acid sequences shown in SEQ ID NOs: 10-262.

[0032] In some embodiments, the amino acid sequence of the Cas9 protein includes the K294R, E480K, E543D, E1219V, and Q1256K mutations in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations in any of the amino acid sequences shown in SEQ ID NOs: 10-262.

[0033] In some embodiments, the amino acid sequence of the Cas9 protein includes the S267G, K294R, E480K, E543D, E1219V, N1224K, and Q1256K mutations in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations in any of the amino acid sequences shown in SEQ ID NOs: 10-262.

[0034] In some embodiments, the amino acid sequence of the Cas9 protein includes the A262T, F405I, S409I, E480K, E543D, M694I, and E1219V mutations in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations in any of the amino acid sequences shown in SEQ ID NOs: 10-262.

[0035] The HNH nuclease domain of Cas9 functions to cleave a DNA strand complementary to the guide RNA (gRNA). Its active site consists of a ββα metal fold, and its histidine 840 activates a water molecule to attack a cleavable phosphate ester, which is electrophilic due to the coordination of a magnesium ion, resulting in the cleavage of the 3'-5' phosphate bond. In some embodiments, the amino acid sequence of the HNH domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of any of the HNH domains of SEQ ID NOs. 9-262. In some embodiments, the amino acid sequence of the HNH domain is identical to the amino acid sequence of any of the HNH domains of SEQ ID NOs. 9-262.

[0036] The RuvC domain of Cas9 cleaves non-target DNA strands. It is encoded at sequence-isolated sites but interacts tertiarily to form the RuvC cleavage domain, which also consists of an RNaseH folded structure. In some embodiments, the amino acid sequence of the RuvC domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of any of the RuvC domains of SEQ ID NOs. 9-262. In some embodiments, the amino acid sequence of the RuvC domain is identical to the amino acid sequence of the RuvC domain of SEQ ID NOs. 9-262.

[0037] In some embodiments, the Cas9 protein contains one or more mutations that affect (e.g., inhibit) Cas9's ability to cleave one or both strands of a DNA double helix. In some embodiments, the Cas9 protein contains the D10A and / or H840A mutation in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10-262. In some embodiments, the Cas9 protein contains the D10X1 and / or H840X2 mutation in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10-262, where X1 is any amino acid other than D and X2 is any amino acid other than H. In some embodiments, the Cas9 protein contains the D10A mutation in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10-262. In some embodiments, the Cas9 protein contains H at amino acid residue 840 of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding residue in any of the amino acid sequences shown in SEQ ID NOs: 10-262. In some embodiments, the Cas9 protein contains the H840A mutation in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation in either of the amino acid sequences shown in SEQ ID NOs: 10-262. In some embodiments, the Cas9 protein contains D at amino acid residue 10 of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding residue in either of the amino acid sequences shown in SEQ ID NOs: 10-262.

[0038] In some embodiments, the Cas9 protein disclosed in the present invention exhibits increased activity (e.g., binding activity) to target sequences that do not contain the classical PAM (5'-NGG-3') at the 3' end, compared to Cas9 of Streptococcus pyogenes shown in SEQ ID NO: 9.

[0039] Some aspects of the disclosure of the present invention provide a recombinant Cas9 protein in which the amino acid sequence of the Cas9 protein is at least 90 percent identical to the amino acid sequence of Cas9 of Streptococcus pyogenes shown in SEQ ID NO: 9, and the amino acid sequence of the Cas9 protein contains at least one, at least two, at least three, at least four, at least five, at least six or at least seven mutations selected from the group consisting of amino acid residues 262, 267, 294, 405, 409, 480, 543, 694, 1219, 1224, 1256 and 1362 of the amino acid sequence shown in SEQ ID NO: 9, and is not identical to the amino acid sequence of the naturally occurring Cas9 protein, and exhibits increased activity against a target sequence that does not contain the classical PAM(5'-NGG-3') at the 3' end compared to Cas9 of Streptococcus pyogenes shown in SEQ ID NO: 9. In some embodiments, Cas9 of Streptococcus pyogenes contains RuvC and HNH domains. In another embodiment, the Cas9 protein contains at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations selected from the group consisting of amino acid residues 122, 137, 182, 262, 294, 409, 480, 543, 660, 694, 1219, and 1329 of the amino acid sequence shown in SEQ ID NO: 9.

[0040] As an example, the Cas9 protein may exhibit enhanced binding to a target sequence, enhanced nuclease activity to a target sequence, or other enhanced activity, depending on whether the Cas9 protein fuses with a further domain such as an enzyme having enzymatic activity. In some embodiments, the enzymatic activity modifies the target DNA. In some embodiments, the enzymatic activity is nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurine activity, oxidation activity, pyrimidine dimerization activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, or glycosylase activity. In some embodiments, the enzymatic activity is nuclease activity. In some embodiments, the nuclease activity cleaves the double strand of the target DNA. In some embodiments, the enzymatic activity modifies the target polypeptide associated with the target DNA. In some embodiments, the enzyme activity is methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity. In some embodiments, the target polypeptide is a histone, and the enzyme activity is methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, or deubiquitination activity.

[0041] In some embodiments, any of the Cas9 proteins fuse with a protein having enzymatic activity. In some embodiments, the enzymatic activity modifies polypeptides related to DNA (e.g., histones). In some embodiments, the enzymatic activity is methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity (i.e., ubiquitinating activity), deubiquitinating activity, adenylating activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, non-myristoylation activity, glycosylation activity (e.g., from O-GlcNAc transferase), or deglycosylation activity. The enzymatic activities listed herein catalyze covalent modifications to proteins. Such modifications to alter the stability or activity of target proteins are known techniques (e.g., phosphorylation by kinase activity can stimulate or quench protein activity depending on the target protein). Histones are particularly well-known as target proteins. Histone proteins are known in the prior art as binding to DNA and forming complexes known as nucleosomes. Modifying histones (e.g., by methylation, acetylation, ubiquitination, or phosphorylation) alters the structure of the surrounding DNA, thereby controlling the accessibility of potentially large portions of DNA to interacting factors (e.g., transcription factors, polymerases, etc.). A single histone can be modified in various ways and in various combinations (e.g., trimethylation of lysine 27 (H3K27) of histone 3 is involved in transcriptional repression of a DNA region, and trimethylation of lysine 4 (H3K4) of histone 3 is involved in active transcription of a DNA region). Thus, site-directed modification polypeptides with histone modification activity offer use for site-directed control of DNA structure and can be used to alter the histone modification pattern of selected regions of target DNA. Such methods can be used for research and clinical applications.

[0042] In some embodiments, the Cas9 protein exhibits activity against target sequences having a 3' end that is not directly adjacent to or does not have a classical PAM sequence (5'-NGG-3'), and this activity is increased by at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 50,000-fold, at least 100,000-fold, at least 500,000-fold, or at least 1,000,000-fold compared to the activity of Streptococcus pyogenes Cas9 shown in SEQ ID NO: 9 against the same target sequences.

[0043] In some embodiments, the 3' end of the target sequence is directly adjacent to AGC, GAG, TTT, GTG, CAA, CAC, GAT, TAA, ACG, CGA, or CGT.

[0044] In some embodiments, Cas9 protein activity is measured by nuclease assays or nucleic acid binding assays, which are well known in the prior art and will be apparent to those skilled in the art. As shown herein, the Cas9 protein can be fused with one or more domains that confer activity to the protein, such as nucleic acid editing activity (e.g., deaminase activity or transcriptional activation activity), and such activity can be measured, for example, by a deaminase assay or transcriptional activation assay. In some embodiments, the Cas9 protein can be fused with a deaminase domain, and its activity can be measured using a deaminase assay. In some embodiments, the Cas9 protein can be fused with a transcriptional activation domain, and its activity can be measured using a transcriptional activation assay (e.g., a reporter activation assay), in which the reporter (e.g., particularly GFP or luciferase) is expressed in response to the binding of Cas9 to a target sequence.

[0045] In some embodiments, the amino acid sequence of the Cas9 protein includes any of the mutations provided herein. In some embodiments, the amino acid sequence of the Cas9 protein includes at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations selected from the group consisting of X262T, X267G, X294R, X405I, X409I, X480K, X543D, X694I, X1219V, N1224K, X1224K, and X1362P of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids. In other embodiments, the mutation may be A262T, S267G, K294R, F405I, S409I, E480K, E543D, M694I, E1219V, N1224K, Q1256K, and L1362P of the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

[0046] In some embodiments, the amino acid sequence of the Cas9 protein includes any of the mutations provided herein. In some embodiments, the amino acid sequence of the Cas9 protein includes at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations selected from the group consisting of X262T, X294R, X409I, X480K, X543D, X694I, and X1219V of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10-262, where X represents any of the amino acids. In other embodiments, the mutations may be A262T, K294R, S409I, E480K, E543D, M694I, or E1219V of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10-262.

[0047] In some embodiments, the amino acid sequence of the Cas9 protein comprises the X1219V mutation or the E1219V mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10-262, where X represents any of the amino acids.

[0048] In some embodiments, the amino acid sequence of the Cas9 protein includes the X480K mutation or the E480K mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10-262, where X represents any of the amino acids.

[0049] In some embodiments, the amino acid sequence of the Cas9 protein comprises the X543D mutation or the E543D mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10-262, where X represents any of the amino acids.

[0050] In some embodiments, the amino acid sequence of Cas9 includes mutations X480K, X543D and X1219V, or mutations E480K, E543D and E1219V of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

[0051] In some embodiments, the amino acid sequence of Cas9 includes mutations X262T, X409I, X480K, X543D, X694I, and X1219V, or mutations A262T, S409I, E480K, E543D, M694I, and E1219V of the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

[0052] In some embodiments, the amino acid sequence of the Cas9 protein includes mutations X294R, X480K, X543D, X1219V, X1256K and X1362P, or mutations K294R, E480K, E543D, E1219V, Q1256K and L1362P of the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10-262, where X represents any of the amino acids.

[0053] In some embodiments, the amino acid sequence of the Cas9 protein includes mutations X294R, X480K, X543D, X1219V and X1256K of the amino acid sequence shown in SEQ ID NO: 9, or mutations K294R, E480K, E543D, E1219V and Q1256K, or corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

[0054] In some embodiments, the amino acid sequence of the Cas9 protein includes mutations X267G, X294R, X480K, X543D, X1219V, X1224K and X1256K, or mutations S267G, K294R, E480K, E543DE1219V, N1224K and Q1256K of the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

[0055] In some embodiments, the amino acid sequence of the Cas9 protein includes mutations X262T, X405I, X409I, X480K, X543D, X694I and X1219V, or mutations A262T, F405I, S409I, E480K, E543D, M694I and E1219V of the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

[0056] In some embodiments, the amino acid sequence of the HNH domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of any of the HNH domains of sequence numbers 9 to 262. In some embodiments, the amino acid sequence of the HNH domain is identical to the amino acid sequence of any of the HNH domains of sequence numbers 9 to 262.

[0057] In some embodiments, the amino acid sequence of the RuvC domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of any of the RuvC domains of SEQ ID NOs. 9 to 262.

[0058] In some embodiments, the Cas9 protein contains the D10A and / or H840A mutation in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10-262. In some embodiments, the Cas9 protein contains the D10X1 and / or H840X2 mutation in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10-262, where X1 is any amino acid other than D and X2 is any amino acid other than H. In some embodiments, the Cas9 protein contains the D10A mutation in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10-262. In some embodiments, the Cas9 protein contains H at amino acid residue 840 of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding residue in any of the amino acid sequences shown in SEQ ID NOs: 10-262. In some embodiments, the Cas9 protein contains the H840A mutation in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10-262. In some embodiments, the Cas9 protein contains D at amino acid residue 10 of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding residue in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

[0059] Some aspects of the disclosure of the present invention provide a fusion protein comprising the Cas9 protein provided herein, which forms a fusion protein by fusing with a second protein. In some embodiments, the second protein is fused to the N-terminus of the Cas9 protein. In some embodiments, the second protein is fused to the C-terminus of the Cas9 protein. In some embodiments, the Cas9 domain and the effector domain are fused via a linker. The linker may be a single covalent bond or a long linker polymerized of many atoms. In certain embodiments, the linker is polypeptide or amino acid-based. In other embodiments, the linker is not peptide-like. In certain embodiments, the linker is a covalent bond (e.g., carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.). In certain embodiments, the linker is a carbon-nitrogen bond via an amide bond. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or linear aliphatic or heteroaliphatic linker. In certain embodiments, the linker is a polymer (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of an aminoalkanoic acid. In certain embodiments, the linker comprises an aminoalkanoic acid (e.g., glycine, ethaneic acid, alanine, β-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of an aminohexanoic acid (Ahx). In certain embodiments, the linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In other embodiments, the linker comprises a polyethylene glycol moiety (PEG). In other embodiments, the linker comprises an amino acid. In certain embodiments, the linker comprises a peptide. In certain embodiments, the linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. The linker may also contain a functionalized moiety that promotes the binding of peptide-derived nucleophiles (e.g., thiols, amino groups) to the linker.Any of the electrophiles may be used as part of the linker. Examples of electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates.

[0060] In some embodiments, the linker comprises a chemical group or molecule that links two molecules or parts, for example, a fusion protein (e.g., two domains of a nuclease-inactive Cas9 domain and an effector domain (e.g., a deaminase domain)). In some embodiments, the linker comprises one or more amino acid residues. For example, the linker may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 25, 30, 35, 40, 45, 50 or more amino acid residues. In some embodiments, the linker is 3, 9, 16, or 21 amino acid long. In some embodiments, the linker is (GGGGS) n (Sequence ID 5)(G) n , (EAAAK) n (Sequence No. 6), (GGS) n , SGSETPGTSESATPES (Sequence ID 7) (also known as XTEN) or (XP) n The linker includes the motif, or any combination thereof, where n is an independent integer from 1 to 30. In some embodiments, the linker includes the (GGS)3 motif or the SGSETPGTSESATPES(SEQ ID NO: 7)(XTEN) motif.

[0061] Some aspects of the disclosure of the present invention provide a fusion protein comprising the Cas9 protein provided in the present invention, which forms a fusion protein by fusing with a second protein. In some embodiments, the second protein is fused to the N-terminus of the Cas9 protein. In some embodiments, the second protein is fused to the C-terminus of the Cas9 protein. In some embodiments, the Cas9 domain and the effector domain are fused via a nuclear localization sequence (NLS) (for example, an NLS comprising the amino acid sequence PKKKRKV (SEQ ID NO: 299), MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 300), or SPKKKRKVEAS (SEQ ID NO: 284)). In some embodiments, the NLS can be bound to any of the above linkers.

[0062] In some embodiments, the effector domain comprises an enzyme domain. In some embodiments, the effector domain comprises a nuclease, nickase, recombinase, deaminase, methyltransferase, methylase, acetylase, acetyltransferase, transcription activator, or transcriptional repressor domain, which may each have nuclease activity, nickase activity, recombinase activity, deaminase activity, methyltransferase activity, methylase activity, acetylase activity, acetyltransferase activity, transcription activator activity, or transcriptional repressor activity. In some embodiments, the effector domain is an effector domain. In some embodiments, the effector domain is a deaminase domain. In some embodiments, the deaminase is a cytosine deaminase or a cytidine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. In some embodiments, the deaminase is an APOBEC1 deaminase. In some aspects, the deaminase is APOBEC2 deaminase. In some aspects, the deaminase is APOBEC3 deaminase. In some aspects, the deaminase is APOBEC3A deaminase. In some aspects, the deaminase is APOBEC3D deaminase. In some aspects, the deaminase is APOBEC3E deaminase. In some aspects, the deaminase is APOBEC3F deaminase. In some aspects, the deaminase is APOBEC3G deaminase. In some aspects, the deaminase is APOBEC3H deaminase. In some aspects, the deaminase is APOBEC4 deaminase. In some aspects, the deaminase is activation-inducing deaminase (AID).In some embodiments, the effector domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with any one deaminase domain of sequence numbers 263-281. In some embodiments, the deaminase is a cytidine deaminase. In some embodiments, the deaminase is a deaminase of the apolipoprotein B messenger RNA-editing complex (APOBEC) family. In some embodiments, the deaminase is a deaminase of the APOBEC1 family. In some embodiments, the deaminase is an activation-inducible cytidine deaminase (AID). In some embodiments, the deaminase is an ACF1 / ASE deaminase. In some embodiments, the deaminase is an adenosine deaminase. In some embodiments, the deaminase is an ADAT lineage deaminase.

[0063] Some aspects of the disclosure of this invention provide fusion proteins comprising a Cas9 protein fused to an effector domain, such as a deaminase and a uracilglycosylase inhibitor (UGI). Some aspects of this disclosure are based on the finding that such fusion proteins exhibit increased nucleic acid editing efficiency compared to fusion proteins that do not contain a UGI domain. Domains such as the deaminase domain and the UGI domain have been previously reported and are within the scope of this disclosure. For example, domains such as the deaminase domain and the UGI domain are described in U.S. Provisional Patent Application No. 62 / 245,828 (filed October 23, 2015), U.S. Provisional Patent Application No. 62 / 279,346 (filed January 15, 2016), U.S. Provisional Patent Application No. 62 / 311,763 (filed March 22, 2016), U.S. Provisional Patent Application No. 62 / 322,178 (filed April 13, 2016), The details are described in U.S. Provisional Patent Application No. 62 / 357,352 (filed June 30, 2016), U.S. Provisional Patent Application No. 62 / 370,700 (filed August 3, 2016), U.S. Provisional Patent Application No. 62 / 398,490 (filed September 22, 2016), and U.S. Provisional Patent Application No. 62 / 408,686 (filed October 14, 2016), the full disclosures of which are incorporated herein by reference. It is naturally understood that the deaminase domains and UGI domains described in the above referenced documents may be fused with any of the Cas9 proteins provided in the present invention within the scope of the disclosures of the present invention.

[0064] In some embodiments, the effector domain of the fusion protein is a nuclease domain. In some embodiments, the nuclease domain is a FokI DNA cleavage domain. In some embodiments, the fusion protein dimerizes. In certain embodiments, the dimer of the fusion protein is active. For example, two FokI DNA cleavage domains can be dimerized to cleave nucleic acids.

[0065] In some embodiments, the Cas9 protein fuses with a second Cas9 protein. In some embodiments, the second Cas9 protein is the Cas9 protein described in any one of claims 1 to 345. In some embodiments, the second Cas9 protein fuses to the N-terminus of the fusion protein. In some embodiments, the second Cas9 protein fuses to the C-terminus of the fusion protein. In some embodiments, the Cas9 protein and the second Cas9 protein fuse via a second linker. In some embodiments, the second linker is (GGGGS) n (Sequence ID 5), (G) n , (EAAAK) n (Sequence No. 6), (GGS) n , SGSETPGTSESATPES (sequence number 7), or (XP) n The second linker includes the motif of (GGS)3, or any combination thereof, where n is an independent integer between 1 and 30. In some embodiments, the second linker includes the motif of (GGS)3.

[0066] Some aspects of this disclosure provide a complex comprising a Cas9 protein or Cas9 fusion protein and a guide RNA bound to the Cas9 protein or Cas9 fusion protein, as provided herein.

[0067] In some embodiments, the target sequence is a DNA base sequence. In some embodiments, the target sequence is a sequence in the mammalian genome. In some embodiments, the target sequence is a sequence in the human genome. In some embodiments, the 3' end of the target sequence is not directly adjacent to the classical PAM sequence (5'-NGG-3').

[0068] Several aspects of this disclosure provide methods using Cas9 proteins, fusion proteins, or complexes provided herein. For example, several aspects of this disclosure provide methods comprising contacting a DNA molecule with a Cas9 protein, Cas9 fusion protein, or Cas9 protein or fusion protein complex (a) with a Cas9 protein or fusion protein and guide RNA provided herein; or (b) with a gRNA provided herein, wherein the guide RNA is approximately 15 to 100 nucleotides long and contains a sequence of at least 10 consecutive nucleotides complementary to the target sequence. In some embodiments, the 3' end of the target sequence is not directly adjacent to a classical PAM sequence (5'-NGG-3'). In some embodiments, the 3' end of the target sequence is directly adjacent to an AGC, GAG, TTT, GTG, or CAA sequence. In some embodiments, the target DNA sequence contains a sequence associated with a disease or disorder. In some embodiments, the target DNA sequence contains a point mutation associated with a disease or disorder. In some embodiments, the activity of the Cas9 protein, Cas9 fusion protein, or complex results in the modification of the point mutation. In some embodiments, the subject is subjected to an in vivo contact step.

[0069] Some aspects of this disclosure provide a kit comprising a nucleic acid construct comprising (a) a nucleotide sequence encoding a Cas9 protein or Cas9 fusion protein provided herein, and (b) a heterologous promoter that drives the expression of the sequence in (a). In some embodiments, the kit further comprises an expression construct encoding a guide RNA backbone, the construct comprising a cloning site positioned to allow the cloning of a nucleic acid sequence identical or complementary to the target sequence into the guide RNA backbone.

[0070] Some aspects of this disclosure provide polynucleotides that encode either a Cas9 protein, a Cas9 fusion protein, or a guide RNA bound to a Cas9 protein or a Cas9 fusion protein, as provided herein. Some aspects of this disclosure provide vectors comprising such polynucleotides. In some embodiments, the vectors include a heterologous promoter that drives the expression of the polynucleotide.

[0071] Some aspects of this disclosure provide cells comprising Cas9 proteins, fusion proteins, nucleic acid molecules, and / or vectors provided herein.

[0072] The above summary is intended to illustrate, in an unspecified manner, some aspects, advantages, features, and uses of the technology disclosed herein. Other aspects, advantages, features, and uses of the technology disclosed herein will be apparent from the modes for carrying out the invention, drawings, examples, and claims. [Brief explanation of the drawing]

[0073] [Figure 1] Figure 1 shows the Cas9 activity of wild-type Streptococcus pyogenes against classical and non-classical PAM libraries.

[0074] [Figure 2] Figure 2 shows the activity of the example post-evolutionary Cas9 clone against the PAM library after directional evolution.

[0075] [Figure 3] Figure 3 shows a comparison of wild-type and evolved Cas9 in a mammalian GFP activation assay.

[0076] [Figure 4]Figures 4A-4B show the binding activity of Cas9 (pJH306) and evolved Cas9 proteins with the 5'-NGG-3' PAM sequence, using GFP as a readout. With the 5'-NGG-3' PAM, many evolved Cas9 proteins showed increased Cas9 binding activity compared to wild-type Cas9, based on an increased GFP fluorescence signal. Figure 4A is a graph showing Cas9 binding activity as a function of the percentage of cells exhibiting higher fluorescence than the background. Figure 4B is a graph showing Cas9 binding activity as a function of the mean fluorescence. The Cas9 proteins used in these experiments were dCas9 proteins fused to the VPR transcription activator.

[0077] [Figure 5] Figures 5A-5B show the binding activity of wild-type dCas9-VPR (pJH306) and evolved dCas9-VPR proteins with NNN PAM sequences, using GFP as a readout. In the NNN PAM library, many evolved Cas9 proteins showed increased Cas9 binding activity compared to wild-type Cas9, based on an increased GFP fluorescence signal. Figure 5A is a graph showing Cas9 binding activity as a function of the percentage of cells showing higher fluorescence than the background. Figure 5B is a graph showing Cas9 binding activity as a function of the mean fluorescence. The Cas9 proteins used in these experiments were dCas9 proteins that fused to VPR.

[0078] [Figure 6] Figure 6 shows the dCas9-VPR for all 64 PAM sequences, indicated by mean fluorescence in transfection cells gated by iRFP fluorescence. The wild-type (WT) dCas9-VPR is pJH306.

[0079] [Figure 7] Figure 7 shows the in vitro cleavage assay. On the gel, WT is wild-type Cas9 (sequence number 9), and 1 is Cas9 with the E1219V mutation (sequence number 9).

[0080] [Figure 8] Figure 8 shows Cas9 fusion proteins that can be used to modulate PAM specificity. It shows one possible configuration for a Cas9-dCas9 ligation system that can be used to increase Cas9 targeting to non-classical PAMs. Binding of dCas9 to 5'-NGG-3' or other PAMs can localize Cas9 to a region near target 2. This localization may facilitate cleavage of PAMs that Cas9 has not previously been able to access.

[0081] [Figure 9] Figures 9A and 9B show the dCas9-VPR binding activity to the NNNNN PAM library. Figure 9A is a graph showing the dCas9-VPR binding activity to the NNNNN PAM library as a function of the percentage of cells showing higher fluorescence than the background. Figure 9B is a graph showing the dCas9-VPR binding activity to the NNNNN PAM library as a function of the mean fluorescence value.

[0082] [Figure 10] Figure 10 shows the cleavage activity of Cas9 using cells (percentage) exhibiting GFP loss as a readout. The Cas9 protein was tested with two sgRNAs, targeting either the classical 5'-NGG-3'PAM or the GAT PAM in the GFP gene.

[0083] [Figure 11] Figure 11 illustrates a double-stranded DNA substrate bound to a Cas9:DNA editing enzyme:sgRNA complex. The DNA editing enzyme may be, but is not limited to, a nuclease, nicasse, recombinase, deaminase, methyltransferase, methylase, acetylase, or acetyltransferase.

[0084] [Figure 12]Figures 12A–12D show the results of the PAM reduction assay. pJH760 was tested in PAM reduction assays against four novel targets: re2 (Figure 12A), VEGF (Figure 12B), CLTA (Figure 12C), and CCR5D (Figure 12D).

[0085] [Figure 13] Figure 13 shows GFP cleavage in mammalian cells.

[0086] [Figure 14] Figure 14 shows the results of a PAM reduction assay that tested the effect of pJH760 (xCas9v1.0) on the re2 target.

[0087] [Figure 15] Figure 15 shows the results of a PAM reduction assay that tested the effect of pJH760(xCas9v1.0) on VEGF targets.

[0088] [Figure 16] Figure 16 shows the results of a PAM reduction assay that tested the effect of pJH760(xCas9v1.0) on the CLTA target.

[0089] [Figure 17] Figure 17 shows the results of a PAM reduction assay that tested the effect of pJH760 (xCas9v1.0) on the CCR5D target.

[0090] [Figure 18] Figure 18 shows the results of a PAM reduction assay that tested the effects of four xCas9v3 mutants on the re2 target.

[0091] [Figure 19] Figure 19 shows the results of a PAM reduction assay that tested the effects of four xCas9v3 variants on the VEGF target.

[0092] [Figure 20]Figure 20 shows the results of a PAM reduction assay that tested the effects of four xCas9v3 variants on the CLTA target. [Modes for carrying out the invention]

[0093] definition As used herein and in the claims, the singular forms "a," "an," and "the" encompass both singular and plural references unless the context clearly indicates otherwise. For example, the reference to "substance" encompasses both a single substance and multiple such substances.

[0094] The term "Cas9" or "Cas9 nuclease" refers to an RNA-inducible nuclease containing the Cas9 protein or a fragment thereof (e.g., a protein containing the active or inactive DNA cleavage domain and / or gRNA-binding domain of Cas9). Cas9 nucleases are sometimes also called cason1 nucleases or CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-associated nucleases. CRISPR is an adaptive immune system that provides defense against mobile genetic elements (viruses, translocation elements, and mating plasmids). CRISPR clusters contain sequences, spacers, that are complementary to the ancestral mobile element and the target entry nucleic acid. CRISPR clusters are transcribed and processed to become CRISPR RNA (crRNA). In the type II CRISPR system, the correct processing of pre-crRNA requires trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and the Cas9 protein. TracrRNA acts as a guide for the processing of pre-crRNA, assisted by ribonuclease-3. Subsequently, Cas9 / crRNA / tracrRNA endoscopically cleaves linear or circular dsDNA targets complementary to the spacer. Target strands not complementary to crRNA are first endoscopically cleaved and then 3'-5' exoscopically trimmed. In nature, DNA binding and cleavage typically require both proteins and both RNAs. However, single guide RNAs ("sgRNAs" or simply "gNRAs") can be manipulated to incorporate aspects of both crRNA and tracrRNA into a single RNA species. See, for example, Jinek M., Chylinski K., Fonfara I, Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012). Its entire content is incorporated here by reference.Cas9 recognizes short motifs (PAM or protospacer-adjacent motifs) within CRISPR repeat sequences, helping to distinguish self from non-self. Cas9 nuclease sequences and structures are well known to those skilled in the art (e.g., "Complete genome sequence of an M1 strain of Streptococcus pyogenes." Ferretti et al., JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C, Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y., Jia HG, Najar FZ, Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW, Roe BA, McLaughlin RE, Proc. Natl. Acad. Sci. USA 98:4658-4663(2001);"CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Deltcheva E., Chylinski K., Sharma CM., Gonzales K., Chao See Y., Pirzada ZA, Eckert MR, Vogel J., Charpentier E., Nature 471:602-607 (2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M., Chylinski K., Fonfara I, Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012). (The entire contents of each are incorporated herein by reference.) Cas9 orthologs have been described for various species, including, but not limited to, S. pyogenes and S. thermophilus.Additional suitable Cas9 nucleases and sequences will be apparent to those skilled in the art based on this disclosure. Such Cas9 nucleases and sequences include Cas9 sequences from organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immune systems" (2013) RNA Biology 10:5, 726-737, the entire contents of which are incorporated herein by reference. In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivating) DNA cleavage domain.

[0095] Nuclease-inactivated Cas9 proteins can interchangeably be referred to as "dCas9" proteins (nuclease-inactive Cas9). Methods for generating Cas9 with an inactive DNA cleavage domain are known (see, for example, Jinek et al., Science. 337:816-821 (2012); Qi et al., "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression" (2013) Cell. 28; 152(5): 1173-83, the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to contain two subdomains: an HNH nuclease subdomain and a RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, while the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can suppress the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science. 337:816-821(2012); Qi et al., Cell. 28; 152(5): 1173-83 (2013)).

[0096] In some embodiments, proteins containing a fragment of Cas9 are provided. For example, in some embodiments, the protein contains one of two Cas9 domains: (1) the gRNA-binding domain of Cas9, or (2) the DNA-cleaving domain of Cas9. In some embodiments, the protein or fragment containing Cas9 is called a "Cas9 variant." The Cas9 variant shares homology with Cas9. For example, the Cas9 variant is at least about 70 percent identical to wild-type Cas9, at least about 80 percent identical, at least about 90 percent identical, at least about 95 percent identical, at least about 96 percent identical, at least about 97 percent identical, at least about 98 percent identical, at least about 99 percent identical, at least about 99.5 percent identical, or at least about 99.9 percent identical. In some embodiments, a Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA-binding domain or a DNA-cleaving domain) which is at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5%, or at least about 99.9% identical to the corresponding fragment of wild-type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid length of the corresponding wild-type Cas9. In some aspects, wild-type Cas9 corresponds to Cas9 derived from Streptococcus pyogenes (NCBI reference sequence: NC_017053.1, SEQ ID NO: 1 (nucleotide); SEQ ID NO: 2 (amino acid)).

number

[0097] In some embodiments, wild-type Cas9 corresponds to or includes SEQ ID NO: 3 (nucleotide) and / or SEQ ID NO: 4 (amino acid):

number

[0098] In some embodiments, wild-type Cas9 corresponds to Cas9 derived from Streptococcus pyogenes (NCBI reference sequence: NC_002737.2, SEQ ID NO: 282 (nucleotide); and Uniport reference sequence: Q99ZW2, SEQ ID NO: 9 (amino acid)).

number

number

[0099] In some embodiments, the Cas9 is derived from Corynebacterium ulcerans (NCBI Refs:NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref:NC_021284.1);Prevotella intermedia(NCBI Ref:NC_017861.1);Spiroplasma taiwanense(NCBI Ref:NC_021846.1);Streptococcus iniae(NCBI Ref:NC_021314.1);Belliella baltica(NCBI Ref:NC_018010.1);Psychroflexus torquisI(NCBI Ref:NC_018721.1);Streptococcus This refers to Cas9 derived from thermophilus (NCBI Ref:YP_820832.1); Listeria innocua (NCBI Ref:NP_472073.1); Campylobacter jejuni (NCBI Ref:YP_002344900.1); or Neisseria meningitidis (NCBI Ref:YP_002342100.1), or from any of the organisms listed in Example 3.

[0100] In some embodiments, dCas9 corresponds to or includes some or all of a Cas9 amino acid sequence having one or more mutations that inactivate Cas9 nuclease activity. For example, in some embodiments, the dCas9 domain includes the D10A and / or H840A mutations. dCas9 (D10A and H840A):

number

number

[0101] In some embodiments, Cas9 corresponds to, or includes in part or in whole, a Cas9 amino acid sequence having one or more mutations that alter the nuclease activity of Cas9. In some embodiments, Cas9 may also be a Cas9 nickase, which is a version of Cas9 that performs single-strand DNA cleavage (rather than double-strand DNA cleavage) based on a gRNA-specific target sequence co-expressed at a particular site. For example, in some embodiments, the Cas9 domain includes the D10A mutation (e.g., SEQ ID NO: 301) and / or the H840A mutation (e.g., SEQ ID NO: 302). Typical Cas9 nickases are shown below. However, it should be understood that further Cas9 nickases that cleave single-strand DNA of the DNA double helix are obvious to those skilled in the art and are within the scope of this disclosure. Cas9 D10A Nikkas:

number

number

number

[0102] In other embodiments, dCas9 variants having mutations other than D10A and H840A are provided, which result in, for example, nuclease-inactivating Cas9 (dCas9). Such mutations include, for example, other amino acid substitutions in D10 and H820, or other substitutions within the nuclease domain of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or RuvC1 subdomain). In some embodiments, variants or homologs of dCas9 (e.g., variants of SEQ ID NO: 9) are provided, which are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to SEQ ID NO: 9. In some embodiments, variants of dCas9 (e.g., variants of SEQ ID NO: 9) are provided, having amino acid sequences that are about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids, or more than SEQ ID NO: 9.

[0103] In some embodiments, the Cas9 fusion proteins described herein include the full-length amino acid sequence of the Cas9 protein (e.g., one of the sequences shown above). However, in other embodiments, the fusion proteins described herein include only fragments of the Cas9 sequence, rather than the full-length sequence. For example, in some embodiments, the Cas9 fusion proteins provided herein include a Cas9 fragment that binds to crRNA and tracrRNA or sgRNA but does not contain a functional nuclease domain (e.g., it includes only a deleted version of the nuclease domain or does not contain a nuclease domain at all). Examples of suitable amino acid sequences of Cas9 domains and Cas9 fragments are shown herein, and further suitable sequences of Cas9 domains and Cas9 fragments will be obvious to those skilled in the art. In some embodiments, the Cas9 fragment has at least 30 percent, at least 35 percent, at least 40 percent, at least 45 percent, at least 50 percent, at least 55 percent, at least 60 percent, at least 65 percent, at least 70 percent, at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, and at least 95 percent of the identity of the corresponding wild-type Cas9 protein, and at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent of the amino acid length. In some embodiments, the Cas9 fragment contains at least 100 amino acids in length. In some embodiments, the Cas9 fragment is at least 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,050, 1,100, 1,150, 1,200, 1,250, 1,300, 1,350, 1,400, 1,450, 1,500, 1,550, or at least 1,600 amino acids of the corresponding wild-type Cas9 protein.In some embodiments, the Cas9 fragment comprises an amino acid sequence having at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical adjacent amino acid residues of the corresponding wild-type Cas9 protein.

[0104] Cas9 In some embodiments, the Cas9 is derived from Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1), Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1), Spiroplasma syrphidicola (NCBI Ref:NC_021284.1), Prevotella intermedia(NCBI Ref:NC 017861.1), Spiroplasma taiwanense(NCBI Ref:NC_021846.1), Streptococcus iniae(NCBI Ref:NC_021314.1), Belliella baltica(NCBI Ref:NC_018010.1), Psychroflexus torquisl(NCBI This refers to Cas9 derived from Streptococcus thermophilus (NCBI Ref: YP_820832.1), Listeria innocua (NCBI Ref: NP_472073.1), Campylobacter jejuni (NCBI Ref: YP_002344900.1), or Neisseria, meningitidis (NCBI Ref: YP_002342100.1).

[0105] As used herein, the terms “deaminase” or “deaminase domain” mean a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase or deaminase domain is a cytidine deaminase that catalyzes a deamination reaction that hydrolyzes cytidine or deoxycytidine to uridine or deoxyuridine, respectively. In some embodiments, the deaminase or deaminase domain is a cytosine deaminase that catalyzes a deamination reaction that hydrolyzes cytosine to uracil. In some embodiments, the deaminase or deaminase domain is a native deaminase derived from an organism such as a human, chimpanzee, gorilla, monkey, cattle, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain is a variant of a native deaminase of a biological origin that does not exist in nature. For example, in some embodiments, the deaminase or deaminase domain has at least 50 percent, at least 55 percent, at least 60 percent, at least 65 percent, at least 70 percent, at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with a naturally occurring deaminase of biological origin.

[0106] As used herein, the term “effective amount” means the amount of a biologically active substance sufficient to produce the desired biological reaction. For example, in some embodiments, the effective amount of a nuclease refers to the amount of nuclease sufficient to induce specific binding and cleavage at the target site to be cleaved by the nuclease. In some embodiments, the effective amount of a fusion protein shown herein (e.g., a fusion protein consisting of a nuclease-inactive Cas9 domain and an effector domain (e.g., a deaminase domain)) refers to the amount of fusion protein sufficient to induce specific binding and editing at the target site to be edited by the fusion protein. As will be understood by those skilled in the art, the effective amount of an agent such as a fusion protein, nuclease, deaminase, recombinase, hybrid protein, protein dimer, complex of a protein (or protein dimer) with a polynucleotide, or polynucleotide may vary depending on various factors, such as the desired biological reaction (e.g., against a specific allele, genome, or target site to be edited), the target cell or tissue, and the agent used.

[0107] The term "directly adjacent" as used in the context of two nucleic acid sequences means that the two sequences are directly linked as part of the same nucleic acid molecule and are not separated by one or more nucleotides. Therefore, the sequences are said to be directly adjacent when the nucleotide at the 3' end of one sequence is directly linked to the nucleotide at the 5' end of the other sequence via a phosphodiester bond.

[0108] As used herein, the term “linker” refers to a chemical group or molecule that links two molecules or parts, such as two domains of a fusion protein (e.g., a nuclease-inactive Cas9 domain and an effector domain (e.g., a deaminase domain)). In some embodiments, a linker links the gRNA-binding domain of an RNA-programmable nuclease, such as the Cas9 nuclease domain, with the catalytic domain of a nucleic acid editing protein. In some embodiments, a linker links dCas9 with a nucleic acid editing protein. Typically, a linker is located between or sandwiched between two groups, molecules, or other parts, and links these two via a covalent bond. In some embodiments, a linker is an amino acid or a group of amino acids (e.g., a peptide or protein). In some embodiments, a linker is an organic molecule, group, polymer, or chemical part. In some embodiments, linkers have an amino acid length of 5 to 100, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200. Longer or shorter linkers are also included.

[0109] As used herein, the term "mutation" refers to the substitution, deletion, or insertion of one or more residues in a sequence (e.g., a nucleic acid or amino acid sequence) by other residues. In this specification, mutations are typically described by first identifying the original residue, then identifying the location of the residue in the sequence, and finally identifying the newly substituted residue. Various methods for constructing the amino acid substitutions (mutations) provided herein are publicly known, for example, as described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)).

[0110] As used herein, the terms “nucleic acid” and “nucleic acid molecule” refer to a compound comprising a nucleic acid base and an acidic moiety (e.g., a nucleoside, nucleotide, or polymer of nucleotides). Typically, polymerized nucleic acids (e.g., a nucleic acid molecule containing three or more nucleotides) are linear molecules, with adjacent nucleotides linked together via phosphodiester bonds. In some embodiments, “nucleic acid” refers to an individual nucleic acid residue (e.g., a nucleotide and / or nucleoside). In some embodiments, “nucleic acid” refers to an oligonucleotide chain containing three or more individual nucleotide residues. As used herein, the terms “oligonucleotide” and “polynucleotide” can be used interchangeably to refer to a polymer of nucleotides (e.g., a chain of at least three nucleotides). In some embodiments, “nucleic acid” encompasses RNA as well as single-stranded and / or double-stranded DNA. Nucleic acids may be naturally occurring, such as genomes, transcripts, mRNA, tRNA, rRNA, siRNA, snRNA, plasmids, cosmids, chromosomes, chromatids, or other naturally occurring nucleic acid molecules. On the other hand, nucleic acid molecules may be, for example, non-naturally occurring recombinant DNA or RNA molecules, artificial chromosomes, designed genomes or fragments thereof, or synthetic DNA, RNA, DNA / RNA hybrids, or may contain non-natural nucleotides or nucleosides. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and / or similar terms also include nucleic acid analogs (e.g., analogs having skeletons other than phosphodiester skeletons). Nucleic acids may be purified from natural sources, optionally purified and produced using recombinant expression systems, or chemically synthesized. If necessary, for example, in the case of chemically synthesized molecules, nucleic acids may include nucleoside analogs (e.g., chemically modified bases or sugars, and analogs having modified skeletons). Unless otherwise specified, nucleic acid sequences are shown in the 5' to 3' direction.In some embodiments, nucleic acids may be or include: natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyluridine) , C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose and hexose), and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoamidite bonds). In some embodiments, RNA is RNA associated with the Cas9 system. For example, RNA may be CRISPR RNA (crRNA), transcoding small RNA (tracrRNA), single guide RNA (sgRNA), or guide RNA (gRNA).

[0111] As used herein, the term “proliferative disorder” refers to any disorder in which the homeostasis of a cell or tissue is disrupted by an abnormally high rate of proliferation of cells or cell populations. Proliferative disorders include hyperproliferative disorders (e.g., preneoplastic hyperplasia and neoplastic disorders). Neoplastic disorders are characterized by abnormal cell proliferation and include benign and malignant neoplasias. Malignant neoplasias are also known as cancer.

[0112] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein and refer to polymers of amino acid residues linked by peptide (amide) bonds. These terms can refer to any protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide is at least three amino acids long. A protein, peptide, or polypeptide may also refer to an individual protein or a group of proteins. The amino acids of one or more proteins, peptides, or polypeptides may be modified, for example, by the addition of chemical moieties (e.g., carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, linkers for conjugates, functionalization, or other modifications). A protein, peptide, or polypeptide may be a single molecule or a complex of multiple molecules. A protein, peptide, or polypeptide may be only fragments of naturally occurring proteins or peptides. A protein, peptide, or polypeptide may be naturally occurring, recombinant, synthetic, or any combination thereof. As used herein, the term “fusion protein” means a hybrid polypeptide containing protein domains derived from at least two different proteins. A single protein may be located at the amino-terminus (N-terminus) of a fusion protein or at the carboxy-terminus (C-terminus) of a protein, thereby forming an "amino-terminus fusion protein" or a "carboxy-terminus fusion protein," respectively. The protein may contain several domains, for example, a nucleic acid-binding domain (e.g., the gRNA-binding domain of Cas9 that facilitates protein binding to a target site) and a nucleic acid-cleaving domain or catalytic domain of a nucleic acid-editing protein. In some embodiments, the protein contains a protein-like moiety, for example, an amino acid sequence constituting the nucleic acid-binding domain and an organic compound (e.g., a compound that can act as a nucleic acid degradation agent). In some embodiments, the protein exists as a complex with or in association with a nucleic acid (e.g., RNA). Any protein provided in the present invention can be produced by any method of the known art.For example, the proteins provided in the present invention may be produced by recombinant protein expression and purification, and are particularly suitable for fusion proteins containing peptide linkers. Methods for recombinant protein expression and purification are known, including the method described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)), which is incorporated by reference in its entirety.

[0113] The terms “RNA programmable nuclease” and “RNA-guided nuclease” are used interchangeably herein to refer to nucleases that form complexes (e.g., bind or associate) with one or more RNAs that are not targets for cleavage. In some embodiments, an RNA programmable nuclease may be referred to as a nuclease:RNA complex when it is a complex with RNA. Typically, the one or more RNAs that bind are referred to as guide RNAs (gRNAs). A gRNA may exist as a complex of two or more RNAs or as a single-stranded RNA molecule. A gRNA existing as a single-stranded RNA molecule is also referred to as a single-stranded guide RNA (sgRNA), although “gRNA” may also refer interchangeably to a guide RNA existing as a single molecule or as a complex of two or more molecules. Typically, a gRNA existing as a single RNA species includes two domains: (1) a domain that shares homology with the target nucleic acid (e.g., one that facilitates the binding of the Cas9 complex to its target), and (2) a domain that binds to the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as tracrRNA and includes a stem-loop structure. For example, in some embodiments, domain (2) is identical to or homologous to the tracrRNA described in Jinek et al, Science 337:816-821 (2012), the full disclosure of which is incorporated herein by reference. Other examples of gRNAs (e.g., those including domain 2) are described in U.S. Provisional Patent Application No. 61 / 874,682 (filed September 6, 2013), “Switchable Cas9 Nuclease and its Use,” and U.S. Provisional Patent Application No. 61 / 874,746 (filed September 6, 2013), the full disclosure of which is incorporated herein by reference. In some embodiments, a gRNA includes two or more domains (1) and (2), which may be referred to as “extended gRNA.” For example, an elongated gRNA binds to two or more Cas9 proteins, as described herein, and binds to a target nucleic acid in two or more different regions.The gRNA contains a nucleotide sequence complementary to the target site, mediates the binding of the nuclease / RNA complex to the target site, and provides sequence specificity for the nuclease:RNA complex. In some embodiments, the RNA programmable nuclease is the (CRISPR-associated) Cas9 endonuclease, for example, Cas9(Csnl) derived from Streptococcus pyogenes (see, for example, "Complete genome sequence of an Ml strain of Streptococcus pyogenes," Ferretti JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C., Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y., Jia HG, Najar FZ, Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW, Roe BA, McLaughlin RE, Proc. Natl. Acad. Sci. USA 98:4658-4663 (2001), "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Deltcheva E., Chylinski K., Sharma CM., Gonzales K., Chao Y., Pirzada ZA, Eckert MR, Vogel J., Charpentier E., Nature 471 : 602-607 (2011), and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek See M., Chylinski K., Fonfara I, Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012).

[0114] RNA programmable nucleases (e.g., Cas9) utilize RNA:DNA hybridization at target DNA cleavage sites; therefore, these proteins can, in principle, target any sequence identified by a guide RNA. Methods using RNA-programmable nucleases (e.g., Cas9) for site-specific cleavage (e.g., genome modification) are known techniques (e.g., Cong, L. et al. Multiplex genome engineering using CRISPR / Cas systems. Science 339, 819-823 (2013), Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013), Hwang, WY et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature biotechnology 31, 227-229 (2013), Jinek, M. et al. RNA-programmed genome editing in human cells. eLife 2, e00471 (2013), Dicarlo, JE et al. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems, which are incorporated herein by reference). see Nucleic acids research (2013), Jiang, W. et al. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature biotechnology 31, 233-239 (2013)).

[0115] As used herein, the term “subject” refers to an individual organism (e.g., an individual mammal). In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, goat, cow, cat, or dog. In some embodiments, the subject is a vertebrate, amphibian, reptile, fish, insect, fly, or nematode. In some embodiments, the subject is an experimental animal. In some embodiments, the subject is a genetically modified, for example, a genetically modified non-human subject. The subject may be of any sex, any age, or any developmental stage.

[0116] The term "target site" refers to a sequence within a nucleic acid molecule that is deaminated by a deaminase or a deaminase-containing fusion protein (for example, the dCas9-deaminase fusion protein provided in this invention).

[0117] The terms “treatment,” “to treat,” and “to treat” refer to a clinical intervention performed to reverse, alleviate, delay the onset of, or inhibit the progression of, or one or more of the signs of, a disease or disorder or one or more of the signs described herein. In some embodiments, treatment may be administered after the onset of one or more symptoms and / or after the disease has been diagnosed. In other embodiments, treatment may be administered even in the absence of signs, for example, to prevent or delay the onset of symptoms or to inhibit the onset or progression of the disease. For example, treatment may be administered to a susceptible individual before signs appear (for example, taking into account the history of symptoms and / or genetic or other susceptibility factors). Treatment may be continued after the symptoms have resolved, for example, to prevent or delay their recurrence.

[0118] As used herein in relation to proteins or nucleic acids, the term “recombinant” refers to proteins or nucleic acids that do not occur naturally but are artificially engineered products. For example, in some embodiments, recombinant proteins or nucleic acid molecules contain an amino acid or nucleotide sequence that contains at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations compared to any naturally occurring sequence.

[0119] As used herein, the term “nucleic acid editing enzyme” means a nucleic acid or a protein capable of modifying one or more nucleotide bases in a nucleic acid. For example, in some embodiments, a nucleic acid editing enzyme is a deaminase capable of catalyzing a change from C to T or from G to A. Other suitable nucleic acid editing enzymes that can be used in accordance with the disclosure herein include, but are not limited to, nucleases, nickases, recombinases, deaminases, methyltransferases, methylases, acetylases, or acetyltransferases.

[0120] Detailed description of specific embodiments of the present invention: Some aspects of the disclosure of the present invention relate to the provision of recombinant Cas9 proteins that efficiently target DNA sequences that do not contain a classical PAM sequence (5'-NGG-3', where N is any nucleotide (e.g., A, T, G, or C)) at the 3' end. In some embodiments, the Cas9 proteins provided in the present invention contain one or more mutations identified in directional evolution experiments using a target sequence library containing randomized PAM sequences. The recombinant PAM-unrestricted Cas9 proteins provided in the present invention are useful for targeting DNA sequences that do not contain a classical PAM sequence at the 3' end, thereby greatly enhancing the utility of Cas9 technology for gene editing.

[0121] Some aspects of the disclosure of the present invention relate to the provision of a fusion protein comprising a Cas9 protein and an effector domain (e.g., a DNA editing domain), such as a deaminase domain. Deamination of nucleic acid bases by deaminase can result in point mutations at specific residues, which are referred to in the present invention as nucleic acid editing. A fusion protein comprising a Cas9 protein or a variant thereof and a DNA editing domain can therefore be used for targeted editing of nucleic acid sequences. Such a fusion protein is useful for targeted mutagenesis, for example, for the creation of mutant cells or animals, for example, for the repair of intracellular gene deletions (e.g., in cells derived from a subject, which are subsequently reintroduced into the same or a different subject) ex vivo, and for targeted mutagenesis, for example, for the repair of gene deletions in disease-related genes in a subject in vivo, or for the inactivation of such genes. Typically, the Cas9 protein of the fusion protein described herein does not have any nuclease activity, but instead is a Cas9 fragment or a dCas9 protein. Methods for using the Cas9 fusion protein described herein are also provided.

[0122] Non-exclusively exemplary nuclease-inactivating Cas9 proteins are provided herein. One preferred exemplary nuclease-inactivating Cas9 protein is the D10A / H840A Cas9 protein mutation:

[0123] Additional suitable nuclease-inactive Cas9 domains will be apparent to those skilled in the art based on this disclosure. Such additional exemplary suitable nuclease-inactive Cas9 proteins include, but are not limited to, D10A, D839A, H840A, N863A, D10A / D839A, D10A / H840A, D10A / N863A, D839A / H840A, D839A / N863A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A mutant proteins (see, for example, Prashant et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31(9): 833-838, the entire contents of which are incorporated herein by reference).

[0124] Recombinant Cas9 protein Some aspects of the disclosure of the present invention relate to providing a recombinant Cas9 protein comprising an amino acid sequence that is at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent or at least 99.5 percent identical to the amino acid sequence of Cas9 represented by any of SEQ ID NOs: 10 to 262, wherein the Cas9 protein comprises a RuvC and an HNH domain, and the amino acids of the Cas9 protein The acid sequence contains at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations in amino acid residues selected from the group consisting of amino acid residues 122, 137, 182, 262, 294, 409, 480, 543, 660, 694, 1219, and 1329 of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding amino acid residues of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, and the amino acid sequence of the recombinant Cas9 protein is not identical to the amino acid sequence of the naturally occurring Cas9 protein.

[0125] In some embodiments, the amino acid sequence of the Cas9 protein includes at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations selected from the group consisting of X262T, X294R, X409I, X480K, X543D, X694I, and X1219V of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids at the corresponding positions.

[0126] In some embodiments, the amino acid sequence of the Cas9 protein includes at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations selected from the group consisting of A262T, K294R, S409I, E480K, E543D, M694I, or E1219V in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10-262. In some embodiments, the amino acid sequence of the Cas9 protein includes the X1219V mutation in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10-262. In some embodiments, the amino acid sequence of the Cas9 protein includes the E1219V mutation in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10-262.

[0127] In some embodiments, the amino acid sequence of the HNH domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of the HNH domain of SEQ ID NO: 9. In some embodiments, the amino acid sequence of the RuvC domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of the RuvC domain of any of SEQ ID NOs: 10 to 262. In some embodiments, the amino acid sequence of the RuvC domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of the RuvC domain of SEQ ID NO: 9. In some embodiments, the Cas9 protein contains the D10A and / or H840A mutation in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10-262.

[0128] Recombinant Cas9 protein with activity against non-classical PAMs Some aspects of the disclosure of the present invention relate to the provision of recombinant Cas9 comprising an amino acid sequence having at least 90 percent identity with the amino acid sequence of Streptococcus pyogenes Cas9 shown by SEQ ID NO: 9, and comprising the RuvC and HNH domains of SEQ ID NO: 9, wherein 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, or at least seven mutations of amino acid residues selected from the group consisting of amino acid residues 122, 137, 182, 262, 294, 409, 480, 543, 660, 694, 1219, and 1329 of the amino acid sequence shown in SEQ ID NO: 9, wherein the amino acid sequence of the recombinant Cas9 protein is not identical to the amino acid sequence of the naturally occurring Cas9 protein, and the recombinant Cas9 protein comprises the amino acid sequence of Streptococcus pyogenes shown in SEQ ID NO: 9. Compared to Cas9 in pyogenes, it shows increased activity against target sequences that do not contain the classic PAM (5'-NGG-3') at the 3' end.

[0129] In some embodiments, the Cas9 protein exhibits activity against a target sequence having a 3' end that is not directly adjacent to the classical PAM sequence (5'-NGG-3'), and compared to the activity of Streptococcus pyogenes Cas9 shown in SEQ ID NO: 9, it is increased by at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 50,000-fold, at least 100,000-fold, at least 500,000-fold, or at least 1,000,000-fold against the same target sequence. In some embodiments, the 3' end of the target sequence is directly adjacent to an AGC, GAG, TTT, GTG, or CAA sequence. In some embodiments, Cas9 protein activity is measured by a nuclease assay, deamination assay, or transcriptional activation assay. In some embodiments, the transcriptional activation assay is a reporter activation assay (e.g., a GFP activation assay). Typical methods for measuring binding activity (e.g., Cas9) using transcriptional activation assays are known and obvious to those skilled in the art. For example, a method for measuring Cas9 activity using the tripartite activator VPR is described in Chavez A., et al., "Highly efficient Cas9-mediated transcriptional programming." Nature Methods 12, 326-328 (2015), the full disclosure of which is incorporated herein by reference.

[0130] In some embodiments, the amino acid sequence of the Cas9 protein includes at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations selected from the group consisting of X262T, X294R, X409I, X480K, X543D, X694I, and X1219V of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids at the corresponding positions.

[0131] In some embodiments, the amino acid sequence of the Cas9 protein includes at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations selected from the group consisting of A262T, K294R, S409I, E480K, E543D, M694I, or E1219V in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10-262. In some embodiments, the amino acid sequence of the Cas9 protein includes the X1219V mutation in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10-262. In some embodiments, the amino acid sequence of the Cas9 protein includes the X1219V mutation in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10-262.

[0132] In some embodiments, the amino acid sequence of the HNH domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of the HNH domain of any of SEQ ID NOs: 2, 4, or 9. In some embodiments, the amino acid sequence of the RuvC domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of any of SEQ ID NOs: 10-262. In some embodiments, the Cas9 protein contains the D10A and / or H840A mutation in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10-262.

[0133] Some aspects of the disclosure of the present invention relate to the provision of recombinant Cas9 comprising an amino acid sequence having at least 90 percent identity with the amino acid sequence of Cas9 of Streptococcus pyogenes shown by SEQ ID NO: 9, comprising the RuvC and HNH domains of SEQ ID NO: 9, wherein 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, or at least seven mutations of amino acid residues selected from the group consisting of amino acid residues 122, 137, 182, 262, 294, 409, 480, 543, 660, 694, 1219, and 1329 of the amino acid sequence shown in SEQ ID NO: 9, wherein the amino acid sequence of the recombinant Cas9 protein is not identical to the amino acid sequence of the naturally occurring Cas9 protein, and the recombinant Cas9 protein exhibits increased activity against target sequences that do not contain the classical PAM (5'-NGG-3') at the 3' end, compared to Cas9 of Streptococcus pyogenes shown in SEQ ID NO: 9. In some embodiments, the amino acid sequence of the RuvC domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of the RuvC domain of any of SEQ ID NOs. 2, 4, or 9. In some embodiments, the Cas9 protein contains the D10A and H840A mutations in the amino acid sequence shown in SEQ ID NOs. 9, or the corresponding mutations in any of the amino acid sequences shown in SEQ ID NOs. 10-262.

[0134] Some aspects of the disclosure of the present invention relate to the provision of recombinant Cas9 comprising an amino acid sequence having at least 90 percent identity with the amino acid sequence of Cas9 of Streptococcus pyogenes shown by SEQ ID NO: 9, and comprising the RuvC and HNH domains of SEQ ID NO: 9, wherein 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, or at least seven mutations of amino acid residues selected from the group consisting of amino acid residues 122, 137, 182, 262, 294, 409, 480, 543, 660, 694, 1219, and 1329 of the amino acid sequence shown in SEQ ID NO: 9, wherein the amino acid sequence of the recombinant Cas9 protein is not identical to the amino acid sequence of the naturally occurring Cas9 protein, and the recombinant Cas9 protein exhibits increased activity against target sequences that do not contain the classical PAM (5'-NGG-3') at the 3' end, compared to Cas9 of Streptococcus pyogenes shown in SEQ ID NO: 9. In some embodiments, the amino acid sequence of the RuvC domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of the RuvC domain of any of SEQ ID NOs. 2, 4, or 9. In some embodiments, the Cas9 protein contains the D10A mutation in the amino acid sequence shown in SEQ ID NOs. 9, or the corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs. 10-262. In some embodiments, Cas9 further contains a histidine residue at position 840 as shown in SEQ ID NOs. 9, or the corresponding histidine residue in any of the amino acid sequences shown in SEQ ID NOs. 10-262. While not bound by any particular theory, the presence of the catalytic residue H840 enables Cas9 to cleave the non-target strand (i.e., the strand bound by sgRNA).In some embodiments, Cas9 having an amino acid residue other than histidine at position 840 of the amino acid sequence shown in SEQ ID NO: 9, or at the corresponding position in the amino acid sequences shown in SEQ ID NOs: 10-262, may have the amino acid residue 840 of the amino acid sequence shown in SEQ ID NO: 9, or at the corresponding position in the amino acid sequences shown in SEQ ID NOs: 10-262, changed or mutated to histidine.

[0135] Some aspects of the disclosure of the present invention relate to the provision of recombinant Cas9 comprising an amino acid sequence having at least 90 percent identity with the amino acid sequence of Cas9 of Streptococcus pyogenes shown by SEQ ID NO: 9, and comprising the RuvC and HNH domains of SEQ ID NO: 9, wherein 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, or at least seven mutations in amino acid residues selected from the group consisting of amino acid residues 262, 267, 294, 405, 409, 480, 543, 694, 1219, 1224, and 1256 of the amino acid sequence shown in SEQ ID NO: 9, wherein the recombinant Cas9 protein is not identical to the amino acid sequence of the naturally occurring Cas9 protein, and the recombinant Cas9 protein exhibits increased activity against target sequences that do not contain the classical PAM (5'-NGG-3') at the 3' end, compared to Cas9 of Streptococcus pyogenes shown in SEQ ID NO: 9.

[0136] In some embodiments, the Cas9 protein exhibits activity against target sequences having a 3' end that is not directly adjacent to the classical PAM sequence (5'-NGG-3'), and is at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 50,000-fold, at least 100,000-fold, at least 500,000-fold, or at least 1,000,000-fold increased compared to the activity of Streptococcus pyogenes Cas9 shown in SEQ ID NO: 9 against the same target sequences. In some embodiments, the 3' end of the target sequence is directly adjacent to an AAA, AAC, AAG, AAT, CAA, CAC, CAG, CAT, GAA, GAC, GAG, GAT, TAA, TAC, TAG, TAT, ACA, ACC, ACG, ACT, CCA, CCC, CCG, CCT, GCA, GCC, GCG, GCT, TCA, TCC, TCG, TCT, AGA, AGC, AGT, CGA, CGC, CGT, GGA, GGC, GGT, TGA, TGC, TGT, ATA, ATC, ATG, ATT, CTA, CTC, CTG, CTT, GTA, GTC, GTG, GTT, TTA, TTC, TTG, or TTT PAM sequence.

[0137] In some embodiments, the amino acid sequence of the Cas9 protein includes at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations selected from the group consisting of X262T, X267G, X294R, X405I, X409I, X480K, X543D, X694I, X1219V, X1224K, X1256K, and X1362P of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids at the corresponding positions.

[0138] In some embodiments, the amino acid sequence of the Cas9 protein includes at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations selected from the group consisting of A262T, S267G, K294R, F405I, S409I, E480K, E543D, M694I, E1219V, N1224K, Q1256K, and L1362P in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations in any of the amino acid sequences shown in SEQ ID NOs: 10-262. In some embodiments, the amino acid sequence of the Cas9 protein includes the X1219V mutation in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10-262. In some embodiments, the amino acid sequence of the Cas9 protein includes the E1219V mutation in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10-262.

[0139] In some embodiments, the amino acid sequence of the Cas9 protein includes the X480K mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of either the amino acid sequence shown in SEQ ID NOs: 10-262. In some embodiments, the amino acid sequence of the Cas9 protein includes the E480K mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of either the amino acid sequence shown in SEQ ID NOs: 10-262.

[0140] In some embodiments, the amino acid sequence of the Cas9 protein includes the X543D mutation in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation in either of the amino acid sequences shown in SEQ ID NOs: 10-262. In some embodiments, the amino acid sequence of the Cas9 protein includes the E543D mutation in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation in either of the amino acid sequences shown in SEQ ID NOs: 10-262.

[0141] In some embodiments, the amino acid sequence of the Cas9 protein includes a combination of mutations selected from the following groups: (X480K, X543D and X1219V), (X262T, X409I, X480K, X543D, X694I and X1219V), (X294R, X480K, X543D, X1219V, X1256K and X1362P) of the amino acid sequence shown in SEQ ID NO: 9 ), (X294R, X480K, X543D, X1219V and X1256K), (X267G, X294R, X480K, X543D, X1219V, X1224K and X1256K), and (X262T, X405I, X409I, X480K, X543D, X694I and X1219V), or any corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs. 10-262. In some embodiments, the amino acid sequence of the Cas9 protein includes a combination of mutations selected from the following group: (E480K, E543D and E1219V), (A262T, S409I, E480K, E543D, M694I and E1219V), (K294R, E480K, E543D, E1219V, Q1256K and L1362 P), (K294R, E480K, E543D, E1219V and Q1256K), (S267G, K294R, E480K, E543D, E1219V, N1224K and Q1256K), and (A262T, F405I, S409I, E480K, E543D, M694I and E1219V), or any corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs. 10-262.

[0142] In some embodiments, the amino acid sequence of the HNH domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of the HNH domain of any of SEQ ID NOs: 2, 4, or 9. In some embodiments, the amino acid sequence of the RuvC domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of the RuvC domain of any of SEQ ID NOs: 2, 4, or 9. In some embodiments, the Cas9 protein contains the D10A and / or H840A mutation in the amino acid sequence shown in SEQ ID NOs: 9, or the corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10-262. In some embodiments, the Cas9 protein contains the D10A and H840A mutations in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations in any of the amino acid sequences shown in SEQ ID NOs: 10-262. In some embodiments, the Cas9 protein contains the D10A mutation in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations in any of the amino acid sequences shown in SEQ ID NOs: 10-262. In some embodiments, the Cas9 protein contains the H840A mutation in the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations in any of the amino acid sequences shown in SEQ ID NOs: 10-262.

[0143] Some aspects of the disclosure of the present invention relate to the provision of a recombinant Cas9 protein comprising an amino acid sequence having at least 90 percent identity with the amino acid sequence of Cas9 of Streptococcus pyogenes shown by SEQ ID NO: 9, and comprising the RuvC and HNH domains of SEQ ID NO: 9, wherein 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, or at least seven mutations of amino acid residues selected from the group consisting of amino acid residues 262, 267, 294, 405, 409, 480, 543, 694, 1219, 1224, 1256, and 1362 of the amino acid sequence shown in SEQ ID NO: 9, wherein the amino acid sequence of the recombinant Cas9 protein is not identical to the amino acid sequence of the naturally occurring Cas9 protein, and the recombinant Cas9 protein exhibits increased activity against target sequences that do not contain the classical PAM (5'-NGG-3') at the 3' end, compared to Cas9 of Streptococcus pyogenes shown in SEQ ID NO: 9. In some embodiments, the amino acid sequence of the RuvC domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of the RuvC domain of any of SEQ ID NOs. 2, 4, or 9. In some embodiments, the Cas9 protein contains the D10A and H840A mutations in the amino acid sequence shown in SEQ ID NOs. 9, or the corresponding mutations in any of the amino acid sequences shown in SEQ ID NOs. 10-262.

[0144] Some aspects of the disclosure of the present invention relate to the provision of a recombinant Cas9 protein comprising an amino acid sequence having at least 90 percent identity with the amino acid sequence of Cas9 of Streptococcus pyogenes shown by SEQ ID NO: 9, and comprising the RuvC and HNH domains of SEQ ID NO: 9, wherein 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, or at least seven mutations of amino acid residues selected from the group consisting of amino acid residues 262, 267, 294, 405, 409, 480, 543, 694, 1219, 1224, 1256, and 1362 of the amino acid sequence shown in SEQ ID NO: 9, wherein the amino acid sequence of the recombinant Cas9 protein is not identical to the amino acid sequence of the naturally occurring Cas9 protein, and the recombinant Cas9 protein exhibits increased activity against target sequences that do not contain the classical PAM (5'-NGG-3') at the 3' end, compared to Cas9 of Streptococcus pyogenes shown in SEQ ID NO: 9. In some embodiments, the amino acid sequence of the RuvC domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of the RuvC domain of any of SEQ ID NOs. 2, 4, or 9. In some embodiments, the Cas9 protein contains the D10A mutation in the amino acid sequence shown in SEQ ID NOs. 9, or the corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs. 10-262. In some embodiments, Cas9 further contains a histidine residue at position 840 as shown in SEQ ID NOs. 9, or the corresponding histidine residue in any of the amino acid sequences shown in SEQ ID NOs. 10-262. While not bound by any particular theory, the presence of the catalytic residue H840 enables Cas9 to cleave the non-target strand (i.e., the strand bound by sgRNA).In some embodiments, Cas9 having an amino acid residue other than histidine at position 840 of the amino acid sequence shown in SEQ ID NO: 9, or at the position in the corresponding amino acid sequence shown in SEQ ID NO: 10-262, may have the amino acid residue 840 of the amino acid sequence shown in SEQ ID NO: 9, or at the position in the corresponding amino acid sequence shown in SEQ ID NO: 10-262, changed or mutated to histidine.

[0145] Cas9 fusion protein Some aspects of the disclosure of the present invention relate to the provision of fusion proteins comprising the Cas9 protein provided herein, wherein the Cas9 protein forms a fusion protein by fusing with a second protein or “fusion partner” (e.g., an effector domain). In some embodiments, the effector domain is fused to the N-terminus of the Cas9 protein. In some embodiments, the effector domain is fused to the C-terminus of the Cas9 protein. In some embodiments, the Cas9 protein and the effector domain are fused to each other via a linker. Preferred strategies for constructing the fusion proteins according to the disclosure of the present invention, with or without the use of a linker, will be obvious to those skilled in the art in light of the description herein and the technical knowledge of the art. For example, Gilbert et al, CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell. 2013; 154(2): 442-51, shows that a C-terminal fusion of Cas9 with VP64, using two NLS (SPKKKRKVEAS, SEQ ID NO: 284) as linkers, can be used for transcriptional activation. Mali et al, CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol. 2013; 31(9): 833-8, reports that a C-terminal fusion with VP64, without linkers, can be used for transcriptional activation. Furthermore, Maeder et al, CRISPR RNA-guided activation of endogenous human genes. Nat Methods. 2013; 10: 977-979, reported that a C-terminal fusion of VP64 using the Gly4Ser (SEQ ID NO: 5) linker can be used as a transcription activator.In recent years, the creation of dCas9-FokI nuclease fusions has been successful, and it has been reported that they exhibit improved enzyme specificity compared to the parent Cas9 enzyme (see Guilinger JP, Thompson DB, Liu DR. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 2014; 32(6): 577-82, and Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V, Reyon D, Goodwin MJ, Aryee MJ, Joung JK. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotechnol. 2014; 32(6): 569-76. PMID:24770325 SGSETPGTSESATPES (SEQ ID NO: 7), or GGGGS). n (Sequence ID 5) The linker is used in each of the FokI-dCas9 fusion proteins. In some embodiments, the linker is (GGGGS) n (Sequence ID 5), (G) n , (EAAAK) n (Sequence No. 6), (GGS) n , SGSETPGTSESATPES (Sequence ID 7) or (XP) n The domain includes a motif, or any combination thereof, where n is an independent integer from 1 to 30. In some embodiments, the effector domain includes an enzyme domain. Preferred effector domains include, but are not limited to, nucleases, nicasses, recombinases, deaminases, methyltransferases, methylases, acetylases, acetyltransferases, transcription activators, and transcription repressors.

[0146] The linker may be a single covalent bond or a polymeric linker consisting of many atoms. In certain embodiments, the linker is polypeptide or amino acid-based. In other embodiments, the linker is not peptide-like. In certain embodiments, the linker is a covalent bond (e.g., carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.). In certain embodiments, the linker is a carbon-nitrogen bond via an amide bond. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker. In certain embodiments, the linker is a polymer (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises an aminoalkanoic acid monomer, dimer, or polymer. In certain embodiments, the linker comprises an aminoalkanoic acid (e.g., glycine, ethaneic acid, alanine, β-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In certain embodiments, the linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In other embodiments, the linker comprises a polyethylene glycol moiety (PEG). In other embodiments, the linker comprises an amino acid. In certain embodiments, the linker comprises a peptide. In certain embodiments, the linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. The linker may also contain a functionalized moiety to facilitate the binding of nucleophiles (e.g., thiols, amino groups) derived from peptides to the linker. Any of the electrophiles may be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates.

[0147] In some embodiments, the effector domain comprises an effector enzyme. Suitable effector enzymes that can be used in accordance with the disclosure of the present invention include nucleases, nicasses, recombinases, and deaminases. However, further effector enzymes are also obvious to those skilled in the art and are within the scope of the present invention. In other embodiments, the effector domain comprises a domain that modulates transcriptional activity. Such transcriptional modulating domains may be, but are not limited to, transcriptional activator domains or transcriptional repressor domains.

[0148] In some aspects, the effector domain is an effector domain. In some aspects, the effector domain is a deaminase domain. In some aspects, the deaminase is a cytosine deaminase or a cytidine deaminase. In some aspects, the deaminase is a deaminase of the apolipoprotein B messenger RNA editing complex (APOBEC) family. In some aspects, the deaminase is an APOBEC1 deaminase. In some aspects, the deaminase is an APOBEC2 deaminase. In some aspects, the deaminase is an APOBEC3 deaminase. In some aspects, the deaminase is an APOBEC3A deaminase. In some aspects, the deaminase is an APOBEC3D deaminase. In some aspects, the deaminase is an APOBEC3E deaminase. In some aspects, the deaminase is an APOBEC3F deaminase. In some aspects, the deaminase is APOBEC3G deaminase. In some aspects, the deaminase is APOBEC3H deaminase. In some aspects, the deaminase is APOBEC4 deaminase. In some aspects, the deaminase is activation-inducing deaminase (AID).

[0149] In some embodiments, the effector domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with any one of the deaminase domains of sequence numbers 263 to 281.

[0150] In some embodiments, the effector domain is a nuclease domain. In some embodiments, the nuclease domain is a FokI DNA cleavage domain. In some embodiments, the disclosure of the present invention relates to providing a dimer of the fusion protein according to the present invention, for example, the dimer of the fusion protein may include a dimerized nuclease domain.

[0151] In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identical to the amino acid sequence of Cas9 shown by any of SEQ ID NOs. 10-262, the Cas9 protein comprises a RuvC and HNH domain, 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, or at least seven mutations in amino acid residues selected from the group consisting of amino acid residues 122, 137, 182, 262, 294, 409, 480, 543, 660, 694, 1219, and 1329 of the amino acid sequence shown in SEQ ID NOs. 9, or the corresponding amino acid residues of any of the amino acid sequences shown in SEQ ID NOs. 10-262, and the amino acid sequence of the recombinant Cas9 protein is not identical to the amino acid sequence of the naturally occurring Cas9 protein. In some embodiments, the Cas9 protein comprises an amino acid sequence having at least 90 percent identity with the amino acid sequence of Streptococcus pyogenes Cas9 shown by SEQ ID NO: 9, and includes the RuvC and HNH domains of SEQ ID NO: 9, and 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, or at least seven mutations in amino acid residues selected from the group consisting of amino acid residues 122, 137, 182, 262, 294, 409, 480, 543, 660, 694, 1219, and 1329 of the amino acid sequence shown by SEQ ID NO: 9, and the recombinant Cas9 protein amino acid sequence is not identical to the amino acid sequence of the naturally occurring Cas9 protein, and the recombinant Cas9 protein exhibits activity against target sequences that do not contain the classical PAM (5'-NGG-3') at the 3' end, compared to Streptococcus pyogenes Cas9 shown by SEQ ID NO: 9.

[0152] In some embodiments, the Cas9 protein comprises an amino acid sequence that is at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent or at least 99.5 percent identical to the amino acid sequence of Cas9 represented by any of SEQ ID NOs: 10-262, the Cas9 protein comprises a RuvC and HNH domain, and the amino acid sequence of the Cas9 protein is a sequence The recombinant Cas9 protein has at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations in amino acid residues selected from the group consisting of amino acid residues 262, 267, 294, 405, 409, 480, 543, 694, 1219, 1224, 1256, and 1362 of the amino acid sequence shown in sequence number 9, or the corresponding amino acid residues of any of the amino acid sequences shown in sequence numbers 10 to 262, and the amino acid sequence of the recombinant Cas9 protein is not identical to the amino acid sequence of the naturally occurring Cas9 protein. In some embodiments, the Cas9 protein comprises an amino acid sequence having at least 90 percent identity with the amino acid sequence of Streptococcus pyogenes Cas9 shown by SEQ ID NO: 9, and includes the RuvC and HNH domains of SEQ ID NO: 9, and 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, or at least seven mutations in amino acid residues selected from the group consisting of amino acid residues 262, 267, 294, 405, 409, 480, 543, 694, 1219, 1224, and 1256 of the amino acid sequence shown by SEQ ID NO: 9, and the amino acid sequence of the recombinant Cas9 protein is not identical to the amino acid sequence of the naturally occurring Cas9 protein, and the recombinant Cas9 protein exhibits increased activity against target sequences that do not contain the classical PAM (5'-NGG-3') at the 3' end, compared to Streptococcus pyogenes Cas9 shown by SEQ ID NO: 9.

[0153] Some aspects of the disclosure of the present invention relate to providing a fusion protein comprising (i) a nuclease-inactive Cas9 protein and (ii) an effector domain. In some embodiments, the effector domain is a DNA editing domain. In some embodiments, the effector domain has deaminase activity. In some embodiments, the effector domain is a deaminase domain or comprises one. In some embodiments, the deaminase is a cytidine deaminase. In some embodiments, the deaminase is a deaminase of the apolipoprotein B messenger RNA editing complex (APOBEC) family. In some embodiments, the deaminase is a deaminase of the APOBEC1 family. In some embodiments, the deaminase is an activation-inducible cytidine deaminase (AID). Several nucleic acid editing domains and Cas9 fusion proteins comprising such domains are described in detail herein. Further preferred effector domains will be obvious to those skilled in the art based on the disclosure herein. In some embodiments, the nucleic acid editing domain is a FokI nuclease domain.

[0154] The disclosure of this invention provides Cas9:effector domain fusion proteins of various three-dimensional structures. In some embodiments, the effector domain is fused to the N-terminus of the Cas9 protein. In some embodiments, the effector domain is fused to the C-terminus of the Cas9 protein. In some embodiments, the Cas9 protein and the effector domain are fused via a linker. In some embodiments, the linker is (GGGGS) n (Sequence ID 5), (G) n , (EAAAK) n (Sequence No. 6), (GGS) n, or the motif of SGSETPGTSESATPES (SEQ ID NO: 7) (referencing herein by reference to the full disclosure; see Guilinger JP, Thompson DB, Liu DR. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 2014; 32(6): 577-82), or (XP) n The linker motif, or any combination thereof, is independently an integer from 1 to 30. In some embodiments, n is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, or one or more linkers or one or more linker motifs are present in any combination thereof. Further preferred linker motifs and linker structures are obvious to those skilled in the art. In some embodiments, preferred linker motifs and structures are those described in Chen et al, Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013; 65(10): 1357-69, the entire disclosure of which is incorporated herein by reference. Further preferred linker arrangements are obvious to those skilled in the art based on the disclosures of the present invention and common technical knowledge in the art.

[0155] In some embodiments, the general structure of an exemplary Cas9 fusion protein provided in the present invention is as follows: [NH2]-[Effector Domain]-[Cas9]-[COOH], or, [NH2]-[Cas9]-[Effector Domain]-[COOH] NH2 is the N-terminus of the fusion protein, and COOH is the C-terminus of the fusion protein. Figure 11 shows a schematic of the Cas9 protein fused with the effector domain (e.g., rAPOBECl) in a complex with sgRNA, and its binding to the target nucleic acid sequence.

[0156] In some embodiments, any of the fusion proteins provided in the present invention may contain one or more nuclear localization sequences (NLS). In this specification, a nuclear localization sequence refers to an amino acid sequence that facilitates the import of a protein (e.g., any of the fusion proteins provided in the present invention having an NLS) into the cell nucleus (e.g., via nuclear transport). Typically, an NLS comprises one or more short amino acid sequences of positively charged lysine or arginine exposed on the protein surface. Nuclear localization sequences are well known in the prior art and are obvious to those skilled in the art. For example, the nuclear localization sequence can be found in Kalderon D., et al. 841-9, Makkerh LP., et al, "Comparative mutagenesis of nuclear localization signals reveals the importance of neutral and acidic amino acids". Curr Biol. (1996) 6 (8): 1025-7, and Ray M., et al., "Quantitative tracking of protein trafficking to the nucleus using cytosolic protein delivery by nanoparticle-stabilized nanocapsules". Bioconjug. Chem. (2015) 26 (6): Further nuclear localization sequences are described in, for example, Plank et al., International Patent Application No. PCT / EP2000 / 011690, and their entire disclosures are incorporated herein by reference.In some embodiments, NLS comprises the amino acid sequence PKKKRKV (SEQ ID NO: 299) or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC (SEQ ID NO: 300).

[0157] Examples of possible features include localization sequences (e.g., nuclear localization sequences), cytoplasmic localization sequences, export sequences (e.g., nuclear export sequences), or other localization sequences, as well as sequence tags useful for solubilization, purification, or detection of fusion proteins. Suitable localization signal sequences and protein tag sequences are shown herein, but are not limited to, biotin carboxylase carrier protein (BCCP) tags, myc tags, calmodulin tags, FLAG tags, hemagglutinin (HA) tags, polyhistidine tags, histidine or His tags, maltose-binding protein (MBP) tags, nus tags, glutathione S-transferase (GST) tags, green fluorescent protein (GFP) tags, thioredoxin tags, S tags, Softag (e.g., Softag1, Softag3), strep tags, biotin ligase tags, FlAsH tags, V5 tags, and SBP tags. Further suitable sequences will be obvious to those skilled in the art and are within the scope of the disclosure of this invention.

[0158] Any of the nuclear localization sequences provided in the present invention can fuse with the fusion protein at any preferred position, for example, to facilitate the translocation of the fusion protein to the cell nucleus without impairing the function of the fusion protein. In some embodiments, the NLS fuses to the N-terminus of the Cas9 protein of the fusion protein. In some embodiments, the NLS fuses to the C-terminus of the Cas9 protein of the fusion protein. In some embodiments, the NLS fuses to the N-terminus of the effector domain of the fusion protein. In some embodiments, the NLS fuses to the C-terminus of the effector domain of the fusion protein.

[0159] In some embodiments, the effector domain is a deaminase. For example, in some embodiments, a typical Cas9 fusion protein having a deaminase domain may have the following structure: [NH2]-[NLS]-[Cas9]-[deaminase]-[COOH], [NH2]-[NLS]-[deaminase]-[Cas9]-[COOH], [NH2]-[Cas9]-[NLS]-[deaminase]-[COOH], [NH2]-[deaminase]-[NLS]-[Cas9]-[COOH], [NH2]-[deaminase]-[Cas9]-[NLS]-[COOH], or [NH2]-[Cas9]-[deaminase]-[NLS]-[COOH] Here, NLS is a nuclear localization signal, NH2 is the N-terminus of the fusion protein, and COOH is the C-terminus of the fusion protein. In some embodiments, the linker is inserted between the Cas9 protein and the deaminase domain. In some embodiments, ]-[ may be one or more linkers. In some embodiments, the NLS is located at the C-terminus of the deaminase and / or Cas9 domain. In some embodiments, the NLS is located between the deaminase and the Cas9 domain. Further features (e.g., sequence tags) may be present.

[0160] Examples of suitable effector domains include, for instance, the APOBEC family of cytosine deaminases. The apolipoprotein B messenger RNA editing complex (APOBEC) family of cytosine deaminase enzymes includes 11 proteins useful for controlled and advantageous mutagenesis. (29) Activation-inducible cytidine deaminase (AID), a member of the family, is involved in antibody maturation by converting cytosine in ssDNA to uracil in a transcription-dependent, strand-biased manner. (30) The apolipoprotein B editing complex 3 (APOBEC3) enzyme protects human cells by deaminating cytosine from reverse-transcribed viral ssDNA in response to certain HIV-1 strains. (31) These proteins, for catalytic activity, use Zn 2+ Coordination motif (His-X-Glu-X 23~26 -Pro-Cys-X2~4 -Cys (SEQ ID NO: 283) and all bound water molecules are required. The Glu residue activates the water molecule to form zinc hydroxide for nucleophilic attack in the deamination reaction. Each family selectively deaminates its own specific "hot spot," such as WRC (W is A or T, and R is A or G) in hAID and TTC in hAPOBEC3F. (32) Recent crystal structure analyses of the catalytic domain of APOBEC3G have revealed a secondary structure consisting of a five-stranded α-sheet core flanked by six α-helices, and this structure is thought to be conserved across the entire family. (33) The active site loop has been shown to be involved in both binding to ssDNA and determining the identity of the "hotspot." (34) Overexpression of these enzymes is associated with genomic instability and cancer, thus highlighting the importance of sequence-specific targeting. (35) .

[0161] Some aspects of the disclosure of the present invention relate to providing a systematic series of fusions between Cas9 and deaminase domains (e.g., cytosine deaminase enzymes such as APOBEC enzyme or adenosine deaminase enzymes such as ADAT enzyme), which, upon construction, allow the enzymatic activity of these deaminases to act on specific sites in genomic DNA. The advantages of using Cas9 as a recognition agent are twofold: (1) the sequence specificity of Cas9 can be easily altered simply by changing the sgRNA sequence, and (2) Cas9 binds to its target sequence by denaturing dsDNA, resulting in the DNA being extended, which, being single-stranded, becomes a substrate for the deaminase. It will be understood that fusion proteins with Cas9 can be constructed using other catalytic domains or catalytic domains derived from other deaminases, and in this regard, the disclosure is not limited to this specification.

[0162] Some aspects of the disclosure of this invention are based on the finding that the Cas9:deaminase fusion protein can efficiently deaminate nucleotides at positions 3–11 in Figure 11. Those skilled in the art can, of course, design suitable guide RNAs to target the fusion protein to target sequences containing the deaminated nucleotides. Both the PAM-dependent Cas9 protein and the Cas9 protein capable of targeting PAM-free target sequences, as described herein, can be used for the deamination of target nucleotides.

[0163] Several suitable nucleic acid editing domains (e.g., deaminase and deaminase domains) that can be fused with the Cas9 domain relating to aspects of the disclosure of the present invention are shown below. Typically, the deaminase is Zn 2+ Coordination motif (His-X-Glu-X 23~26 -Pro-Cys-X 2~4 -Cys (SEQ ID NO: 283) and bound water molecules are required for catalytic activity. The Glu residue activates water molecules to form zinc hydroxide for nucleophilic attack in the deamination reaction. In some embodiments, it will be understood that the active domain of each sequence can be a domain without a localization signal (nuclear localization signal, no nuclear export signal, cytoplasmic localization signal), for example.

[0164] Human AID:

number

[0165] Mouse AID:

number

[0166] Dog AID:

number

[0167] Bovine AID:

number

[0168] Mouse APOBEC-3:

number

[0169] Rat APOBEC-3:

number

[0170] Rhesus macaque APOBEC-3G:

number

[0171] Chimpanzee APOBEC-3G:

number

[0172] Green monkey APOBEC-3G:

number

[0173] Human APOBEC-3G:

number

number

[0174] Human APOBEC-3F:

number

[0175] Human APOBEC-3B:

number

[0176] Human APOBEC-3C:

number

[0177] Human APOBEC-3A:

number

[0178] Human APOBEC-3H:

number

[0179] Human APOBEC-3D:

number

[0180] Human APOBEC-1: MTSEKGPSTGDPTLRRRIEPWEFDVFYDPRELRKEACLLYEIKWGMSRKIWRSSGKNTTNHVEVNFIKKFTSERDFHPSMSCSITWFLSWSPCWECSQAIREFLSRHPGVTLVIYVARLFWHMDQQNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCIILSLPPCLKISRRWQNHLTFFRLHLQNCHYQTIPPHILLATGLIHPSVAWR(Sequence ID 279)

[0181] Mouse APOBEC-1: MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSVWRHTSQNTSNHVEVNFLEKFTTERYFRPNTRCSITWFLSWSPCGECSRAITEFLSRHPYVTLFIYIARLYHHTDQRNRQGLRDLISSGVTIQIMTEQEYCYCWRNFVNYPPSNEAYWPRYPHLWVKLYVLELYCIILGLPPCLKILRRKQPQLTFFTITLQTCHYQRIPPHLLWATGLK (Sequence ID 280)

[0182] Rat APOBEC-1: MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK (Sequence ID 281)

[0183] In some embodiments, the fusion proteins described herein include the full-length amino acids of the effector domain (e.g., any of the sequences shown above). However, in other embodiments, the fusion proteins described herein include only fragments of the effector domain, rather than the full-length sequence. For example, in some embodiments, the fusion proteins provided herein include a Cas9 protein and a fragment of the effector domain, the fragment comprising the effector domain. Examples of amino acid sequences of the effector domain are shown in italics above, but further preferred sequences of such domains will be obvious to those skilled in the art.

[0184] Further preferred nucleic acid editing domains (e.g., deaminase domain sequences) that can be used in accordance with aspects of the disclosure of the present invention and can be fused with, for example, nuclease-inactive Cas9 proteins are obvious to those skilled in the art based on the disclosure of the present invention. In some embodiments, such further domain sequences include deaminase domain sequences having at least 70 percent, at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, or at least 99 percent similarity to the sequences shown herein. Further preferred Cas9 proteins, variants, and sequences are obvious to those skilled in the art. Further suitable examples of such Cas9 proteins include, but are not limited to, Cas9 proteins having the following mutations: D10A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A (see, for example, Prashant et al, CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31(9): 833-838, the full disclosure is incorporated herein by reference).

[0185] Further preferred strategies for constructing fusion proteins comprising the Cas9 protein and an effector domain (e.g., a DNA editing domain) are obvious to those skilled in the art, in combination with common technical knowledge, based on the disclosures of this invention. Preferred strategies for constructing the fusion proteins according to the disclosures of this invention, with or without the use of a linker, are obvious to those skilled in the art, in view of the descriptions herein and the technical knowledge of the art. For example, Gilbert et al, CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. 2013; 154(2): 442-51, show that a C-terminal fusion of Cas9 with VP64, using two NLS (SPKKKRKVEAS, SEQ ID NO: 284) as linkers, can be used for transcriptional activation. Mali et al, "Cas9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering." Nat Biotechnol. 2013; 31(9): 833-8, reports that a C-terminal fusion with VP64, without the use of a linker, can be used for transcriptional activation. Additionally, Maeder et al, "CRISPR RNA-guided activation of endogenous human genes." Nat Methods. 2013; 10: 977-979, reports that a C-terminal fusion with VP64 using the Gly4Ser (SEQ ID NO: 5) linker can be used as a transcriptional activator.In recent years, the creation of dCas9-FokI nuclease fusions has been successful, and it has been reported that they exhibit improved enzyme specificity compared to the parent Cas9 enzyme (Guilinger JP, Thompson DB, Liu DR. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 2014; 32(6): 577-82, and Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V, Reyon D, Goodwin MJ, Aryee MJ, Joung JK. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat. Biotechnol. 2014; 32(6): 569-76, and PMID:24770325). The SGSETPGTSESATPES (SEQ ID NO: 7) or GGGGS (SEQ ID NO: 5) linker is used in the FokI-dCas9 fusion protein, respectively.

[0186] In some embodiments, the Cas9 fusion protein comprises: (i) the Cas9 protein, and (ii) a transcriptional activator domain. In some embodiments, the transcriptional activator domain comprises VPR, which is the VP64-SV40-P65-RTA ternary activator. In some embodiments, the VPR comprises a VP64 amino acid sequence encoded by the nucleic acid sequence of Sequence ID No. 292:GAGGCCAGCGGTTCCGGACGGGCTGACGCATTGGACGATTTTGATCTGGATATGCTGGGAAGTGACGCCCTCGATGATTTTGACCTTGACATGCTTGGTTCGGATGCCCTTGATGACTTTGACCTCGACATGCTCGGCAGTGACGCCCTTGATGATTTCGACCTGGACATGCTGATTAACTCTAGATAG (Sequence ID No. 292).

[0187] In some embodiments, VPR comprises the VP64 amino acid sequence shown in SEQ ID NO: 293:EASGSGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLINSR (SEQ ID NO: 293).

[0188] In some embodiments, VPR is sequence number 294:

[0189] In some embodiments, VPR is sequence number 295: It contains the VP64-SV40-P65-RTA amino acid sequence shown in (Sequence ID 295).

[0190] Some aspects of the disclosure of the present invention relate to the provision of a fusion protein comprising a transcription activator. In some embodiments, the transcription activator is VPR. In some embodiments, the VPR comprises wild-type VPR or the VPR described in SEQ ID NO: 293. In some embodiments, the VPR protein provided in the present invention comprises fragments of VPR, as well as proteins homologous to VPR or VPR fragments. For example, in some embodiments, the VPR comprises a fragment of the amino acid sequence described in SEQ ID NO: 293. In some embodiments, the VPR comprises an amino acid sequence homologous to the amino acid sequence described in SEQ ID NO: 293, or an amino acid sequence homologous to a fragment of the amino acid sequence described in SEQ ID NO: 293. In some embodiments, a protein comprising VPR or a fragment of VPR, or a protein homologous to VPR or a fragment of VPR, is referred to as a "VPR variant." A VPR variant shares homology with VPR or a fragment thereof. For example, a VPR variant is at least about 70 percent identical, at least about 80 percent identical, at least about 90 percent identical, at least about 95 percent identical, at least about 96 percent identical, at least about 97 percent identical, at least about 98 percent identical, at least about 99 percent identical, at least about 99.5 percent identical, or at least about 99.9 percent identical to the wild-type VPR or the VPR described in SEQ ID NO: 293. In some embodiments, the VPR variant comprises a fragment of VPR, which is at least about 70 percent identical, at least about 80 percent identical, at least about 90 percent identical, at least about 95 percent identical, at least about 96 percent identical, at least about 97 percent identical, at least about 98 percent identical, at least about 99 percent identical, at least about 99.5 percent identical, or at least about 99.9 percent identical to the wild-type VPR or the corresponding fragment of VPR described in SEQ ID NO: 293. In some embodiments, the VPR comprises the amino acid sequence described in SEQ ID NO: 293.In some embodiments, the VPR comprises an amino acid sequence encoded by the nucleotide sequence described in SEQ ID NO: 292.

[0191] In some embodiments, VPR is a three-element activator of VP64-SV40-P65-RTA. In some embodiments, VP64-SV40-P65-RTA comprises VP64-SV40-P65-RTA as shown in SEQ ID NO: 295. In some embodiments, the VP64-SV40-P65-RTA protein provided in the present invention comprises a fragment of VP64-SV40-P65-RTA, as well as proteins homologous to VP64-SV40-P65-RTA and the VP64-SV40-P65-RTA fragment. For example, in some embodiments, VP64-SV40-P65-RTA comprises a fragment of the amino acid sequence described in SEQ ID NO: 295. In some embodiments, VP64-SV40-P65-RTA comprises an amino acid sequence homologous to the amino acid sequence described in SEQ ID NO: 295, or an amino acid sequence homologous to a fragment of the amino acid sequence described in SEQ ID NO: 295. In some embodiments, proteins containing VP64-SV40-P65-RTA or a fragment of VP64-SV40-P65-RTA, or proteins homologous to VP64-SV40-P65-RTA or a fragment of VP64-SV40-P65-RTA, are referred to as "VP64-SV40-P65-RTA variants." VP64-SV40-P65-RTA variants share homology with VP64-SV40-P65-RTA or a fragment of it. For example, the VP64-SV40-P65-RTA variant is at least approximately 70 percent identical, at least approximately 80 percent identical, at least approximately 90 percent identical, at least approximately 95 percent identical, at least approximately 96 percent identical, at least approximately 97 percent identical, at least approximately 98 percent identical, at least approximately 99 percent identical, at least approximately 99.5 percent identical, or at least approximately 99.9 percent identical to the VP64-SV40-P65-RTA described in Sequence ID No. 295.In some embodiments, the VP64-SV40-P65-RTA variant comprises a fragment of VP64-SV40-P65-RTA that is at least about 70 percent identical, at least about 80 percent identical, at least about 90 percent identical, at least about 95 percent identical, at least about 96 percent identical, at least about 97 percent identical, at least about 98 percent identical, at least about 99 percent identical, at least about 99.5 percent identical, or at least about 99.9 percent identical to the corresponding fragment of VP64-SV40-P65-RTA described in SEQ ID NO: 295. In some embodiments, VP64-SV40-P65-RTA comprises the amino acid sequence described in SEQ ID NO: 295. In some embodiments, VP64-SV40-P65-RTA comprises the amino acid sequence encoded by the nucleotide sequence described in SEQ ID NO: 294.

[0192] Some aspects of the disclosure of the present invention relate to providing a fusion protein comprising (i) a Cas9 protein and (ii) an effector domain. In some aspects, the fusion protein provided in the present invention further comprises (iii) a DNA-binding protein (e.g., a Zn finger domain, TALE, or a second Cas9 protein). Without being bound by any particular theory, fusing a DNA-binding protein (e.g., a second Cas9 protein) to a fusion protein comprising (i) the protein and (ii) the effector domain may be useful to improve the specificity of the fusion protein to a target nucleic acid sequence, or to improve the specificity or binding ability of the fusion protein in binding to a target nucleic acid that does not contain the classical PAM(5'-NGG-3') sequence. In some embodiments, the second Cas9 protein is any of the Cas9 proteins provided in the present invention. In some embodiments, the second Cas9 protein is fused to the N-terminus of the Cas9 protein of the fusion protein. In some embodiments, the second Cas9 protein is fused to the C-terminus of the Cas9 protein of the fusion protein. In some embodiments, the Cas9 protein and the second Cas9 protein fuse via a linker.

[0193] The present invention further provides a complex comprising one of the fusion proteins provided in the present invention, a first guide RNA that binds to the Cas9 protein of the fusion protein, and a second guide RNA that binds to the second Cas9 protein of the fusion protein. In some embodiments, the first guide RNA is about 15 to 100 nucleotides long and contains at least 10 consecutive nucleotides complementary to a first target sequence, and the second guide RNA is about 15 to 100 nucleotides long and contains at least 10 consecutive nucleotides complementary to a second target sequence. In some embodiments, the first guide RNA and / or the second guide RNA are 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long. In some embodiments, the first guide RNA and the second guide RNA are different. In some embodiments, the first guide RNA contains 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 consecutive nucleotides complementary to the first target sequence, and the second guide RNA contains 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 consecutive nucleotides complementary to the second target sequence. In some embodiments, the first and second target sequences are different. In some embodiments, the first and second target sequences are DNA base sequences. In some embodiments, the first and second target sequences are present in the mammalian genome. In some embodiments, the first and second target sequences are present in the human genome. In some embodiments, the first target sequence is located within 30 nucleotides of the second target sequence. In some embodiments, the 3' end of the first target sequence is not directly adjacent to the classical PAM sequence (5'-NGG-3').In some embodiments, the 3' end of the second target sequence is not directly adjacent to the classical PAM sequence (5'-NGG-3').

[0194] In some embodiments, the general structure of the exemplary Cas9 fusion protein provided in the present invention is: [NH2]-[effector domain]-[Cas9]-[second Cas9 protein]-[COOH]; [NH2]-[Second Cas9 protein]-[Cas9]-[Effector domain]-[COOH]; [NH2]-[Cas9]-[effector domain]-[second Cas9 protein]-[COOH]; [NH2]-[Second Cas9 protein]-[Effector or domain]-[Cas9]-[COOH]; [NH2]-[UGI]-[effector domain]-[Cas9]-[second Cas9 protein]-[COOH]; [NH2]-[UGI]-[Second Cas9 protein]-[Cas9]-[Effector domain]-[COOH]; [NH2]-[UGI]-[Cas9]-[Effector Domain]-[Second Cas9 Protein]-[COOH]; [NH2]-[UGI]-[Second Cas9 protein]-[Effector domain]-[Cas9]-[COOH]; [NH2]-[effector domain]-[Cas9]-[second Cas9 protein]-[UGI]-[COOH]; [NH2]-[Second Cas9 protein]-[Cas9]-[Effector domain]-[UGI]-[COOH]; [NH2]-[Cas9]-[effector domain]-[second Cas9 protein]-[UGI]-[COOH]; or It has the structure of [[NH2]]-[the second Cas9 protein]-[effector domain]-[Cas9]-[UGI]-[COOH], where [[NH2]] is the N-terminus of the fusion protein and [[COOH]] is the C-terminus of the fusion protein. In some embodiments, the "]-[ " used in the above general composition indicates the presence of any linker sequence. In other examples, the general composition of the exemplary Cas9 fusion proteins provided by the present invention is [[NH2]]-[effector domain]-[Cas9]-[the second Cas9 protein]-[COOH]; [[NH2]]-[the second Cas9 protein]-[Cas9]-[effector domain]-[COOH]; [[NH2]]-[Cas9]-[effector domain]-[the second Cas9 protein]-[COOH]; [[NH2]]-[the second Cas9 protein]-[effector domain]-[Cas9]-[COOH]; [[NH2]]-[UGI]-[effector domain]-[Cas9]-[the second Cas9 protein]-[COOH]; [[NH2]]-[UGI]-[the second Cas9 protein]-[Cas9]-[effector domain]-[COOH];[[ID=1,3]] [[NH2]]-[UGI]-[Cas9]-[effector domain]-[the second Cas9 protein]-[COOH]; [[NH2]]-[UGI]-[the second Cas9 protein]-[effector domain]-[Cas9]-[COOH]; [[NH2]]-[effector domain]-[Cas9]-[the second Cas9 protein]-[UGI]-[COOH]; [[NH2]]-[the second Cas9 protein]-[Cas9]-[effector domain]-[UGI]-[COOH]; [[NH2]]-[Cas9]-[effector domain]-[the second Cas9 protein]-[UGI]-[COOH]; or It has the structure of [[NH2]]-[the second Cas9 protein]-[effector domain]-[Cas9]-[UGI]-[[COOH]], where [[NH2]] is the N-terminus of the fusion protein and [[COOH]] is the C-terminus of the fusion protein. In some embodiments, the "-" used in the above general composition indicates the presence of any linker sequence. In some embodiments, the second Cas9 is a dCas9 protein. In some embodiments, the general composition of the exemplary Cas9 fusion proteins provided by the present invention includes the structure shown in FIG. 8. It is understood that any of the proteins shown in the general composition of the exemplary Cas9 fusion proteins can be linked by one or more linkers provided by the present invention. In some embodiments, the linkers are identical. In some embodiments, the linkers are different. In some embodiments, one or more of the proteins shown in any of the general compositions of the exemplary Cas9 fusion proteins do not fuse via a linker. In some embodiments, the fusion protein further includes a nuclear targeting sequence (e.g., a nuclear localization sequence). In some embodiments, the fusion proteins provided by the present invention further include a nuclear localization sequence (NLS). In some embodiments, the NLS fuses to the N-terminus of the fusion protein. In some embodiments, the NLS fuses to the C-terminus of the fusion protein. In some embodiments, the NLS fuses to the N-terminus of the second Cas9 protein. In some embodiments, the NLS fuses to the C-terminus of the second Cas9 protein. In some embodiments, the NLS fuses to the N-terminus of the Cas9 protein. In some embodiments, the NLS fuses to the C-terminus of the Cas9 protein. In some embodiments, the NLS fuses to the N-terminus of the effector domain. In some embodiments, the NLS fuses to the C-terminus of the effector domain. In some embodiments, the NLS fuses to the N-terminus of the UGI protein. In some embodiments, the NLS fuses to the C-terminus of the UGI protein. In some embodiments, the NLS fuses to the fusion protein via one or more linkers. In some embodiments, the NLS fuses to the fusion protein without a linker.

[0195] Uracil glycosylase inhibitor fusion protein Some aspects of the disclosure of the present invention provide fusion proteins comprising a Cas9 protein fused to an effector domain, such as a deaminase and a uracilglycosylase inhibitor (UGI). In some embodiments, the fusion protein comprises the following structure: [Deaminase]-[Arbitrary linker sequence]-[Cas9]-[Arbitrary linker sequence]-[UGI]; [Deaminase]-[Arbitrary linker sequence]-[UGI]-[Arbitrary linker sequence]-[Cas9]; [UGI]-[arbitrary linker sequence]-[deaminase]-[arbitrary linker sequence]-[Cas9]; [UGI]-[arbitrary linker sequence]-[Cas9]-[arbitrary linker sequence]-[deaminase]; [Cas9]-[any linker sequence]-[deaminase]-[any linker sequence]-[UGI]; or [Cas9]-[arbitrary linker sequence]-[UGI]-[arbitrary linker sequence]-[deaminase]. In some embodiments, the fusion protein does not contain a linker sequence. In some embodiments, one or both of any linker sequences are present.

[0196] In some embodiments, the fusion protein further comprises a second Cas9 protein. For example, the second Cas9 protein may be any of the Cas9 proteins provided in the present invention. In some embodiments, the fusion protein comprises the following structure: [deaminase]-[Cas9]-[UGI]; [deaminase]-[UGI]-[Cas9]; [UGI]-[deaminase]-[Cas9]; [UGI]-[Cas9]-[deaminase]; [Cas9]-[deaminase]-[UGI]; [Cas9]-[UGI]-[deaminase]; [Second Cas9]-[deaminase]-[Cas9]-[UGI]; [Second Cas9]-[deaminase]-[UGI]-[Cas9]; [Second Cas9]-[UGI]-[deaminase]-[Cas9]; [Second Cas9]-[UGI]-[Cas9]-[deaminase]; [Second Cas9]-[Cas9]-[deaminase]-[UGI]; [Second Cas9]-[Cas9]-[UGI]-[deaminase]; [Deaminase]-[Second Cas9]-[Cas9]-[UGI]; [Deaminase]-[Second Cas9]-[UGI]-[Cas9]; [UGI]-[Second Cas9]-[Deaminase]-[Cas9]; [UGI]-[Second Cas9]-[Cas9]-[Deaminase]; [Cas9]-[Second Cas9]-[Deaminase]-[UGI]; [Cas9]-[Second Cas9]-[UGI]-[Deaminase]; [Deaminase]-[Cas9]-[Second Cas9]-[UGI]; [Deaminase]-[UGI]-[Second Cas9]-[Cas9]; [UGI]-[deaminase]-[second Cas9]-[Cas9]; [UGI]-[Cas9]-[Second Cas9]-[Deaminase]; [Cas9]-[deaminase]-[second Cas9]-[UGI]; [Cas9]-[UGI]-[Second Cas9]-[Deaminase]; [Deaminase]-[Cas9]-[UGI]-[Second Cas9]; [Deaminase]-[UGI]-[Cas9]-[Second Cas9]; [UGI]-[deaminase]-[Cas9]-[second Cas9]; [UGI]-[Cas9]-[deaminase]-[second Cas9]; [Cas9]-[deaminase]-[UGI]-[second Cas9]; or [Cas9]-[UGI]-[deaminase]-[second Cas9]. In some embodiments, the "-" used in the above general configuration indicates the presence of an arbitrary linker sequence. In some embodiments, the fusion protein containing UGI further includes a nuclear targeting sequence (e.g., a nuclear localization sequence). In some embodiments, the fusion protein provided in the present invention further includes a nuclear localization sequence (NLS). In some embodiments, the NLS is fused to the N-terminus of the fusion protein. In some embodiments, the NLS is fused to the C-terminus of the fusion protein. In some embodiments, the NLS is fused to the N-terminus of the UGI protein. In some embodiments, the NLS is fused to the C-terminus of the UGI protein. In some embodiments, the NLS is fused to the N-terminus of the Cas9 protein. In some embodiments, the NLS is fused to the C-terminus of the Cas9 protein. In some embodiments, the NLS is fused to the N-terminus of the deaminase. In some embodiments, the NLS is fused to the C-terminus of the deaminase. In some embodiments, the NLS is fused to the N-terminus of a second Cas9 protein. In some embodiments, the NLS is fused to the C-terminus of a second Cas9 protein. In some embodiments, the NLS fuses to the fusion protein via one or more linkers. In some embodiments, the NLS fuses to the fusion protein without the use of linkers.

[0197] In some embodiments, UGI includes wild-type UGI or the UGI shown in Sequence ID No. 553. In some embodiments, the UGI protein provided in the present invention includes a fragment of UGI, as well as a protein homologous to UGI or a UGI fragment. For example, in some embodiments, UGI includes a fragment of the amino acid sequence described in Sequence ID No. 553. In some embodiments, UGI includes an amino acid sequence homologous to the amino acid sequence described in Sequence ID No. 553, or an amino acid sequence homologous to a fragment of the amino acid sequence described in Sequence ID No. 553. In some embodiments, a protein containing UGI or a UGI fragment, or a homolog of UGI or a UGI fragment, is referred to as a "UGI variant." UGI variants share homology with UGI or a fragment thereof. For example, a UGI variant is at least about 70 percent identical, at least about 80 percent identical, at least about 90 percent identical, at least about 95 percent identical, at least about 96 percent identical, at least about 97 percent identical, at least about 98 percent identical, at least about 99 percent identical, at least about 99.5 percent identical, or at least about 99.9 percent identical to the wild-type UGI or the UGI described in SEQ ID NO: 553. In some embodiments, the UGI variant comprises a fragment of UGI, which is at least about 70 percent identical, at least about 80 percent identical, at least about 90 percent identical, at least about 95 percent identical, at least about 96 percent identical, at least about 97 percent identical, at least about 98 percent identical, at least about 99 percent identical, at least about 99.5 percent identical, or at least about 99.9 percent identical to the wild-type UGI or the corresponding fragment of UGI described in SEQ ID NO: 553. In some embodiments, the UGI comprises the following amino acid sequence: >sp|P14739|UNGI_BPPB2 Uracil-DNA glycosylase inhibitor MTLSDIIEKETGKQLVIQESILMLPEEVEEVIGKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGEKIKML (Sequence ID 553).

[0198] Suitable UGI proteins and nucleotide sequences are provided herein, and further suitable UGI sequences are also well known to those skilled in the art, for example, Wang et al., Uracil-DNA glycosylase inhibitor gene of bacteriophage PBS2 encodes a binding protein specific for uracil-DNA glycosylase. J. Biol. Chem. 264: 1163-1171 (1989); Lundquist et al., Site-directed mutagenesis and characterization of uracil-DNA glycosylase inhibitor protein. Role of specific carboxylic amino acids in complex formation with Escherichia coli uracil-DNA glycosylase. J. Biol. Chem. 272:21408-21419 (1997); Ravishankar et al., X-ray analysis of a complex of Escherichia coli uracil DNA glycosylase (EcUDG) with a proteinaceous inhibitor. The structure elucidation of a Examples include prokaryotic UDG. Nucleic Acids Res. 26: 4880-4887 (1998), and Putnam et al., Protein mimicry of DNA from crystal structures of the uracil-DNA glycosylase inhibitor protein and its complex with Escherichia coli uracil-DNA glycosylase. J. Mol. Biol. 287:331-346 (1999), the full disclosures of which are incorporated herein by reference.

[0199] Naturally, the additional protein may also be a uracilglycosylase inhibitor. For example, other proteins that can inhibit (e.g., sterically block) uracil-DNA glycosylase base-cleavage repair enzyme are also within the scope of the disclosure of this invention. In some embodiments, the uracilglycosylase inhibitor is a protein that binds to DNA. In some embodiments, the uracilglycosylase inhibitor is a protein that binds to single-stranded DNA. For example, the uracilglycosylase inhibitor may be a single-strand binding protein of Erwinia tasmaniensis. In some embodiments, the single-strand binding protein comprises an amino acid sequence (SEQ ID NO: 303). In some embodiments, the uracilglycosylase inhibitor is a protein that binds to uracil. In some embodiments, the uracilglycosylase inhibitor is a protein that binds to uracil in DNA. In some embodiments, the uracilglycosylase inhibitor is a catalytically inactive uracil DNA-glycosylase protein. In some embodiments, the uracil glycosylase inhibitor is a catalytically inactive uracil DNA-glycosylase protein that does not excise uracil from DNA. For example, the uracil glycosylase inhibitor is UdgX. In some embodiments, UdgX comprises the amino acid sequence (SEQ ID NO: 304). As another example, the uracil glycosylase inhibitor is a catalytically inactive UDG. In some embodiments, the catalytically inactive UDG comprises the amino acid sequence (SEQ ID NO: 305). Naturally, other uracil glycosylase inhibitors are obvious to those skilled in the art and fall within the scope of the disclosure of this invention. SSB (thermal-stable single-stranded DNA-binding protein) of Erwinia tasmaniensis MASRGVNKVILVGNLGQDPEVRYMPNGGAVANITLATSESWRDKQTGETKEKTEWHRVVLFGKLAEVAGEYLRKGSQVYIEGALQTRKWTDQAGVEKYTTEVVVNVGGTMQMLGGRSQGGGASAGGQNGGSNNGWGQPQQPQGGNQFSGGAQQQARPQQQPQQNNAPANNEPPIDFDDDIP(Sequence ID 303) UdgX (binds to uracil in DNA but does not excise it) MAGAQDFVPHTADLAELAAAAGECRGCGLYRDATQAVFGAGGRSARFMIGEQPGDKEDLAGLPFVGPAGRLLDRALEAADIDRDALYVTNAVKHFKFTRAAGGKRRIHKTPSRTEVVACRPWLIAEMTSVEPDVVVLLGATAAKALLGNDFRVTQFIRGEVLHVDDVPGDPALVATVHPSSLLRGPKEERESAFAGLVDDLRVAADVRP (Sequence ID 304)UDG (catalytically inactive human UDG, which binds to uracil in DNA but does not excise it) MIGQKTLYSFFSPSPARKRHAPSPEPAVQGTGVAGVPEESGDAAAIPAKKAPAGQEEPGTPPSSPLSAEQLDRIQRNKAAALLRLAARNVPVGFGESWKKHLSGEFGKPYFIKLMGFVAEERKHYTVYPPPHQVFTWTQMCDIKDVKVVILGQEPYHGPNQAHGLCFSVQRPVPPPPSLENIYKELSTDIEDFVHPGHGDLSGWAKQGVLLLNAVLTVRAHQANSHKERGWEQFTDAVVSWLNQNSNGLVFLLWGSYAQKKGSAIDRKRHHVLQTAHPSPLSVYRGFFGCRHFSKTNELLQKSGKKPIDWKEL (Sequence ID 305)

[0200] High-fidelity Cas9 Some aspects of the disclosure of the present invention relate to providing highly accurate Cas9 proteins. In some embodiments, highly accurate Cas9 proteins have lower electrostatic interactions between the Cas9 protein and the sugar-phosphate backbone of DNA compared to the wild-type Cas9 domain. In some embodiments, any of the Cas9 proteins provided in the present invention include one or more mutations that reduce the association between the Cas9 protein and the sugar-phosphate backbone of DNA. In some embodiments, any of the Cas9 proteins provided in the present invention include one or more mutations that reduce the association between the Cas9 protein and the sugar-phosphate backbone of DNA by at least 5 percent, at least 10 percent, at least 15 percent, at least 20 percent, at least 25 percent, at least 30 percent, at least 35 percent, at least 40 percent, at least 45 percent, at least 50 percent, at least 60 percent, at least 70 percent, at least 80 percent, at least 90 percent, or at least 95 percent. In some embodiments, any of the Cas9 proteins provided in the present invention include one or more N497X, R661X, Q695X and / or Q926X mutations in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10-262, where X is any of the amino acids. In some embodiments, any of the Cas9 proteins provided in the present invention include one or more N497A, R661A, Q695A and / or Q926A mutations in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10-262. In some embodiments, the Cas9 protein includes the D10A mutation in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10-262. In some embodiments, the Cas9 protein includes the amino acid sequence shown in SEQ ID NO: 306. More accurate Cas9 proteins are described in the prior art literature and are obvious to those skilled in the art.For example, highly accurate Cas9 proteins are described in Kleinstiver, B.P., et al. "High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects." Nature 529, 490-495 (2016), and Slaymaker, I.M., et al. "Rationally engineered Cas9 nucleases with improved specificity." Science 351, 84-88 (2015), the entire disclosures of each of which are incorporated herein by reference. Of course, based on the disclosure of the present invention and common technical knowledge, any Cas9 protein can be mutated to create a highly accurate Cas9 protein with reduced electrostatic interaction between the Cas9 protein and the sugar-phosphate backbone of DNA as compared with the wild-type Cas9 domain. The mutation sites of the Cas9 domain with respect to Cas9 of SEQ ID NO: 9 are shown in bold and underlined.

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[0201] Cas9 domain with reduced PAM exclusivity Several aspects of this disclosure provide Cas9 proteins with different PAM specificities. Typically, Cas9 proteins, e.g., Cas9 from S. pyogenes (spCas9), require classical NGG PAM sequences to bind to specific nucleic acid regions. Their ability to bind to nucleotide sequences that do not contain classical (e.g., NGG) PAM sequences may be limited. For example, Cas9 proteins that bind to non-classical PAM sequences are described in Kleinstiver, BP, et al., "Engineered CRISPR-Cas9 nucleases with altered PAM specificities" Nature 523, 481-485 (2015); and Kleinstiver, BP, et al., "Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition" Nature Biotechnology 33, 1293-1298 (2015); the entire contents of each are incorporated herein by reference.

[0202] In some embodiments, the Cas9 protein is the Cas9 protein from Staphylococcus aureus (SaCas9). In some embodiments, the SaCas9 protein is nuclease-active SaCas9, nuclease-inactive SaCas9 (SaCas9d), or SaCas9 niccas (SaCas9n). In some embodiments, SaCas9 comprises the amino acid sequence SEQ ID NO: 307. In some embodiments, SaCas9 comprises the N579X mutation in SEQ ID NO: 307, or the corresponding mutation in any of the amino acid sequences provided by SEQ ID NOs: 9-262, where X is any amino acid other than N. In some embodiments, SaCas9 comprises the N579A mutation in SEQ ID NO: 307, or the corresponding mutation in any of the amino acid sequences provided by SEQ ID NOs: 10-262. In some embodiments, the SaCas9 protein, SaCas9d protein, or SaCas9n protein can bind to nucleic acid sequences having a non-classical PAM. In some embodiments, the SaCas9 protein, SaCas9d protein, or SaCas9n protein may bind to a nucleic acid sequence having an NNGRRT PAM sequence. In some embodiments, the SaCas9 protein contains one or more E781X, N967X, or R1014X mutations of SEQ ID NO: 307, or a corresponding mutation in any of the amino acid sequences provided by SEQ ID NOs: 10-262, where X is any of the amino acids. In some embodiments, the SaCas9 protein contains one or more E781K, N967K, or R1014H mutations of SEQ ID NO: 307, or one or more corresponding mutations in any of the amino acid sequences provided by SEQ ID NOs: 10-262. In some embodiments, the SaCas9 protein contains the E781K, N967K, and R1014H mutations of SEQ ID NO: 307, or a corresponding mutation in any of the amino acid sequences provided by SEQ ID NOs: 10-262. It should be understood that these mutations may be combined with any of the other mutations provided herein.

[0203] In some embodiments, the Cas9 protein of any of the fusion proteins provided herein contains an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of SEQ ID NOs. 307-309. In some embodiments, the Cas9 protein of any of the fusion proteins provided herein contains an amino acid sequence of any one of SEQ ID NOs. 307-309. In some embodiments, the Cas9 protein of any of the fusion proteins provided herein consists of an amino acid sequence of any one of SEQ ID NOs. 307-309. Example SaCas9 sequence

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[0204] In some embodiments, the Cas9 protein is the Cas9 protein from Streptococcus pyogenes (SpCas9). In some embodiments, the SpCas9 protein is nuclease-active SpCas9, nuclease-inactive SpCas9 (SpCas9d), or SpCas9 nickase (SpCas9n). In some embodiments, SpCas9 comprises the amino acid sequence SEQ ID NO: 9. In some embodiments, SpCas9 comprises the D10X mutation in SEQ ID NO: 9, or the corresponding mutation in any of the amino acid sequences provided by SEQ ID NOs: 10-262, where X is any amino acid other than D. In some embodiments, SpCas9 comprises the D10A mutation in SEQ ID NO: 9, or the corresponding mutation in any of the amino acid sequences provided by SEQ ID NOs: 10-262. In some embodiments, the SpCas9 protein, SpCas9d protein, or SpCas9n protein can bind to nucleic acid sequences having a non-classical PAM. In some embodiments, the SpCas9 protein, SpCas9d protein, or SpCas9n protein can bind to nucleic acid sequences having NGG, NGA, or NGCG PAM sequences. In some embodiments, the SpCas9 protein contains one or more D1135X, R1335X, and T1337X mutations of SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences provided by SEQ ID NOs: 10-262, where X is any of the amino acids. In some embodiments, the SpCas9 protein contains one or more D1135E, R1335Q, and T1335R mutations of SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences provided by SEQ ID NOs: 10-262. In some embodiments, the SpCas9 protein contains the D1135E, R1335Q, and T1337R mutations of SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences provided by SEQ ID NOs: 10-262. In some embodiments, the SpCas9 protein contains one or more of the D1135X, R1335X, and T1338X mutations of SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences provided by SEQ ID NOs: 10-262, where X is any of the amino acids.In some embodiments, the SpCas9 protein contains one or more D1135V, R1335Q, and T1337R mutations of SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences provided by SEQ ID NOs: 10-262. In some embodiments, the SpCas9 protein contains one or more D1135X, G1218X, R1335X, and T1337R mutations of SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences provided by SEQ ID NOs: 10-262. In some embodiments, the SpCas9 protein contains one or more D1135X, G1218X, R1335X, and T1337X mutations of SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences provided by SEQ ID NOs: 10-262, where X is any of the amino acids. In some embodiments, the SpCas9 protein contains one or more of the D1135V, G1218R, R1335Q, and T1337R mutations of SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences provided by SEQ ID NOs: 10-262. It should be understood that these mutations may be combined with any of the other mutations provided herein.

[0205] In some embodiments, the Cas9 protein of any of the fusion proteins provided herein contains an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any one of SEQ ID NOs: 9, 310-313. In some embodiments, the Cas9 domain of any of the fusion proteins provided herein contains an amino acid sequence that is at least one of SEQ ID NOs: 9, 310-313. In some embodiments, the Cas9 domain of any of the fusion proteins provided herein consists of an amino acid sequence that is at least one of SEQ ID NOs: 9, 310-313. Example of SpCas9 Example SpCas9n Example: SpEQR Cas9

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[0206] The residues V1134, R1217, Q1334, and R1336 of sequence number 313 can be mutated from D1134, G1217, R1334, and T1336 of sequence number 9 to produce SpVRER Cas9, and these are underlined and in bold.

[0207] Cas9 complex with guide RNA Several aspects of this disclosure provide complexes comprising a Cas9 protein or Cas9 fusion protein as provided herein. In some embodiments, the guide RNA is about 15 to 100 nucleotides long and contains a sequence of at least 10 consecutive nucleotides complementary to the target sequence. In some embodiments, the guide RNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long. In some embodiments, the guide RNA contains a sequence of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides that are complementary to the target sequence. In some embodiments, the target sequence is a DNA sequence. In some embodiments, the target sequence is a sequence in the mammalian genome. In some embodiments, the target sequence is a sequence in the human genome. In some embodiments, the 3' end of the target sequence is not immediately adjacent to the classical PAM sequence (5'-NGG-3').

[0208] In some aspects of the disclosure of the present invention, a complex is provided comprising a first guide RNA bound to the Cas9 protein of a fusion protein and a second guide RNA bound to a second Cas9 protein of a fusion protein. In some embodiments, the first guide RNA is about 15 to 100 nucleotides long and contains at least 10 consecutive nucleotides complementary to a first target sequence, and the second guide RNA is about 15 to 100 nucleotides long and contains at least 10 consecutive nucleotides complementary to a second target sequence. In some embodiments, the first guide RNA and / or the second guide RNA are 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long. In some embodiments, the first guide RNA and the second guide RNA are different. In some embodiments, the first guide RNA contains 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 consecutive nucleotides complementary to the first target sequence, and the second guide RNA contains 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 consecutive nucleotides complementary to the second target sequence.

[0209] In some embodiments, the first target sequence and the second target sequence are different. In some embodiments, the first target sequence and the second target sequence are DNA base sequences. In some embodiments, the first target sequence and the second target sequence are present in the mammalian genome. In some embodiments, the first target sequence and the second target sequence are present in the human genome. In some embodiments, the first target sequence is present within at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, or 200 nucleotides of the second target sequence. In some embodiments, the 3' end of the first target sequence is not directly adjacent to the classical PAM sequence (5'-NGG-3'). In some embodiments, the 3' end of the second target sequence is not directly adjacent to the classical PAM sequence (5'-NGG-3'). How to use Cas9 fusion protein:

[0210] Method using Cas9 fusion protein Some aspects of this disclosure provide methods using Cas9 proteins, fusion proteins, or complexes provided herein. For example, some aspects of this disclosure provide methods comprising contacting a DNA molecule with a Cas9 protein, Cas9 fusion protein, or Cas9 protein or fusion protein complex with (a) one of the Cas9 proteins or fusion proteins provided herein and at least one guide RNA; or (b) at least one gRNA provided herein, wherein the guide RNA is approximately 15 to 100 nucleotides long and contains a sequence of at least 10 consecutive nucleotides complementary to the target sequence. In some embodiments, the 3' end of the target sequence is not immediately adjacent to a classical PAM sequence (5'-NGG-3'). In some embodiments, the 3' end of the target sequence is immediately adjacent to an AGC, GAG, TTT, GTG, or CAA sequence.

[0211] In some embodiments, the target DNA sequence includes a sequence associated with a disease or abnormality. In some embodiments, the target DNA sequence includes a point mutation associated with a disease or abnormality. In some embodiments, the activity of a Cas9 protein, Cas9 fusion protein, or complex results in the modification of a point mutation. In some embodiments, the target DNA sequence includes a T→C point mutation associated with a disease or abnormality, and deamination of the mutant C base results in a sequence not associated with the disease or abnormality. In some embodiments, the target DNA sequence codes for a protein, and the point mutation is in a codon, resulting in a change in the amino acid encoded by the mutant codon compared to the wild-type codon. In some embodiments, deamination of mutant C results in a change in the amino acid encoded by the mutant codon. In some embodiments, deamination of mutant C results in a codon encoding a wild-type amino acid. In some embodiments, contact is in vivo with respect to the subject. In some embodiments, the subject has or has been diagnosed with a disease or abnormality. In some embodiments, the disease or abnormality is a neoplasm associated with cystic fibrosis, phenylketonuria, exfoliative hyperkeratosis (EHK), Charcot-Marie-Tooth disease type 4J, neuroblastoma (NB), von Willebrand disease (vWD), congenital myotonia, hereditary renal amyloidosis, dilated cardiomyopathy (DCM), hereditary lymphedema, familial Alzheimer's disease, HIV, prion disease, chronic infantile neurocutaneous arthral syndrome (CINCA), desmin-associated myopathy (DRM), mutant PI3KCA protein, mutant CTNNB1 protein, mutant HRAS protein, or mutant p53 protein.

[0212] Several embodiments provide methods for using the Cas9 DNA editing fusion protein provided herein. In some embodiments, the fusion protein is used to introduce a point mutation into a nucleic acid by deaminating a target nucleic acid base, such as a C residue. In some embodiments, deamination of the target nucleic acid base results in the correction of a gene deletion, such as the correction of a point mutation leading to loss of function of a gene product. In some embodiments, the gene deletion is associated with a disease or abnormality, such as lysosomal storage abnormalities or metabolic disorders such as type 1 diabetes. In some embodiments, the methods provided herein are used to introduce an inactivating point mutation into a gene or allele encoding a gene product associated with the disease or abnormality. For example, in some embodiments, methods are provided herein for using the Cas9 DNA editing fusion protein to introduce an inactivating point mutation into an oncogene (e.g., for the treatment of proliferative disorders). In some embodiments, the inactivating mutation may create an immature stop codon in the coding sequence, which results in the expression of a truncated gene product, such as a truncated protein lacking the function of a full-length protein.

[0213] In some embodiments, the objective of the methods provided herein is to restore the function of a dysfunctional gene by genome editing. The Cas9 deaminase fusion proteins provided herein can be validated in vitro for gene-editing-based human therapies, for example, by correcting disease-associated mutations in human cell cultures. It will be understood by those skilled in the art that the fusion proteins provided herein, for example, fusion proteins comprising a Cas9 domain and a nucleic acid deaminase domain, can be used to correct any single T→C or A→G point mutation. In the first case, deamination from mutant C back to U corrects the mutation, and in the latter case, deamination of C that is base-paired with mutant G, followed by one round of replication, corrects the mutation.

[0214] An exemplary disease-related mutation that can be modified in vitro or in vivo by the provided fusion protein is the H1047R(A3140G) polymorphism of the PI3KCA protein. The phosphoinositide-3-kinase catalytic alpha subunit (PI3KCA) protein works to phosphorylate the 3-OH group of the inositol ring of phosphatidylinositol. The PI3KCA gene has been found to be mutated in many different cancers, and therefore it is considered a potent oncogene. (50) In fact, the A3140G mutation is present in several NCI-60 cancer cell lines, such as HCT116, SKOV3, and T47D cell lines, which are readily available from the American Type Culture Collection (ATCC). (51) .

[0215] In some embodiments, cells containing a mutation to be corrected, such as a point mutation, for example, an A3140G point mutation in exon 20 of the PI3KCA gene resulting in an H1047R substitution in the PI3KCA protein, are brought into contact with an expression construct encoding a Cas9 deaminase fusion protein and a suitably designed sgRNA that targets the fusion protein to the respective mutation site in the encoding PI3KCA gene. Control experiments may be performed, in which the sgRNA is designed to target the fusion enzyme to a non-C residue within the PI3KCA gene. Genomic DNA from the treated cells is extracted, and the relevant sequence of the PI3KCA gene is PCR amplified and sequenced, and the activity of the fusion protein can be evaluated in human cell cultures.

[0216] It will be understood that the example of correcting a point mutation in PI3KCA is provided for illustrative purposes only and is not intended to limit the disclosure. Those skilled in the art will understand that the DNA editing fusion proteins of this disclosure may be used to correct other point mutations and mutations associated with other cancers and non-cancer diseases, including other proliferative disorders.

[0217] Successful modification of point mutations in disease-related genes and alleles opens up new strategies for gene modification with applications in therapy and basic research. Site-directed single-nucleotide alteration systems, such as the disclosed fusions of Cas9 with deaminase enzymes or domains, also have applications in "reverse" gene therapy, where certain gene functions are deliberately suppressed or lost. In those cases, site-directed mutations of Trp (TGG), Gln (CAA and CAG), or Arg (CGA) residues to immature stop codons (TAA, TAG, TGA) can be used to cause loss of protein function in vitro, ex vivo, or in vivo.

[0218] This disclosure provides methods for treating subjects diagnosed with diseases associated with or caused by point mutations that can be corrected by the Cas9 DNA editing fusion proteins provided herein. For example, in some embodiments, methods are provided for subjects having such diseases, e.g., cancer associated with the PI3KCA point mutation described above, comprising administering an effective amount of a Cas9 deaminase fusion protein that corrects the point mutation or introduces an inactivating mutation into a disease-related gene. In some embodiments, the disease is a proliferative disorder. In some embodiments, the disease is a genetic disorder. In some embodiments, the disease is a neoplasm. In some embodiments, the disease is a metabolic disorder. In some embodiments, the disease is a lysosomal storage disorder. Other diseases that can be treated by correcting point mutations or introducing inactivating mutations into disease-related genes will be known to those skilled in the art. This disclosure is not limited in this respect.

[0219] This disclosure provides methods for treating additional diseases or abnormalities, such as those related to or caused by point mutations that can be corrected by deaminase-mediated gene editing. Several such diseases are described herein, and additional suitable diseases that can be treated by the strategies and fusion proteins provided herein will be apparent to those skilled in the art based on this disclosure. Exemplary suitable diseases and abnormalities are listed below. It will be understood that the numbering of specific positions or residues in each sequence depends on the specific protein and the numbering scheme used. Numbering may differ, for example, between the precursor of a mature protein and the mature protein itself, and interspecies sequence differences may affect the numbering. Those skilled in the art will have the ability to identify any homologous protein and each residue in the nucleic acid they each encode by methods well known in the art, for example, by sequence alignment and homologous residue determination. Exemplary suitable diseases and abnormalities include, without limitation, cystic fibrosis (see, e.g., Schwank et al., Functional repair of CFTR by CRISPR / Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell stem cell. 2013; 13: 653-658; and Wu et al., Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell stem cell. 2013; 13: 659-662; neither of these uses a deaminase fusion protein to correct the gene defect); phenylketonuria—e.g., mutations from phenylalanine to serine (T>C mutations) at position 835 (mouse) or 240 (human) or homologous residue in the phenylalanine hydroxylase gene—e.g., McDonald et al., Genomics.See 1997; 39:402-405; Bernard-Soulier syndrome (BSS) - e.g., a mutation from phenylalanine to serine at position 55 or homologous residue of platelet glycoprotein IX, or a mutation from cysteine ​​to arginine (T>C mutation) at residue 24 or homologous residue - see e.g., Noris et al., British Journal of Haematology. 1997; 97: 312-320 and Ali et al., Hematol. 2014; 93: 381-384; Exfoliative hyperkeratosis (EHK) - e.g., a mutation from leucine to proline (T>C mutation) at position 160 or 161 (counting the initiating methionine) or homologous residue of keratin 1 - see e.g., Chipev et al., Cell. 1992; 70: See 821-828, also see accession number P04264 in the UNIPROT database at www.uniprot.org; Chronic obstructive pulmonary disease (COPD) - e.g., leucine-to-proline mutation (T>C mutation) at position 54 or 55 (counting the initiating methionine) or homologous residue in the processed form of α1-antitrypsin, or at residue 78 or homologous residue in the unprocessed form - e.g., see Poller et al., Genomics. 1993; 17: 740-743, also see accession number P01011 in the UNIPROT database; Charcot-Marie-Tooth disease type 4J - e.g., isoleucine-to-threonine mutation (T>C mutation) at position 41 or homologous residue in FIG4 - e.g., Lenk et al, PLoS Genetics. 2011; 7: See e1002104; neuroblastoma (NB) - e.g., leucine-to-proline mutations (T>C mutations) at position 197 or homologous residue of caspase 9 - e.g., Kundu et al., 3 Biotech.See 2013, 3:225-234; von Willebrand disease (vWD) - e.g., a cysteine-to-arginine mutation (T>C mutation) at position 509 or a homologous residue of the processed form of the von Willebrand factor, or at position 1272 or a homologous residue of the unprocessed form of the von Willebrand factor - e.g., see Lavergne et al., Br. J. Haematol. 1992;82: 66-72, also see accession number P04275 in the UNIPROT database; congenital myotonia - e.g., a cysteine-to-arginine mutation (T>C mutation) at position 277 or a homologous residue of the muscle chloride ion channel gene CLCN1 - e.g., Weinberger et al., The J. of Physiology. 2012; 590: See 3449-3464; Hereditary renal amyloidosis - e.g., mutations from a stop codon to arginine at position 78 or a homologous residue in the processed form of apolipoprotein AII, or at position 101 or a homologous residue in the unprocessed form (T>C mutations) - e.g., see Yazaki et al., Kidney Int. 2003; 64: 11-16; Dilated cardiomyopathy (DCM) - e.g., mutations from tryptophan to arginine at position 148 or a homologous residue in the FOXD4 gene (T>C mutations), e.g., see Minoretti et al., Int. J. of Mol. Med. 2007; 19: 369-372; Hereditary lymphedema - e.g., mutations from histidine to arginine at position 1035 or a homologous residue in the VEGFR3 tyrosine kinase (A>G mutations), e.g., Irrthum et al, Am. See J. Hum. Genet. 2000; 67: 295-301; Familial Alzheimer's disease—e.g., isoleucine-to-valine mutations (A>G mutations) at position 143 or homologous residue of presenilin 1, e.g., Gallo et. al., J. Alzheimer's disease.See 2011; 25: 425-431; prion diseases—e.g., mutations from methionine to valine (A>G mutations) at position 129 or a homologous residue in the prion protein—e.g., see Lewis et. al., J. of General Virology. 2006; 87: 2443-2449; chronic infantile neurocutaneous arthral syndrome (CINCA)—e.g., mutations from tyrosine to cysteine ​​(A>G mutations) at position 570 or a homologous residue in cryopyrin—e.g., see Fujisawa et. al. Blood. 2007; 109: 2903-2911; and desmin-related myopathy (DRM)—e.g., mutations from arginine to glycine (A>G mutations) at position 120 or a homologous residue in αβ-crystallin—e.g., Kumar et al., J. Biol. Chem. 1999; 274: See and include 24137-24141. All references and the entire contents of the database entries are incorporated herein by reference.

[0220] The disclosure of this invention provides a list of genes containing pathogenic T>C or A>G mutations, which can be modified using any of the Cas9 fusion proteins provided herein. Hereinafter, the names of these genes, their respective sequence numbers, their IDs, and the sequence numbers adjacent to the mutation site are indicated. See Tables 4 and 5. While not bound by any particular theory, the mutations shown in Tables 4 and 5 can be modified using the Cas9 fusions provided herein that can bind to target sequences lacking classical PAM sequences. In some embodiments, the Cas9-deaminase fusion protein exhibits activity against non-classical PAMs and can therefore modify all pathogenic T>C or A>G mutations listed in Tables 4 and 5 (sequence numbers 674-2539 and 3144-5083, respectively). In some embodiments, the Cas9-deaminase fusion protein recognizes classical PAMs and can therefore modify pathogenic T>C or A>G mutations having classical PAMs (e.g., 5'-NGG-3'). Naturally, those skilled in the art will understand how to design RNA (e.g., gRNA) and use either the Cas9 protein or fusion protein provided herein to target any target sequence and modify any of the mutations shown herein, such as those shown in Tables 4 and 5. It will be apparent to those skilled in the art that, in order to target the Cas9 / effector domain fusion protein disclosed herein to a target site, such as a site containing a point mutation to be edited, it is typically necessary to co-express the Cas9 / effector domain fusion protein together with a guide RNA, such as sgRNA. As described in more detail elsewhere herein, the guide RNA typically comprises a tracrRNA framework that allows Cas9 binding and a guide sequence that confers sequence specificity to the Cas9 / effector domain fusion protein. In some embodiments, the guide RNA comprises the structure 5'-[guide sequence]-guuuuagagcuagaaauagcaaguuaaaauaaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuu-3' (SEQ ID NO: 285), where the guide sequence comprises a sequence complementary to the target sequence.Typically, the guide sequence is 20 nucleotides long. Suitable guide RNA sequences for targeting Cas9 / effector domain fusion proteins to specific genomic target sites will be apparent to those skilled in the art based on this disclosure. Typically, such suitable guide RNA sequences include a guide sequence that is complementary to a nucleic acid sequence within 50 nucleotides upstream or downstream of the target nucleotide to be edited. Several exemplary guide RNA sequences suitable for targeting Cas9: nucleic acid editing enzyme / domain fusion proteins to specific target sequences are provided below.

[0221] Kits, vectors, cells Some aspects of this disclosure provide a kit comprising a nucleic acid construct comprising (a) a nucleotide sequence encoding a Cas9 protein or Cas9 fusion protein provided herein, and (b) a heterologous promoter that drives the expression of the sequence in (a). In some embodiments, the kit further comprises an expression construct encoding a guide RNA backbone, the construct comprising a cloning site positioned to allow the cloning of a nucleic acid sequence identical or complementary to the target sequence into the guide RNA backbone.

[0222] Some aspects of this disclosure provide polynucleotides encoding the Cas9 protein of the fusion protein provided herein. Some aspects of this disclosure provide vectors comprising such polynucleotides. In some embodiments, the vectors include heterologous promoters that drive the expression of the polynucleotides.

[0223] Some aspects of this disclosure provide cells comprising Cas9 proteins, fusion proteins, nucleic acid molecules, and / or vectors as provided herein.

[0224] The exemplary embodiments of reporter systems described herein are provided for illustrative purposes only and are not intended to limit them. Additional reporter systems, such as variations of the exemplary systems described in detail above, are also included in this disclosure.

[0225] Example 1: PACE evolution of Cas9 without PAM sequence suppression Building a PAM Library Four different protospacer target sequences were synthesized: Doench 1 5'-AAGAGAGACAGTACATGCCC-3' (SEQ ID NO: 286), Doench 2 5'-GGAGCCCACCGAGTACCTGG-3' (SEQ ID NO: 287), G7' 5'-AGTCTCCTCAGCAAAACGAA-3' (SEQ ID NO: 288), and VEGF Target 2 5'-GACCCCCTCCACCCCGCCTC-3' (SEQ ID NO: 289). For each protospacer target sequence, a 3'-NNN PAM library was constructed. The classical PAM sequence is 5'-NGG-3' (e.g., [Doench 1]-[ Classic PAM The target sequence is 5'-[AAGAGAGACAGTACATGCCC]-[ NGG ]-3' (sequence number 291), while the 3'-NNN PAM library for each protospacer target sequence contained completely random PAM sequences (e.g., Doench 1 5'-AAGAGAGACAGTACATGCCC). NNN -3' (Sequence ID 290, where N represents any nucleotide). Therefore, the NNN PAM library contained all possible combinations of PAM sequences at the 3' end of each protospacer target sequence.

[0226] Activity testing of Cas9 against PAM library in ω-dCas9 luciferase assay Cas9 activity was tested using a bacterial luciferase activation assay. In this assay, a fusion protein of dCas9 and the ω subunit of E. coli RNA polymerase (rpoZ) (see, e.g., Bikard et al., Nucleic Acids Res. 2013 Aug; 41(15): 7429-7437) promotes the production of luciferase encoded by a nucleic acid under the control of a weak promoter containing a sequence that can be targeted by sgRNA. Each PAM library was cloned into a plasmid containing such a weak promoter, where the [target sequence]—[PAM library] nucleic acid sequence functions as a sequence that can be targeted by sgRNA. The PAM libraries were cloned into promoters, and ω-dCas9 assays were performed against all four protospacer targets for both classical and random PAM libraries. Figure 1 shows the activity of wild-type S. pyogenes Cas9 against the PAM libraries.

[0227] The evolution of Cas9 in PAM libraries The dCas9 of S. pyogenes was fused to the ω unit of RNA polymerase. The resulting ω-dCas9 fusion protein was cloned into an M13 phage-based selected phagemide (SP) containing the entire M13 phage genome except for a functional version of the gene encoding pIII (the gene required for the production of infectious phage particles). The phage gene encoding pIII was inserted into a separate plasmid (accessory plasmid, AP) under the control of a promoter whose transcription is activated by ω-dCas9. The PAM library was cloned into the promoter region of the accessory plasmid. The accessory plasmid was introduced into host cells used for directional evolution of the Cas9 protein without PAM repression. This made it so that the amount of infectious phage particles produced by a given host cell upon infection by the selected phage depends on the activity of the ω-dCas9 fusion protein required for the production of the pIII protein, which is encoded by the selected phage and controlled by the promoter of the accessory plasmid. Therefore, accessory plasmids give selective advantages to their selected phages encoding ω-dCas9 fusion protein variants with increased activity against different non-classical PAM sequences.

[0228] A lagoon was conjugated, and a flow of host cells containing accessory plasmids was generated along the lagoon. The host cells were brought into contact with selected phagemids, resulting in a population of selected phages that diffused into the host cell flow along the lagoon. The phage-infected host cells were removed from the lagoon, and new, uninfected host cells were introduced into the lagoon at a predetermined rate. As a result, the host cells remaining in the lagoon, on average, had a shorter time between cell divisions of host cells but a longer lifetime than the average lifetime of M13 phages.

[0229] To generate Cas9 mutants during directional evolution experiments, host cells in a lagoon were incubated under conditions that increased the mutation rate. The host cells contained a mutagenesis plasmid (MP), which increased the mutation rate, thereby introducing mutations into the ω-dCas9 fusion protein encoded by the selected phagemide during the phage's lifetime. Due to the flow rate of host cells along the lagoon, the average time between host cell divisions is shorter for host cells remaining in the lagoon than the average time between host cell divisions. Therefore, host cells in the lagoon cannot accumulate mutations in their genome or in accessory plasmids resulting from the increased mutation rate conferred by the mutagenesis plasmid. However, the selected phages replicate in the lagoon amid the flow of host cells and thus accumulate mutations over time, resulting in the creation of novel, evolved ω-dCas9 fusion protein mutants.

[0230] If any of these evolved ω-dCas9 fusion protein variants contain mutations that increase their activity against accessory plasmids, including the PAM library, this directly leads to further production of pIII by host cells infected with the selected phages encoding each ω-dCas9 fusion protein variant. The increased production of pIII then results in the creation of more infectious selected phage particles, which, over time, give selected phages without such mutations a competitive advantage over the mutant selected phages with such advantageous mutations. After a predetermined time, the application of selection pressure by the accessory plasmids positions the selected phages with the advantageous mutations as the dominant species, meaning they can replicate in the host cell flow, while selected phages without mutations or with unfavorable mutations are washed away from the lagoon.

[0231] Because the activity of the ω-dCas9 fusion protein against the PAM library was very low at the start of the experiment, selected phagesimides were grown multiple times overnight in host cells containing accessory plasmids with the PAM library to evolve Cas9 mutants with increased activity against non-classical PAM sequences. After the directional evolution experiment was completed, the evolved population of selected phages was isolated from the lagoon, and several representative clones were subjected to assays to detect Cas9 mutants with advantageous mutations. While all observed mutations resulted in advantageous phenotypes, mutations shared by multiple clones, or by all clones, were of particular interest.

[0232] A mutation from Cas9 PACE From directional evolution experiments using accessory plasmids from the above-mentioned PAM library, many selected phage clones were isolated. Mutations identified in the Cas9 amino acid sequence of several exemplary clones are shown in Table 1 below (residue numbering follows Sequence ID No. 9): [Table 1]

[0233] Clones 1-4 were tested in the ω-dCas9 luciferase activation assay described above. When the entire PAM library was tested, various clones showed elevated luciferase expression (Figure 2 - Cas9 activity of exemplary evolved clones relative to the PAM library after directional evolution).

[0234] Improvement of Cas9 activity against non-classical PAM sequences The activity of the evolved Cas9 protein against target sequences containing non-classical PAMs was further evaluated. The relative activity of clone 4 (containing I122, D182, and E1219V mutations) was tested against various [Doench 2 5'-GGAGCCCACCGAGTACCTGG-3', SEQ ID NO: 287]-[PAM] target sequences using an ω-dCas9 luciferase activation assay and compared to the activity of wild-type dCas9.

[0235] Improvement of Cas9 activity against non-classical PAM sequences The activity of the evolved Cas9 protein against target sequences containing non-classical PAMs was further evaluated. The relative activity of clone 4 (containing I122, D182, and E1219V mutations) was tested against various [Doench 2 5'-GGAGCCCACCGAGTACCTGG-3', SEQ ID NO: 287]-[PAM] target sequences using an ω-dCas9 luciferase activation assay and compared to the activity of wild-type dCas9. The data are shown in Table 2. [Table 2]

[0236] Example 2: Cas9 PACE evolution without any PAM sequence suppression Due to the very low activity of the ω-dCas9 fusion protein against the NNN-PAM library, a second round of PACE experiments was performed. In this round, the first phase of diversification of the ω-dCas9 fusion protein population was carried out in the absence of selective pressure by supplying a source of pIII independent of ω-dCas9 fusion protein activity. The initial diversification stage resulted in mutations that would not be obtained in PACE experiments where selective pressure is applied throughout the experiment.

[0237] Selected phages containing an ω-dCas9 fusion protein with the dCas9 sequence shown as SEQ ID NO: 8, bearing the D10A and H840A mutations, were grown in 1030 host cells in the presence of arabinose along with an MP6 mutagenerating plasmid to construct a library of mutant selected phages encoding a library of ω-dCas9 fusion protein variants. PIII was expressed from individual plasmids in the host cells during this initial diversification stage. After overnight (12-hour) diversification, 1030 host cells containing an accessory plasmid as a guide RNA target sequence, along with a mutagenesis plasmid containing an NNN PAM library cloned under weak promoter control, were logarithmically grown and used as a source of host cells for a flow of host cells along a lagoon. Cells in the lagoon were infected with the diversified selected phages obtained from the overnight culture. Host cells in the lagoon were brought into contact with arabinose to maintain high levels of expression of mutagenic genes from the mutagenesis plasmid.

[0238] The initial phage titer is approximately 10 8 The pfu / mL level was pfu / mL. As described above, PACE experiments were performed on each of the four NNN-PAM libraries ([Doench 1]-[NNN-PAM], [Doench 2]-[NNN-PAM], [G7]-[NNN-PAM], and [VEGF target]-[NNN-PAM]) cloned into accessory plasmids expressing pIII under weak promoter control. Phage titers were monitored during the PACE experiments. Gradually increased to 10 4 A decrease in phage titer to pfu / mL was observed. At that point, the phage population was isolated from the lagoon and grown in 2208 host cells containing another source of pIII (psp-induced pIII). After this low-stringency growth period, a 1:100 dilution of the supernatant was added to new host cells containing the accessory plasmid as the sole source of pIII in the new lagoon, and the PACE experiment was continued. No decrease in phage titer was observed after this low-stringency culture in 2208 cells.

[0239] In one experimental example, a PACE experiment was performed for 72 hours. After that time, 24 viable clones were isolated from the lagoon, sequenced, and analyzed. Identified mutations included A262T, K294R, S409I, M694I, E480K, E543D, and E1219V (following the amino acid numbering in SEQ ID NO: 9). In another experimental example, viable clones were isolated after 15 days of culture. The activity of the identified dVas9 mutants was analyzed using an ω-dCas9 luciferase assay. The clones with the highest ω-dCas9 fusion protein activity against non-classical PAM target sequences had the following mutations: E480K, E543D, E1219V, and T1329.

[0240] Cas9-mediated activation of GFP in mammals Both wild-type dCas9 (SEQ ID NO: 9) and evolved Cas9 clones were tested in a dCas9-GFP assay in Hek293T cells. Cells were contacted with a reporter construct that expressed a GFP coding sequence under weak promoter control, containing a [gRNA target sequence]-[PAM] sequence. Fusion proteins of dCas9 (wild-type and PACE mutants) bound to the transcriptional activator VP64-p65-Rta (VPR) were constructed, and various dCas9-VPR mutants were tested for their ability to activate the GFP reporter in HEK293 cells.

[0241] Hek293T cells were transfected with each of the following four plasmids: a dCas9-VPR expression plasmid, a plasmid expressing an sgRNA targeting the gRNA target sequence of a GFP reporter plasmid, a GFP reporter plasmid, and an iRFP transfection control. In one experiment, HEK293 cells were exposed to a GFP reporter containing TAA PAM, and in another experiment, HEK293 cells were exposed to a population of reporter plasmids containing an NNN PAM library. Cells were harvested 48 hours after transfection, and GFP-expressing cells were quantified using a BD LSR-FORTESSA cell analyzer.

[0242] Figure 3 - Cas9-mediated GFP activation in mammals Compared to wild-type Cas9, evolved Cas9s showed significantly higher activity against TAA PAM (21.08 percent compared to 0.60 percent of the negative control cells mentioned above) and NNN PAM libraries (22.76 percent compared to 3.38 percent of the negative control cells mentioned above).

[0243] Cleavage activity of evolved Cas9 against target sequences with non-classical PAMs To demonstrate that PACE mutations universally confer Cas9 activity without PAM suppression, nuclease-active Cas9 proteins were constructed based on sequences containing various PACE mutations but lacking the D10A and H840A mutations. Evolutionary Cas9 mutants were tested in a Cas9 GFP assay, evaluating their ability to inactivate the emGFP gene integrated into the HEK293 cell genome using guide RNA targeting non-classical PAM sequences. Observations showed that 6.45 percent of cells exhibited decreased GFP expression upon contact with wild-type nuclease-active Cas9 (SEQ ID NO: 9), while 54.55 percent of cells exhibited decreased GFP expression upon contact with evolutionary Cas9 (E480K, E543D, E1219V, and T1329).

[0244] Example 3: Cas9 mutant without PAM suppression We mapped advantageous mutations that confer Cas9 activity to non-classical PAM sequences to the wild-type sequence of S. pyogenes. An exemplary Cas9 sequence (Cas9 of S. pyogenes with D10 and H840 residues indicated by asterisks, according to each amino acid residue of SEQ ID NO: 9) is shown below. By muting the D10 and H840 residues of SEQ ID NO: 9, it is possible to create nuclease-inactive Cas9 (e.g., D10A and H840A) or nickase Cas9 (e.g., having D10A and H840, or D10 and H840A). The HNH domain (bold and underlined) and RuvC domain (enclosed text) are shown. Mutant residues present in clones isolated from various PACE experiments include amino acid residues 122, 137, 182, 262, 294, 409, 480, 543, 660, 694, 1219, and 1329, which are identified by asterisks based on each amino acid residue.

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[0245] We mapped advantageous mutations that confer Cas9 activity to non-classical PAM sequences to further exemplary wild-type Cas9 sequences. The HNH domain (bold and underlined) and RuvC domain (enclosed) are shown. Amino acid residues 122, 137, 182, 262, 294, 409, 480, 543, 660, 694, 1219, and 1329 are identified with asterisks based on each amino acid residue as homologous to the mutant residue in Sequence ID No. 9. Furthermore, amino acid residues 10 and 840, which are mutated in dCas9 protein mutants, are also identified with asterisks.

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[0246] The disclosure of the present invention relates to the provision of Cas9 mutants having one or more amino acid residues identified by asterisks as described herein. In some embodiments, the D10 and H840 residues are mutated, for example, to alanine residues, and furthermore, the Cas9 mutant has one or more amino acid residues indicated by asterisks as shown in this specification. In some embodiments, the D10 residue is mutated, for example, to alanine residue, and furthermore, the Cas9 mutant has one or more amino acid residues indicated by asterisks as shown herein.

[0247] Multiple Cas9 sequences from various species were aligned to determine whether corresponding homologous amino acid residues were identified in other Cas9 proteins and whether the generation of Cas9 variants with corresponding mutations in homologous amino acid residues was possible. Alignment was performed using the NCBI Constraint-based Multiple Alignment Tool (COBALT, accessible at st-va.ncbi.nlm.nih.gov / tools / cobalt) with the following parameters: Alignment parameters: Gap Penalties -11, -1; End-Gap Penalties -5, -1. CDD Parameters: Use RPS BLAST ON; Blast E-value 0.003; Find Conserved columns and Recompute ON. Query Clustering Parameters: Use query clusters ON; Word Size 4; Max cluster distance 0.8; Alphabet Regular.

[0248] An exemplary alignment of four Cas9 sequences is provided below. The Cas9 sequences in the alignment are: Sequence 1 (S1): SEQ ID NO: 10 | WP_010922251 | gi 499224711 | Type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus pyogenes]; Sequence 2 (S2): SEQ ID NO: 11 | WP_039695303 | gi 746743737 | Type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus gallolyticus]; Sequence 3 (S3): SEQ ID NO: 12 | WP_045635197 | gi 782887988 | Type II CRISPR RNA-guided endonuclease Cas9 [Streptococcus mitis]; Sequence 4 (S4): SEQ ID NO: 13 | 5AXW_A | gi 924443546 | Staphylococcus aureus Cas9. The HNH domain (bold and underlined) and the RuvC domain (boxed) have been identified for each of the four sequences. Homologous amino acids in the sequences aligned with amino acid residues 10, 122, 137, 182, 262, 294, 409, 480, 543, 660, 694, 840, 1219, and 1329 in S1 are identified by the following asterisk after each amino acid residue.

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[0249] Alignment demonstrates that amino acid sequences and amino acid residues homologous to a reference Cas9 amino acid sequence or amino acid residue can be identified from Cas9 sequence variants that include, but are not limited to, Cas9 sequences from different species, by identifying amino acid sequences or residues that align with a reference sequence or reference residue using alignment programs and algorithms known in the art. This disclosure provides a Cas9 variant in which one or more amino acid residues identified by asterisks in SEQ ID NO: 9 (e.g., S1, S2, S3, and S4, respectively) are mutated as described herein. Residues in Cas9 sequences other than SEQ ID NO: 9 that correspond to residues identified by asterisks in SEQ ID NO: 9 are referred to herein as “homologous” or “corresponding” residues. Such homologous residues can be identified by sequence alignment, for example, as described above, by identifying sequences or residues that align with a reference sequence or residue. Similarly, mutations in Cas9 sequences other than SEQ ID NO: 9 that correspond to the identified mutations in SEQ ID NO: 9, such as the mutations in residues 10, 122, 137, 182, 262, 294, 409, 480, 543, 660, 694, 840, 1219, and 1329 in SEQ ID NO: 9 are referred to herein as “homologous” or “corresponding” mutations. For example, for the four aligned sequences above, the mutation corresponding to S1 is D10A in S2, D9A in S3, and D13A in S4; the mutation corresponding to H840A in S1 is H850A in S2, H842A in S3, and H560 in S4; the mutation corresponding to X1219 in S1 is X1228V in S2, X1226 in S3, and X903V in S4, etc.

[0250] A total of 250 Cas9 sequences (sequence numbers 10–262) from different species were aligned using the same algorithm and alignment parameters outlined above. Homologous amino acid residues to residues 10, 122, 137, 182, 262, 294, 409, 480, 543, 660, 694, 840, 1219, and 1329 were identified in the same manner outlined above. The alignments are provided below. The HNH domain (bold and underlined) and the RuvC domain (boxed) are identified for each of the four sequences. The amino acid residues corresponding to 10, 122, 137, 182, 262, 294, 409, 480, 543, 660, 694, 840, 1219, and 1329 of SEQ ID NO: 9 are enclosed in a box in SEQ ID NO: 10 during the alignment, allowing for the identification of the corresponding amino acid residues in the aligned sequence.

[0251] The present invention provides Cas9 mutants having one or more mutations in amino acid residues homologous to amino acid residues 122, 137, 182, 262, 294, 409, 480, 543, 660, 694, 1,219, and 1,329 of SEQ ID NO: 9. In some embodiments, the Cas9 mutants provided in the present invention include mutations corresponding to the D10A and H840A mutations of SEQ ID NO: 9 that result in nuclease-inactive dCas9, and mutations in at least one, at least two, at least three, at least four, at least five, at least six, or at least seven amino acid residues homologous to amino acid residues 122, 137, 182, 262, 294, 409, 480, 543, 660, 694, 1219, and 1329 of SEQ ID NO: 9.

[0252] The present invention provides Cas9 mutants having one or more mutations in amino acid residues homologous to amino acid residues 122, 137, 182, 262, 294, 409, 480, 543, 660, 694, 1219, and 1329 of SEQ ID NO: 9. In some embodiments, the Cas9 mutants provided in the present invention include a mutation corresponding to the D10A mutation of SEQ ID NO: 9, which results in a partially nuclease-inactive dCas9 that nicks in the non-target chain but not in the target chain, and at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations in amino acid residues homologous to amino acid residues 122, 137, 182, 262, 294, 409, 480, 543, 660, 694, 1219, and 1329 of SEQ ID NO: 9.

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[0253] Further preferred Cas9 sequences in which amino acid residues homologous to residues 10, 122, 137, 182, 262, 294, 409, 480, 543, 660, 694, 840, 1219 and / or 1329 of SEQ ID NO: 9 can be identified are known to those skilled in the art. See, for example, Supplementary Table S2 and Supplementary Figure S2 of Fonfara et al., Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems, Nucl. Acids Res. 2013, doi: 10.1093 / nar / gktl074 (the entire disclosure is incorporated herein by reference). Cas9 variants having sequences shown herein or known in the prior art, which include one or more mutations in one or more amino acid residues homologous to amino acid residues 10, 122, 137, 182, 262, 294, 409, 480, 543, 660, 694, 840, 1219 and / or 1329 of SEQ ID NO: 9 (for example, at least one, at least two, at least three, at least four, at least five, at least six or at least seven mutations shown herein), are provided by the disclosure of the present invention as Cas9 variants including, for example, A262T, K294R, S409I, E480K, E543D, M694I and / or E1219V mutations.

[0254] Example 4: Evolution of Cas9 with broad PAM specificity Using PACE, evolution of Cas9 from S. pyogenes against an NNN PAM library yielded evolved Cas9s with extended PAM specificity, exhibiting high activity against many non-classical PAMs. These Cas9s still retain their native DNA binding and cleavage activity and are usable with all currently available tools. It is hypothesized that modifying Cas9's interaction with DNA can improve and extend its PAM specificity. Other Cas9s, such as those from Staphylococcus aureus, can be designed, and their PAM specificity modified and extended according to this method. For example, by using methods to modify DNA binding, such as target-specific mutagenesis of the Cas9 protein, fusion with DNA-binding proteins, or the use of multiple linked Cas9 proteins, it is possible to expand the range of targetable PAMs.

[0255] The evolution of Cas9 After evolution using phages with mutagenesis plasmids (MPs) and overnight proliferation, we used phages with the above mutations due to PACE mutations. 24 phages were sequenced from PACE runs. The mutations present in the Cas9 gene are shown in Table 3 below. The Cas9 gene containing these mutations was cloned out from the phages, cloned into plasmids, and tested for DNA binding and cleavage activity. Table 3: Cas9 mutations [Table 3]

[0256] GFP activation in human cultured cells We conducted tests on reporters containing GFP reporters activated by dCas9-VPR. The tests were first performed on the 5'-NGG-3' PAM (Figures 4A and 4B), and then on a library of GFP reporters (Figures 5A and 5B) all containing the same Cas9 target site but with NNN at the PAM position. These tests showed that dCas9-VPR activates the GFP signal. All of the following Cas9 mutations were tested: wild-type Cas9 (pJH306), Cas9 evolved by perinaval proliferation (pJH562), and Cas9 evolved by PACE (pJH599-pJH605). The results for each Cas9 mutation are shown in Table 3 above.

[0257] dCas9-VPR for all 64 PAM sequences We tested all 64 PAM sequences using pJH306 (wild-type dCas9-VPR) and pJH599 (wild-type dCas9-VPR with mutations A262T, S409I, E480K, E543D, M694I, and E1219V) (Figure 6). Using dCas9-VPR, GFP was activated as described above, and different reporter plasmids were used in each well to confirm the activation efficiency for all 64 different PAM sequences. The average GFP fluorescence was measured for all transfected cells (gated by iRFP signal). Compared to pJH306, pJH599 showed similar to to higher activation levels for all PAM sequences.

[0258] In vitro cutting assay The ability of expressed and purified wild-type Cas9 (WT) and Cas9 with the E1219V mutation (1) to cleave DNA with different PAMs was tested (Figure 7). Cas9 was incubated with dsDNA containing the target site. Cleavage ability was measured by gel electrophoresis of the DNA and comparing the amount of uncleaved material with the amount of cleaved product (rapid electrophoresis was performed due to the small size). The E1219V mutation was found to increase the cleavage activity of Cas9 against non-classical PAMs while maintaining activity against 5'-NGG-3' PAMs.

[0259] Evolution of various systems In addition to evolving Cas9 in S. pyogenes, it is possible to evolve other Cas9 systems (e.g., S. aureus, S. thermophilus, N. meningitidis, and T. denticola) and improve and extend their PAM specificity. The data shows that Cas9 in S. aureus, including phages, can be evolved using a system similar to that used for Cas9 evolution in S. pyogenes, and its PAM specificity can be extended.

[0260] Regulation of PAM specificity Cas9 can be modified to expand the range of targetable PAMs by mutating neutral and positively charged amino acids to positively charged amino acids. Generally, introducing mutations that increase the positive charge into the Cas9 protein improves the DNA binding affinity of Cas9. In combination with the Cas9 mutations provided in this invention, further residues that can be mutated to improve the PAM targeting ability of S. pyogenes Cas9 are those identified as modifying PAM specificity (D1135, G1218, R1333, R1335, T1337). (38) And there are residues (S845 and L847) that can improve Cas9 activity. (37) . Residues that improve Cas9 specificity (for example, mutations of arginine, histidine, and lysine to alanine that have been identified so far) (37) , and the previously identified mutations of asparagine, arginine, and glutamine to alanine (39) These mutations likely reduce tolerance to non-classical PAMs because they impair the interaction between Cas9 and DNA.

[0261] Fusion for regulating PAM specificity By fusing programmable DNA-binding proteins (e.g., Zn finger domains, TALEs, and other Cas9 proteins) to Cas9, it is possible to improve its targeting ability (e.g., activity, specificity, or efficiency) to nucleotide sequences with classical or non-classical PAMs. Fusing nuclease-deleted (null) dCas9 with nuclease-active Cas9 can improve the ability of nuclease-active Cas9 to target various PAM sequences. An example of nuclease-deleted dCas9 fused to nuclease-active Cas9 is shown in Figure 8. Such fusions may be useful in improving the targeting ability to nucleotide sequences with classical or non-classical PAMs. Cas9s may originate from the same species or different species. Furthermore, both Cas9s may be nuclease-deficient dCas9s and may be further fused with effector proteins such as VP64, VP64-p65-Rta, FokI, GFP, and other fluorescent proteins, deaminases, or any of the effector proteins provided in this invention.

[0262] Localization of other nucleases and other DNA-binding proteins using Cas9 By using Cas9, it becomes possible to overcome the innate binding specificity of other proteins through localization to their DNA targets. DNA nucleases, recombinases, deaminases, and other effectors typically possess intrinsic DNA specificity. By fusing Cas9 to these proteins, it becomes possible to overcome and extend their innate DNA specificity. gRNAs target Cas9 to sites adjacent to the target sites of DNA effectors, assisting their localization to those target sites.

[0263] dCas9-VPR for NNNNN PAM Library To test whether evolved Cas9s possess specificity to the fourth and fifth PAM sites, dCas9-VPR was tested against the NNNNN PAM library. As observed in the NNN library, most constructs (e.g., pJH562, pJ559, pJH600, pJH601, pJH602, pJH603, and pJH605) showed enhanced activity. pJH599 consistently showed an increased percentage of cells exhibiting GFP activation (Figure 9A) and an increased mean fluorescence in cells exhibiting GFP activation (Figure 9B).

[0264] GFP cleavage by Cas9 Wild-type Cas9 (pJH407) was compared with nuclease-positive evolved Cas9 (pJH760, Figure 10). pJH760 contains the same mutations as pJH599 but does not contain the nuclease-inactivating mutations D10A and H480A, and is not fused to VPR. The GFP gene integrated into the genome was cleaved with Cas9, and activity was measured using the loss of GFP signaling in cells as an indicator. Both pJH407 and pJH760 showed good activity at sites containing the 5'-NGG-3' PAM. At sites containing the GAT PAM, pJH760 showed a significant improvement in activity compared to pJH407.

[0265] Example 5: Cas9: DNA editing enzyme fusion protein The present invention further relates to providing Cas9 fused with a DNA editing enzyme for targeted editing of DNA base sequences. Figure 11 illustrates a double-stranded DNA substrate bound to a DNA editing enzyme-dCas9:sgRNA complex. The DNA editing enzyme shown is a deaminase. The structure in Figure 11 is identified in its sequence (36 bp: underlined, sgRNA target sequence: bold, PAM: enclosed text, 21 bp: italicized).

[0266] Example 6: PAM reduction assay A library of E. coli cells containing plasmids encoding PAM sequences and containing antibiotic resistance genes was obtained. If Cas9 can cleave the PAM sequence on the plasmid, the plasmid is lost without replication, and only the uncleaved plasmids remain in the population. High-throughput sequencing, which involves sequencing the initial and final plasmid populations, allows for the measurement of plasmids cleaved by Cas9. The ratio of libraries consisting of each PAM sequence was obtained by dividing the number of reads containing PAM sequences by the total number of reads. Next, the reduction score was calculated by dividing the ratio of libraries containing PAM sequences before selection by the ratio of libraries containing selected PAM sequences. A high reduction score indicates high cleavage activity of a particular PAM sequence by Cas9. The results of the PAM reduction assay are shown in Figure 12.

[0267] Many PAM sequences that were not cleaved by wild-type Cas9 were cleaved by evolved Cas9 (xCas9 v1.0, pJH760). In particular, all PAM sequences of NGN and NNG, as well as GAA and GAT, showed more than a 10-fold reduction compared to xCas9. The single G at a second or third PAM location may be sufficient for cleavage by the newly evolved Cas9, significantly opening up sequence space for target sites that can be targeted using Cas9. The PAM reduction scores are shown in Table 4. Table 4. PAM reduction score [Table 4-1] [Table 4-2] [Table 4-3]

[0268] Example 7: GFP-cleaved mammalian cells Mammalian cells containing a genome-integrated GFP gene were transfected with wild-type or evolved Cas9 and gRNAs. Different gRNAs targeted different sites with different PAM sequences, and Cas9-mediated GFP cleavage resulted in loss of GFP signaling. GFP signaling was quantified by flow cytometry after 5 days. As shown in Figure 13, evolved Cas9 cleaved GFP in mammalian cells, which is consistent with the results of GFP activation assays and PAM reduction assays. High-throughput sequencing around the cleavage sites confirmed the results observed by flow cytometry and was consistent with the percentage of cleavage observed by flow cytometry.

[0269] Example 8: Further evolution of the HHH library Because SpCas9 exhibits selectivity for G residues in the second and third bases, evolution was continued using an endpoint from the final evolution against the HHH(H=A, C, or T)PAM library. After evolution, 13 colonies were sequenced, and many novel mutations were identified. Three mutations, E1219V, E480K, and E543D, were present in all clones. Many clones had either the S267G / K294R / Q1256K mutation or the A262T / S409I mutation, but these mutations never coexisted, suggesting that the clones followed two different differentiation pathways in line with the evolutionary landscape. The novel mutations are shown in Table 5. Table 5. Novel mutations [Table 5-1] [Table 5-2]

[0270] Example 9: Further evolved Cas9 protein for gene editing pJH760 (described in Example 6) was used to test PAM reduction assays against many novel targets. Four novel targets were selected: re2:GGGGCCACTAGGGACAGGAT (SEQ ID NO: 314), a synthetic target conventionally used for GFP activation in mammalian cells; VEGF:GGGTGGGGGGAGTTTGCTCC (SEQ ID NO: 315), a target in the VEGF gene; CLTA:GCAGATGTAGTGTTTCCACA (SEQ ID NO: 316), a target in the CLTA gene; and CCR5D:TCACTATGCTGCCGCCCAGT (SEQ ID NO: 317), a target in the CCR5D gene. The results of the PAM reduction assays are shown in Figures 14-17. It was found that PAM reduction indicated cleavage not only of classical NGG sequences but also of most NGNs and some NNG sequences.

[0271] The HHH (H=A, C, or T) PAM library was further evolved using endpoints from the last evolutionary stage. After evolution, 13 colonies were sequenced, and many novel mutations were identified. Three mutations, E1219V, E480K, and E543D, were present in all clones. Many clones had either the K294R / Q1256K mutation or the A262T / S409I mutation, but these mutations never coexisted, suggesting that the clones followed two different differentiation pathways along the evolutionary landscape. The novel mutations are shown in Tables 8 and 9 below.

[0272] As expected, variations in activity were observed due to differences in targets. PAM reduction assay scores are shown in Table 10. NGN consistently showed cleavage activity with several targets. Variation was observed particularly in the xCas9 3.x mutant, which exhibited the highest activity. Specifically, xCas9 3.3 contained the K294R / Q1256K mutant series, while the other three mutants (3.6, 3.7, and 3.8) contained the A262T / S409I mutant series. xCas9 3.6 and 3.7 outperformed 3.8. In most cases, 3.3 appeared to have the highest activity, but 3.6 and 3.7 functioned well against specific PAM sequences. The results of PAM reduction assays against the three novel targets mentioned above are shown in Figures 18-20.

[0273] An NNNNN PAM-depleted library was constructed. Assays were performed to test the specificity for the fourth or fifth base. The initial results of the PAM-depleted assay showed, as expected, no selectivity for the fourth and fifth bases.

[0274] In summary, E1219V was found to be one of the earliest established mutations in evolution. It is located near the PAM sequence in the crystal structure. E480K and E543D are also observed in all clones obtained from the early stages of evolution and are considered important. K294R / Q1256K and A262T / S409I are considered to be two different differentiation pathways and are considered important. Their PAM sequence profiles appear to be slightly different, which implies their importance in relation to the determination of PAM activity. Table 6. Diseases / Disorders Containing a T-to-C Change. The table includes human gene mutations that may be modified by changing cytosine (C) to thymine (T). Gene name, gene symbol, and Gene ID are shown. [Table 6-1] [Table 6-2] Table 6-3 Table 6-4 Table 6-5 Table 6-6 Table 6-7 Table 6-8 Table 6-9 Table 6-10 Table 6-11 Table 6-12 Table 6-13 Table 6-14 Table 6-15 Table 6-16 Table 6-17 Table 6-18 Table 6-19 Table 6-20 Table 6-21 Table 6-22 Table 6-23 Table 6-24 Table 6-25 Table 6-26 Table 6-27 Table 6-28 Table 6-29 Table 6-30 Table 6-31 Table 6-32 Table 6-33 Table 6-34 Table 6-35 Table 6-36 Table 6-37 Table 6-38 Table 6-39 Table 6-40 Table 6-41 Table 6-42 Table 6-43 Table 6-44 Table 6-45 Table 6-46 Table 6-47 Table 6-48 Table 6-49 Table 6-50 Table 6-51 Table 6-52 Table 6-53 Table 6-54 Table 6-55 Table 6-56 Table 6-57 Table 6-58 Table 6-59 Table 6-60 Table 6-61 Table 6-62 Table 6-63 Table 6-64 Table 6-65 Table 6-66 Table 6-67 Table 6-68 Table 6-69 Table 6-70 Table 6-71 Table 6-72 Table 6-73 Table 6-74 Table 6-75 Table 6-76 Table 6-77 Table 6-78 Table 6-79 Table 6-80 Table 6-81 Table 6-82 Table 6-83 Table 6-84 Table 6-85 Table 6-86 Table 6-87 [Table 6-88] [Table 6-89] [Table 6-90] Table 7. Diseases / Disorders Containing Changes from A to C. The table includes human gene mutations that may be modified by changing guanine (G) to adenine (A). Gene name, gene symbol, and Gene ID are shown. [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6] [Table 7-7] [Table 7-8] [Table 7-9] [Table 7-10] [Table 7-11] [Table 7-12] Table 7-13 Table 7-14 Table 7-15 Table 7-16 Table 7-17 Table 7-18 Table 7-19 Table 7-20 Table 7-21 Table 7-22 Table 7-23 Table 7-24 Table 7-25 Table 7-26 Table 7-27 Table 7-28 Table 7-29 Table 7-30 Table 7-31 Table 7-32 Table 7-33 Table 7-34 Table 7-35 Table 7-36 Table 7-37 Table 7-38 Table 7-39 Table 7-40 Table 7-41 Table 7-42 Table 7-43 Table 7-44 Table 7-45 Table 7-46 Table 7-47 Table 7-48 Table 7-49 Table 7-50 Table 7-51 Table 7-52 Table 7-53 Table 7-54 Table 7-55 Table 7-56 Table 7-57 Table 7-58 Table 7-59 Table 7-60 Table 7-61 Table 7-62 Table 7-63 Table 7-64 Table 7-65 Table 7-66 Table 7-67 Table 7-68 Table 7-69 Table 7-70 Table 7-71 Table 7-72 Table 7-73 Table 7-74 Table 7-75 Table 7-76 Table 7-77 Table 7-78 Table 7-79 Table 7-80 [Table 7-81] [Table 7-82] [Table 7-83] [Table 7-84] [Table 7-85] [Table 7-86] [Table 7-87] [Table 7-88] [Table 7-89] [Table 7-90] [Table 7-91] [Table 7-92] [Table 7-93] Table 8. xCas9v3 mutations (K294R / Q1256K series) [Table 8] Table 9. xCas9v3 mutations (A262T / S409I series) [Table 9] Table 10. PAM reduction score (xCas9v3.0~3.6 mutations) [Table 10-1] [Table 10-2] [Table 10-3] Table 10 continued. PAM reduction score (xCas9v3.7~3.12 mutations) [Table 10-4] [Table 10-5] [Table 10-6] References 1. Humbert O, Davis L, Maizels N. Targeted gene therapies: tools, applications, optimization. Crit Rev Biochem Mol.2012; 47(3):264-81. PMID: 22530743. 2. Perez-Pinera P, Ousterout DG, Gersbach CA. Advances in targeted genome editing. Curr Opin Chem Biol. 2012; 16(3-4):268-77. PMID: 22819644. 3. Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD. Genome editing with engineered zinc finger nucleases. Nat Rev Genet. 2010; 11(9):636-46. PMID: 20717154. 4. Joung JK, Sander JD. TALENs: a widely applicable technology for targeted genome editing. Nat Rev Mol Cell Biol. 2013; 14(1):49-55. PMID: 23169466. 5. Charpentier E, Doudna JA. Biotechnology: Rewriting a genome. Nature. 2013; 495, (7439):50-1. PMID: 23467164. 6. Pan Y, Xia L, Li AS, Zhang X, Sirois P, Zhang J, Li K. Biological and biomedical applications of engineered nucleases. Mol Biotechnol. 2013; 55(1):54-62. PMID: 23089945. 7. De Souza, N. Primer: genome editing with engineered nucleases. Nat Methods. 2012; 9(1):27. PMID: 22312638. 8. Santiago Y, Chan E, Liu PQ, Orlando S, Zhang L, Urnov FD, Holmes MC, Guschin D, Waite A, Miller JC, Rebar EJ, Gregory PD, Klug A, Collingwood TN. Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases. Proc Natl Acad Sci U S A. 2008; 105(15):5809-14. PMID: 18359850. 9. Cargill M, Altshuler D, Ireland J, Sklar P, Ardlie K, Patil N, Lane CR, Lim EP, Kalyanaraman N, Nemesh J, Ziaugra L, Friedland L, Rolfe A, Warrington J, Lipshutz R, Daley GQ, Lander ES. Characterization of single-nucleotide polymorphisms in coding regions of human genes. Nat Genet. 1999; 22(3):231-8. PMID: 10391209. 10. Jansen R, van Embden JD, Gaastra W, Schouls LM. Identification of genes that are associated with DNA repeats in prokaryotes. Mol Microbiol. 2002; 43(6):1565-75. PMID: 11952905. 11. Mali P, Esvelt KM, Church GM. Cas9 as a versatile tool for engineering biology. Nat Methods. 2013; 10(10):957-63. PMID: 24076990. 12. Jore MM, Lundgren M, van Duijin E, Bultema JB, Westra ER, Waghmare SP, Wiedenheft B, Pul U, Wurm R, Wagner R, Beijer MR, Barendregt A, Shou K, Snijders AP, Dickman MJ, Doudna JA, Boeke EJ, Oost van Heder AJ, JJ, Oost SJ. Structural basis for CRISPR RNA-guided DNA recognition by Cascade. Nat Struct Mol Biol. 2011; 18(5):529-36. PMID: 21460843. 13. Horvath P, Barrangou R. CRISPR / Cas, the immune system of bacteria and archaea. Science. 2010; 327(5962):167-70. PMID: 20056882. 14. Wiedenheft B, Sternberg SH, Doudna JA. RNA-guided genetic silencing systems in bacteria and archaea. Nature. 2012; 482(7385):331-8. PMID: 22337052. 15. Gasiunas G, Siksnys V. RNA-dependent DNA endonuclease Cas9 of the CRISPR system: Holy Grail of genome editing? Trends Microbiol. 2013; 21(11):562-7. PMID: 24095303. 16. Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell. 2013; 152(5):1173-83. PMID: 23452860. 17. Perez-Pinera P, Kocak DD, Vockley CM, Adler AF, Kabadi AM, Polstein LR, Thakore PI, Glass KA, Ousterout DG, Leong KW, Guilak F, Crawford GE, Reddy TE, Gersbach CA. RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat Methods. 2013; 10(10):973-6. PMID: 23892895. 18. Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, Yang L, Church GM. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol. 2013; 31(9):833-8. PMID: 23907171. 19. Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, Stern-Ginossar N, Brandman O, Whitehead EH, Doudna JA, Lim WA, Weissman JS, Qi LS. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell. 2013; 154(2):442-51. PMID: 23849981. 20. Larson MH, Gilbert LA, Wang X, Lim WA, Weissman JS, Qi LS. CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nat Protoc. 2013; 8(11):2180-96. PMID: 24136345. 21. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. RNA-guided human genome engineering via Cas9. Science. 2013; 339(6121):823-6. PMID: 23287722. 22. Cole-Strauss A, Yoon K, Xiang Y, Byrne BC, Rice MC, Gryn J, Holloman WK, Kmiec EB. Correction of the mutation responsible for sickle cell anemia by an RNA-DNA oligonucleotide. Science. 1996; 273(5280):1386-9. PMID: 8703073. 23. Tagalakis AD, Owen JS, Simons JP. Lack of RNA-DNA oligonucleotide (chimeraplast) mutagenic activity in mouse embryos. Mol Reprod Dev. 2005; 71(2):140-4. PMID: 15791601. 24. Ray A, Langer M. Homologous recombination: ends as the means. Trends Plant Sci. 2002; 7(10):435-40. PMID 12399177. 25. Britt AB, May GD. Re-engineering plant gene targeting. Trends Plant Sci. 2003; 8(2):90-5. PMID: 12597876. 26. Vagner V, Ehrlich SD. Efficiency of homologous DNA recombination varies along the Bacillus subtilis chromosome. J Bacteriol. 1988; 170(9):3978-82. PMID: 3137211. 27. Saleh-Gohari N, Helleday T. Conservative homologous recombination preferentially repairs DNA double-strand breaks in the S phase of the cell cycle in human cells. Nucleic Acids Res. 2004; 32(12):3683-8. PMID: 15252152. 28. Lombardo A, Genovese P, Beausejour CM, Colleoni S, Lee YL, Kim KA, Ando D, Urnov FD, Galli C, Gregory PD, Holmes MC, Naldini L. Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery. Nat Biotechnol. 2007; 25(11):1298-306. PMID: 17965707. 29. Conticello SG. The AID / APOBEC family of nucleic acid mutators. Genome Biol. 2008; 9(6):229. PMID: 18598372. 30. Reynaud CA, Aoufouchi S, Faili A, Weill JC. What role for AID: mutator, or assembler of the immunoglobulin mutasome? Nat Immunol. 2003; 4(7):631-8. 31. Bhagwat AS. DNA-cytosine deaminases: from antibody maturation to antiviral defense. DNA Repair (Amst). 2004; 3(1):85-9. PMID: 14697763. 32. Navaratnam N, Sarwar R. An overview of cytidine deaminases. Int J Hematol. 2006; 83(3):195-200. PMID: 16720547. 33. Holden LG, Prochnow C, Chang YP, Bransteitter R, Chelico L, Sen U, Stevens RC, Goodman MF, Chen XS. Crystal structure of the anti-viral APOBEC3G catalytic domain and functional implications. Nature. 2008; 456(7218):121-4. PMID: 18849968. 34. Chelico L, Pham P, Petruska J, Goodman MF. Biochemical basis of immunological and retroviral responses to DNA-targeted cytosine deamination by activation-induced cytidine deaminase and APOBEC3G. J Biol Chem. 2009; 284(41). 27761-5. PMID: 19684020. 35. Pham P, Bransteitter R, Goodman MF. Reward versus risk: DNA cytidine deaminases triggering immunity and disease. Biochemistry. 2005; 44(8):2703-15. PMID 15723516. 36. Chen X, Zaro JL, Shen WC. Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013; 65(10):1357-69. PMID: 23026637. 37. Slaymaker, I. M. et al. Rationally engineered Cas9 nucleases with improved specificity. Science, doi:10.1126 / science.aad5227 (2015). 38. Kleinstiver, BP et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523, 481-485, doi:10.1038 / nature14592 (2015). 39. Pattanayak, V. et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nature Biotechnology 31, 839-843, doi:10.1038 / nbt.2673 (2013). 40. Shcherbakova, DM & Verkhusha, VV Near-infrared fluorescent proteins for multicolor in vivo imaging. Nature Methods 10, 751-754, doi:10.1038 / nmeth.2521 (2013). 41. Kleinstiver, et al., High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490-495 doi:10.1038 / nature16526 (2016).

[0275] In this specification, all publications, patents, patent applications, publications, and database entries (e.g., sequence database entries) referenced, for example, in the sections of Background Art, Abstract, Detailed Description of the Invention, Examples, and / or References, are incorporated herein by reference in their entirety, just as individual publications, patents, patent applications, publications, and database entries are specifically and individually cited herein. In the event of any inconsistency, this application (including definitions herein) shall prevail.

[0276] Equal objects and range Those skilled in the art will recognize many equivalents of the embodiments described herein, or will be able to verify them by conventional experimental work alone. The scope of this disclosure is not intended to be limited to what is stated above, but rather as set forth in the appended claims.

[0277] Articles such as “a,” “an,” and “the” can mean one or more unless otherwise indicated or otherwise evident from the context. A claim or statement containing “or” between two or more members of a group is deemed satisfied if one, more than one, or all of the members of the group are present, unless otherwise indicated or otherwise evident from the context. Disclosures of groups containing “or” between two or more members of a group provide embodiments of the presence of exactly one member of the group, embodiments of the presence of more than one member of the group, and embodiments of the presence of all of the members of the group. For the sake of brevity, these embodiments are not written out individually in this specification, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or waived.

[0278] It should be understood that the present invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, or descriptive terms are introduced into another claim from one or more claims or from one or more relevant parts of the specification. For example, a claim dependent on another claim may be modified to encompass one or more limitations found in any other claim dependent on the same basic claim. Furthermore, unless otherwise indicated, or unless it is obvious to those skilled in the art that a contradiction or inconsistency would arise, where a claim describes a composition, it should be understood that, as appropriate, methods of making or using the composition are encompassed, either according to any of the methods of making or using disclosed herein or according to methods known in the art.

[0279] Where elements are presented as a list, for example in Markush group format, it should be understood that all possible subgroups of the elements are also disclosed, and that any element or subgroup of an element may be removed from the group. It should also be noted that the term “includes” is intended to be open and allows for the inclusion of additional elements or steps. Generally, where a certain aspect, product, or method is said to include a specific element, feature, or step, it should be understood that a similar aspect, product, or method consisting of or essentially comprising such element, feature, or step is also provided. For the sake of brevity, these aspects are not individually listed herein, but it should be understood that each of these aspects is provided herein and may be specifically claimed or waived.

[0280] Where a range is given, the endpoints are encompassed. Furthermore, in some embodiments, unless otherwise indicated or otherwise obvious from the context and / or the understanding of those skilled in the art, the values ​​expressed as a range may take any particular value within the given range, down to one-tenth of the lower limit unit of the range, unless the context clearly states otherwise. For the sake of brevity, the values ​​within each range are not individually listed herein, but it should be understood that each of those values ​​is provided herein and may be specifically claimed or waived. Unless otherwise indicated or otherwise obvious from the context and / or the understanding of those skilled in the art, the values ​​expressed as a range may take any subrange within a given range, and the endpoints of the subranges may be expressed with the same degree of precision as one-tenth of the lower limit unit of the range.

[0281] In addition, it will be understood that any specific aspect of the present invention may be expressly excluded from any one or more claims. Where a range is given, any value within that range may be expressly excluded from any one or more claims. Any aspect, element, feature, use, or aspect of any composition and / or method of the present invention may be excluded from any one or more claims. For the sake of brevity, not all aspects from which one or more elements, features, uses, or aspects have been excluded are expressly shown herein.

Claims

1. It contains an amino acid sequence that is at least 80 percent identical to the amino acid sequence of Cas9 represented by any one of sequence numbers 9 to 262, The amino acid sequence of the Cas9 protein contains at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations in an amino acid residue selected from the group consisting of amino acid residues 262, 267, 294, 405, 409, 480, 543, 694, 1219, 1224, 1256, and 1362 of the amino acid sequence shown in SEQ ID NO: 9, or in a corresponding amino acid residue of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262. Recombinant Cas9 proteins in which the amino acid sequence of the recombinant Cas9 protein is not identical to that of naturally occurring Cas9 proteins.

2. The Cas9 protein according to claim 1, comprising an amino acid sequence that is at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identical to the amino acid sequence of Cas9 represented by any one of sequence numbers 9 to 262.

3. The Cas9 protein according to claim 1, wherein the Cas9 protein comprises a RuvC and an HNH domain.

4. The Cas9 protein according to claim 1, wherein 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, or at least seven mutations selected from the group consisting of X262T, X267G, X294R, X405I, X409I, X480K, X543D, X694I, X1219V, X1224K, X1256K, and X1362P of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, and X represents any of these amino acids.

5. The Cas9 protein according to claim 1, wherein the amino acid sequence of the Cas9 protein includes at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations selected from the group consisting of A262T, S267G, K294R, F405I, S409I, E480K, E543D, M694I, E1219V, N1224K, Q1256K, and L1362P of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

6. The Cas9 protein according to claim 1, wherein the amino acid sequence of the Cas9 protein includes the X1219V mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

7. The Cas9 protein according to claim 1, wherein the amino acid sequence of the Cas9 protein includes the E1219V mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

8. The Cas9 protein according to claim 1, wherein the amino acid sequence of the Cas9 protein includes the X294R mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

9. The Cas9 protein according to claim 1, wherein the amino acid sequence of the Cas9 protein includes the K294R mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

10. The Cas9 protein according to claim 1, wherein the amino acid sequence of the Cas9 protein includes the X1256K mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

11. The Cas9 protein according to claim 1, wherein the amino acid sequence of the Cas9 protein includes a Q1256K mutation in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

12. The Cas9 protein according to claim 1, wherein the amino acid sequence of the Cas9 protein includes the X694I mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

13. The Cas9 protein according to claim 1, wherein the amino acid sequence of the Cas9 protein includes the M694I mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

14. The Cas9 protein according to claim 1, wherein the amino acid sequence of the Cas9 protein includes the X543D mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

15. The Cas9 protein according to claim 1, wherein the amino acid sequence of the Cas9 protein includes the E543D mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

16. The Cas9 protein according to claim 1, wherein the amino acid sequence of the Cas9 protein includes the X480K mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

17. The Cas9 protein according to claim 1, wherein the amino acid sequence of the Cas9 protein includes the E480K mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

18. The Cas9 protein according to claim 1, wherein the amino acid sequence of the Cas9 protein includes the X409I mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

19. The Cas9 protein according to claim 1, wherein the amino acid sequence of the Cas9 protein includes the S409I mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

20. The Cas9 protein according to claim 1, wherein the amino acid sequence of the Cas9 protein includes the X262T mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

21. The Cas9 protein according to claim 1, wherein the amino acid sequence of the Cas9 protein includes the A262T mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

22. It contains an amino acid sequence that is at least 80 percent identical to the amino acid sequence of Cas9 represented by any one of sequence numbers 9 to 262, The amino acid sequence of the Cas9 protein contains at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations in amino acid residues selected from the group consisting of amino acid residues 262, 267, 294, 405, 409, 480, 543, 694, 1219, 1224, 1256, and 1362 of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding amino acid residues of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262. Recombinant Cas9 proteins in which the amino acid sequence of the recombinant Cas9 protein is not identical to that of naturally occurring Cas9 proteins.

23. The Cas9 protein according to claim 22, comprising an amino acid sequence that is at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identical to the amino acid sequence of Cas9 shown in any one of SEQ ID NOs. 9 to 262.

24. The Cas9 protein according to claim 22 or 23, wherein the Cas9 protein comprises a RuvC and an HNH domain.

25. The Cas9 protein according to any one of claims 22 to 24, wherein 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, or at least seven mutations selected from the group consisting of X262T, X267G, X294R, X405I, X409I, X480K, X543D, X694I, X1219V, X1224K, X1256K, and X1362P of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, and X represents any of these amino acids.

26. The Cas9 protein according to any one of claims 22 to 25, wherein the amino acid sequence of the Cas9 protein includes at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations selected from the group consisting of A262T, S267G, K294R, F405I, S409I, E480K, E543D, M694I, E1219V, N1224K, Q1256K, and L1362P of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

27. The Cas9 protein according to any one of claims 22 to 26, wherein the amino acid sequence of the Cas9 protein includes the X1219V mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

28. The Cas9 protein according to any one of claims 22 to 27, wherein the amino acid sequence of the Cas9 protein comprises the E1219V mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

29. The Cas9 protein according to any one of claims 22 to 28, wherein the amino acid sequence of the Cas9 protein includes the X480K mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

30. The Cas9 protein according to any one of claims 22 to 29, wherein the amino acid sequence of the Cas9 protein includes the E480K mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

31. The Cas9 protein according to any one of claims 22 to 30, wherein the amino acid sequence of the Cas9 protein includes the X543D mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

32. The Cas9 protein according to any one of claims 22 to 31, wherein the amino acid sequence of the Cas9 protein includes the E543D mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

33. The Cas9 protein according to any one of claims 22 to 32, wherein the amino acid sequence of the Cas9 protein comprises the X480K, X543D, and X1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

34. The Cas9 protein according to any one of claims 22 to 32, wherein the amino acid sequence of the Cas9 protein includes the X262T, X409I, X480K, X543D, X694I, and X1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

35. The Cas9 protein according to any one of claims 22 to 32, wherein the amino acid sequence of the Cas9 protein includes the X294R, X480K, X543D, X1219V, X1256K, and X1362P mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

36. The Cas9 protein according to any one of claims 22 to 32, wherein the amino acid sequence of the Cas9 protein comprises the X294R, X480K, X543D, X1219V, and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

37. The Cas9 protein according to any one of claims 22 to 32, wherein the amino acid sequence of the Cas9 protein includes the X267G, X294R, X480K, X543D, X1219V, X1224K, and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

38. The Cas9 protein according to any one of claims 22 to 32, wherein the amino acid sequence of the Cas9 protein includes the X262T, X405I, X409I, X480K, X543D, X694I, and X1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

39. The Cas9 protein according to any one of claims 22 to 32, wherein the amino acid sequence of the Cas9 protein comprises the E480K, E543D, and E1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

40. The Cas9 protein according to any one of claims 22 to 32, wherein the amino acid sequence of the Cas9 protein comprises the A262T, S409I, E480K, E543D, M694I, and E1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

41. The Cas9 protein according to any one of claims 22 to 32, wherein the amino acid sequence of the Cas9 protein comprises the K294R, E480K, E543D, E1219V, Q1256K and L1362P mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

42. The Cas9 protein according to any one of claims 22 to 32, wherein the amino acid sequence of the Cas9 protein comprises the K294R, E480K, E543D, E1219V, and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

43. The Cas9 protein according to any one of claims 22 to 32, wherein the amino acid sequence of the Cas9 protein comprises the S267G, K294R, E480K, E543D, E1219V, N1224K, and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

44. The Cas9 protein according to any one of claims 22 to 32, wherein the amino acid sequence of the Cas9 protein comprises the A262T, F405I, S409I, E480K, E543D, M694I, and E1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

45. The Cas9 protein according to any one of claims 24 to 44, wherein the amino acid sequence of the HNH domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of the HNH domain of any one of sequence numbers 9 to 262.

46. The Cas9 protein according to any one of claims 24 to 45, wherein the amino acid sequence of the HNH domain is identical to the amino acid sequence of the HNH domain of SEQ ID NO:

9.

47. The Cas9 protein according to any one of claims 24 to 46, wherein the amino acid sequence of the RuvC domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of the RuvC domain of any of SEQ ID NOs. 9 to 262.

48. The Cas9 protein according to any one of claims 24 to 47, wherein the amino acid sequence of the Ruvc domain is identical to the amino acid sequence of the Ruvc domain of SEQ ID NO:

9.

49. The Cas9 protein according to any one of claims 22 to 48, wherein the Cas9 protein comprises a DIOA and / or H840A mutation in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

50. The Cas9 protein has the D1OX amino acid sequence shown in SEQ ID NO:

9. １ and / or H840X ２ A mutation, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10-262, X １ is any amino acid other than D, and X ２ The Cas9 protein according to any one of claims 22 to 48, wherein is any amino acid other than H.

51. The Cas9 protein according to any one of claims 22 to 50, wherein the Cas9 protein comprises a D10A mutation in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

52. The Cas9 protein according to claim 51, wherein the Cas9 protein comprises H at amino acid residue 840 of the amino acid sequence shown in SEQ ID NO: 9, or a corresponding residue in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

53. The Cas9 protein according to any one of claims 22 to 51, wherein the Cas9 protein comprises an H840A mutation in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

54. The Cas9 protein according to claim 53, wherein the Cas9 protein comprises D in amino acid residue 10 of the amino acid sequence shown in SEQ ID NO: 9, or a corresponding residue in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

55. The Cas9 protein according to any one of claims 22 to 54, wherein the Cas9 protein exhibits increased activity against target sequences that do not contain a classical PAM (5'-NGG-3') at the 3' end, compared to Cas9 of Streptococcus pyogenes shown in SEQ ID NO:

9.

56. It contains an amino acid sequence that is at least 90 percent identical to the amino acid sequence or fragment of Cas9 of Streptococcus pyogenes shown in Sequence ID No. 9, The amino acid sequence of the Cas9 protein contains at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations in amino acid residues selected from the group consisting of amino acid residues 262, 267, 294, 405, 409, 480, 543, 694, 1219, 1224, 1256, and 1362 of the amino acid sequence shown in Sequence ID No.

9. The amino acid sequence of the recombinant Cas9 protein is not identical to the amino acid sequence of the naturally occurring Cas9 protein. Recombinant Cas9 protein exhibits increased activity against target sequences that do not contain the classical PAM (5'-NGG-3') at the 3' end, compared to Cas9 from Streptococcus pyogenes shown in SEQ ID NO:

9.

57. The Cas9 protein according to claim 56, wherein the Cas9 protein comprises a RuvC and an HNH domain.

58. The Cas9 protein according to claim 56 or 57, wherein the Cas9 protein exhibits activity against a target sequence having a 3' end that is not directly adjacent to the classical PAM sequence (5'-NGG-3'), and the activity against the same target sequence is increased by at least 5, at least 10, at least 50, at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, at least 50,000, at least 100,000, at least 500,000, or at least 1,000,000 compared to the activity of Cas9 of Streptococcus pyogenes shown in SEQ ID NO:

9.

59. The Cas9 protein according to claim 58, wherein the 3' end of the target sequence is directly adjacent to an AGC, GAG, TTT, GTG, or CAA sequence.

60. The Cas9 protein according to claim 58 or 59, wherein the 3' end of the target sequence is directly adjacent to a sequence selected from the group consisting of CAC, GAT, TAA, ACG, CGA, and CGT.

61. The Cas9 protein according to any one of claims 56 to 60, wherein the activity of the Cas9 protein is measured by a nuclease assay, a deamination assay, or a transcriptional activation assay.

62. The Cas9 protein according to claim 61, wherein the transcriptional activation assay is a GFP activation assay.

63. The Cas9 protein according to any one of claims 56 to 62, wherein 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, or at least seven mutations selected from the group consisting of X262T, X267G, X294R, X405I, X409I, X480K, X543D, X694I, X1219V, X1224K, X1256K, and X1362P of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, and X represents any of these amino acids.

64. The Cas9 protein according to any one of claims 56 to 63, wherein the amino acid sequence or fragment of the Cas9 protein comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations selected from the group consisting of A262T, S267G, K294R, F405I, S409I, E480K, E543D, M694I, E1219V, N1224K, Q1256K, and L1362P of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

65. The Cas9 protein according to any one of claims 56 to 64, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X1219V mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

66. The Cas9 protein according to any one of claims 56 to 65, wherein the amino acid sequence or fragment of the Cas9 protein comprises the E1219V mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

67. The Cas9 protein according to any one of claims 56 to 66, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X480K mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

68. The Cas9 protein according to any one of claims 56 to 67, wherein the amino acid sequence or fragment of the Cas9 protein comprises the E480K mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

69. The Cas9 protein according to any one of claims 56 to 68, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X543D mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

70. The Cas9 protein according to any one of claims 56 to 69, wherein the amino acid sequence or fragment of the Cas9 protein comprises the E543D mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

71. The Cas9 protein according to any one of claims 56 to 70, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X480K, X543D and X1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

72. The Cas9 protein according to any one of claims 56 to 70, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X262T, X409I, X480K, X543D, X694I and X1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

73. The Cas9 protein according to any one of claims 56 to 70, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X294R, X480K, X543D, X1219V, X1256K and X1362P mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

74. The Cas9 protein according to any one of claims 56 to 70, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X294R, X480K, X543D, X1219V, and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

75. The Cas9 protein according to any one of claims 56 to 70, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X267G, X294R, X480K, X543D, X1219V, X1224K and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

76. The Cas9 protein according to any one of claims 56 to 70, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X262T, X405I, X409I, X480K, X543D, X694I and X1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

77. The Cas9 protein according to any one of claims 56 to 70, wherein the amino acid sequence or fragment of the Cas9 protein comprises the E480K, E543D and E1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

78. The Cas9 protein according to any one of claims 56 to 70, wherein the amino acid sequence or fragment of the Cas9 protein comprises the A262T, S409I, E480K, E543D, M694I and E1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

79. The Cas9 protein according to any one of claims 56 to 70, wherein the amino acid sequence or fragment of the Cas9 protein comprises the K294R, E480K, E543D, E1219V, Q1256K and L1362P mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

80. The Cas9 protein according to any one of claims 56 to 70, wherein the amino acid sequence or fragment of the Cas9 protein comprises the K294R, E480K, E543D, E1219V and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

81. The Cas9 protein according to any one of claims 56 to 70, wherein the amino acid sequence or fragment of the Cas9 protein comprises the S267G, K294R, E480K, E543D, E1219V, N1224K and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

82. The Cas9 protein according to any one of claims 56 to 70, wherein the amino acid sequence or fragment of the Cas9 protein comprises the A262T, F405I, S409I, E480K, E543D, M694I and E1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

83. The Cas9 protein according to any one of claims 57 to 82, wherein the amino acid sequence of the HNH domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of the HNH domain of any one of SEQ ID NOs: 9 to 262.

84. The Cas9 protein according to any one of claims 57 to 83, wherein the amino acid sequence of the HNH domain is identical to the amino acid sequence of the HNH domain of SEQ ID NO:

9.

85. The Cas9 protein according to any one of claims 57 to 84, wherein the amino acid sequence of the Ruvc domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of the Ruvc domain of any one of SEQ ID NOs. 9 to 262.

86. The Cas9 protein according to any one of claims 57 to 85, wherein the amino acid sequence of the Ruvc domain is identical to the amino acid sequence of the Ruvc domain of SEQ ID NO:

9.

87. The Cas9 protein according to any one of claims 56 to 86, wherein the Cas9 protein or a fragment thereof comprises a D10A and / or H840A mutation in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

88. The Cas9 protein or a fragment thereof has the D10X amino acid sequence shown in SEQ ID NO:

9. １ and / or H840X ２ A mutation, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10-262, X １ is any amino acid other than D, and X ２ The Cas9 protein according to any one of claims 56 to 86, wherein is any amino acid other than H.

89. The Cas9 protein or fragment thereof comprises a D10A mutation in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, according to any one of claims 56 to 88.

90. The Cas9 protein according to claim 89, wherein the Cas9 protein or a fragment thereof comprises H at amino acid residue 840 of the amino acid sequence shown in SEQ ID NO: 9, or a corresponding residue in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

91. The Cas9 protein according to any one of claims 56 to 89, wherein the Cas9 protein or a fragment thereof comprises an H840A mutation in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

92. The Cas9 protein according to claim 91, wherein the Cas9 protein or a fragment thereof comprises D at amino acid residue 10 of the amino acid sequence shown in SEQ ID NO: 9, or a corresponding residue in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

93. It contains an amino acid sequence that is at least 80 percent identical to the amino acid sequence of Cas9 represented by any one of sequence numbers 9 to 262, The amino acid sequence of the Cas9 protein contains at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations in an amino acid residue selected from the group consisting of amino acid residues 122, 137, 182, 262, 294, 409, 480, 543, 660, 694, 1219, and 1329 of the amino acid sequence shown in SEQ ID NO: 9, or in a corresponding amino acid residue of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262. Recombinant Cas9 proteins in which the amino acid sequence of the recombinant Cas9 protein is not identical to that of naturally occurring Cas9 proteins.

94. The Cas9 protein according to claim 93, wherein the Cas9 protein comprises a RuvC and an HNH domain.

95. The Cas9 protein according to claim 93 or 94, wherein 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, or at least seven mutations selected from the group consisting of X262T, X294R, X409I, X480K, X543D, X694I, and X1219V of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, and X represents any of the amino acids.

96. The Cas9 protein according to any one of claims 93 to 95, wherein the amino acid sequence of the Cas9 protein includes at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations selected from the group consisting of A262T, K294R, S409I, E480K, E543D, M694I, or E1219V of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

97. The Cas9 protein according to any one of claims 93 to 96, wherein the amino acid sequence of the Cas9 protein includes the X1219V mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

98. The Cas9 protein according to any one of claims 93 to 97, wherein the amino acid sequence of the Cas9 protein includes the E1219V mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

99. The Cas9 protein according to any one of claims 94 to 98, wherein the amino acid sequence of the HNH domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of the HNH domain of any one of SEQ ID NOs: 9 to 262.

100. The Cas9 protein according to any one of claims 94 to 99, wherein the amino acid sequence of the HNH domain is identical to the amino acid sequence of the HNH domain of SEQ ID NO:

9.

101. The Cas9 protein according to any one of claims 94 to 100, wherein the amino acid sequence of the Ruvc domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of the Ruvc domain of any one of SEQ ID NOs. 9 to 262.

102. The Cas9 protein according to any one of claims 94 to 101, wherein the amino acid sequence of the Ruvc domain is identical to the amino acid sequence of the Ruvc domain of SEQ ID NO:

9.

103. The Cas9 protein according to any one of claims 93 to 102, wherein the Cas9 protein comprises a D10A and / or H840A mutation in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

104. The Cas9 protein according to any one of claims 93 to 102, wherein the Cas9 protein comprises a D10A mutation in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

105. The Cas9 protein according to claim 104, wherein the Cas9 protein contains H at amino acid residue 840 of the amino acid sequence shown in SEQ ID NO: 9, or a corresponding residue in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

106. The Cas9 protein according to any one of claims 93 to 105, wherein the Cas9 protein exhibits increased activity against a target sequence that does not contain a classical PAM (5'-NGG-3') at its 3' end, compared to Cas9 of Streptococcus pyogenes shown in SEQ ID NO:

9.

107. It comprises an amino acid sequence having at least 90 percent identity with the amino acid sequence or fragment of Cas9 of Streptococcus pyogenes represented by Sequence ID No. 9, and includes the RuvC and HNH domains of Sequence ID No. 9, The amino acid sequence of the Cas9 protein contains at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations in amino acid residues selected from the group consisting of amino acid residues 122, 137, 182, 262, 294, 409, 480, 543, 660, 694, 1219, and 1329 of the amino acid sequence shown in Sequence ID No.

9. The amino acid sequence of the recombinant Cas9 protein is not identical to the amino acid sequence of the naturally occurring Cas9 protein. Recombinant Cas9 protein exhibits increased activity against target sequences that do not contain the classical PAM (5'-NGG-3') at the 3' end, compared to Cas9 from Streptococcus pyogenes shown in SEQ ID NO:

9.

108. The Cas9 protein according to claim 107, wherein the Cas9 protein comprises a RuvC and an HNH domain.

109. The Cas9 protein exhibits activity against target sequences that have a 3' end that is not directly adjacent to the classical PAM sequence (5'-NGG-3'). The Cas9 protein according to claim 107 or 108, which is increased by at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 50,000-fold, at least 100,000-fold, at least 500,000-fold, or at least 1,000,000-fold compared to the activity of Cas9 of Streptococcus pyogenes shown in Sequence ID No. 9 for the same target sequence.

110. The Cas9 protein according to claim 109, wherein the 3' end of the target sequence is directly adjacent to an AGC, GAG, TTT, GTG, or CAA sequence.

111. The Cas9 protein according to any one of claims 107 to 110, wherein the activity of the Cas9 protein is measured by a nuclease assay, a deamination assay, or a transcriptional activation assay.

112. The Cas9 protein according to claim 111, wherein the transcriptional activation assay is a GFP activation assay.

113. The Cas9 protein according to any one of claims 107 to 112, wherein 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, or at least seven mutations selected from the group consisting of X262T, X294R, X409I, X480K, X543D, X694I, and X1219V of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, and X represents any of the amino acids.

114. The Cas9 protein according to any one of claims 107 to 113, wherein the amino acid sequence or fragment of the Cas9 protein comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations selected from the group consisting of A262T, K294R, S409I, E480K, E543D, M694I, or E1219V of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

115. The Cas9 protein according to any one of claims 107 to 114, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X1219V mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

116. The Cas9 protein according to any one of claims 107 to 115, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X1219V mutation of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

117. The Cas9 protein according to any one of claims 108 to 116, wherein the amino acid sequence of the HNH domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of the HNH domain of any of SEQ ID NOs. 9 to 262.

118. The Cas9 protein according to any one of claims 108 to 117, wherein the amino acid sequence of the HNH domain is identical to the amino acid sequence of the HNH domain of SEQ ID NO:

9.

119. The Cas9 protein according to any one of claims 108 to 118, wherein the amino acid sequence of the Ruvc domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of the Ruvc domain of any of SEQ ID NOs. 9 to 262.

120. The Cas9 protein according to any one of claims 108 to 119, wherein the amino acid sequence of the Ruvc domain is identical to the amino acid sequence of the Ruvc domain of SEQ ID NO:

9.

121. The Cas9 protein according to any one of claims 107 to 120, wherein the Cas9 protein or a fragment thereof comprises a mutation D10A and / or H840A in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

122. The Cas9 protein according to any one of claims 107 to 121, wherein the Cas9 protein comprises a D10A mutation in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

123. The Cas9 protein according to claim 122, wherein the Cas9 protein contains H at amino acid residue 840 of the amino acid sequence shown in SEQ ID NO: 9, or a corresponding residue in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

124. It includes an amino acid sequence that is at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identical to the amino acid sequence of Cas9 represented by any one of sequence numbers 9 to 262, The amino acid sequence of the Cas9 protein contains mutations in 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 ten amino acid residues selected from the group consisting of amino acid residues 23, 108, 115, 141, 180, 230, 257, 262, 267, 284, 294, 324, 409, 455, 466, 474, 480, 543, 554, 654, 694, 711, 727, 763, 1063, 1100, 1219, 1244, 1256, 1289, and 1323 of the amino acid sequence shown in SEQ ID NO: 10 to 262, Recombinant Cas9 proteins in which the amino acid sequence of the recombinant Cas9 protein is not identical to that of naturally occurring Cas9 proteins.

125. The Cas9 protein according to claim 124, wherein the Cas9 protein comprises a RuvC and an HNH domain.

126. The amino acid sequence of the Cas9 protein is X23N, X108G, X115H, X141Q, X180N, X230S, X257N, X262T, X267G, X284N, X294R, X324L, X409I, X455F, X466A, X474I, X480K, X543D, X554R, X654L, X694I, X711E, X727P, X763I, X1063V, X1100I, X1219V, X1244 The Cas9 protein according to claim 124 or 125, comprising 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 ten mutations selected from the group consisting of N, X1256K, X1289Q, and X1323S, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs. 10 to 262, wherein X represents any of the amino acids.

127. The amino acid sequence of the Cas9 protein is D23N, E108G, R115H, K141Q, D180N, P230S, D257N, A262T, S267G, D284N, K294R, R324L, S409I, L455F, T466A, T474I, E480K, E543D, K554R, R654L, M694I, A711E, L727P, M763I, I1063V, V1100I, E1 The Cas9 protein according to any one of claims 124 to 126, comprising 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 ten mutations selected from the group consisting of 219V, K1244N, Q1256K, K1289Q, and A1323S, or corresponding mutations in any of the amino acid sequences shown in SEQ ID NOs. 10 to 262.

128. The Cas9 protein according to any one of claims 124 to 127, wherein the amino acid sequence of the Cas9 protein includes the X115H, X141Q, X267G, X294R, X480K, X543D, X1219V, and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

129. The Cas9 protein according to any one of claims 124 to 127, wherein the amino acid sequence of the Cas9 protein includes the X267G, X294R, X480K, X543D, X1219V, and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

130. The Cas9 protein according to any one of claims 124 to 127, wherein the amino acid sequence of the Cas9 protein includes mutations X23N, X108G, X262T, X409I, X480K, X543D, X694I, X727P, X1219V, and X1289Q of the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

131. The Cas9 protein according to any one of claims 124 to 127, wherein the amino acid sequence of the Cas9 protein includes the X257N, X267G, X294R, X466A, X480K, X543D, X1063V, X1219V, and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

132. The Cas9 protein according to any one of claims 124 to 127, wherein the amino acid sequence of the Cas9 protein comprises the X409I, X455F, X480K, X543D, X654L, X1100I, X1219V, and X1323S mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

133. The Cas9 protein according to any one of claims 124 to 127, wherein the amino acid sequence of the Cas9 protein includes the X267G, X294R, X480K, X543D, X1219V, and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

134. The Cas9 protein according to any one of claims 124 to 127, wherein the amino acid sequence of the Cas9 protein comprises the X262T, X324L, X409I, X480K, X543D, X694I, and X1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

135. The Cas9 protein according to any one of claims 124 to 127, wherein the amino acid sequence of the Cas9 protein includes the X294R, X480K, X543D, X711E, X1219V, and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

136. The Cas9 protein according to any one of claims 124 to 127, wherein the amino acid sequence of the Cas9 protein comprises the X230S, X267G, X294R, X480K, X543D, X1219V, and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

137. The Cas9 protein according to any one of claims 124 to 127, wherein the amino acid sequence of the Cas9 protein includes the X180N, X284N, X474I, X480K, X543D, X554R, X763I, X1219V, and X1244N mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

138. The Cas9 protein according to any one of claims 124 to 127, wherein the amino acid sequence of the Cas9 protein comprises the X262T, X409I, X480K, X543D, X694I, and X1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

139. The Cas9 protein according to any one of claims 124 to 127, wherein the amino acid sequence of the Cas9 protein includes the X267G, X294R, X480K, X543D, X1219V, and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

140. The Cas9 protein according to any one of claims 124 to 127, wherein the amino acid sequence of the Cas9 protein comprises the X230S, X267G, X294R, X480K, X543D, X1219V, and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

141. The Cas9 protein according to any one of claims 124 to 127, wherein the amino acid sequence of the Cas9 protein comprises the X267G, X294R, and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

142. The Cas9 protein according to any one of claims 124 to 127, wherein the amino acid sequence of the Cas9 protein comprises the X262T and X409I mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

143. The Cas9 protein according to any one of claims 124 to 127, wherein the amino acid sequence of the Cas9 protein comprises the R115H, K141Q, S267G, K294R, E480K, E543D, E1219V, and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

144. The Cas9 protein according to any one of claims 124 to 127, wherein the amino acid sequence of the Cas9 protein comprises the S267G, K294R, E480K, E543D, E1219V, and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

145. The Cas9 protein according to any one of claims 124 to 127, wherein the amino acid sequence of the Cas9 protein comprises the D23N, E108G, A262T, S409I, E480K, E543D, M694I, L727P, E1219V, and K1289Q mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

146. The Cas9 protein according to any one of claims 124 to 127, wherein the amino acid sequence of the Cas9 protein comprises the D257N, S267G, K294R, T466A, E480K, E543D, I1063V, E1219V, and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

147. The Cas9 protein according to any one of claims 124 to 127, wherein the amino acid sequence of the Cas9 protein comprises the S409I, L455F, E480K, E543D, R654L, V1100I, E1219V and A1323S mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

148. The Cas9 protein according to any one of claims 124 to 127, wherein the amino acid sequence of the Cas9 protein comprises the S267G, K294R, E480K, E543D, E1219V, and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

149. The Cas9 protein according to any one of claims 124 to 127, wherein the amino acid sequence of the Cas9 protein comprises the A262T, R324L, S409I, E480K, E543D, M694I, and E1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

150. The Cas9 protein according to any one of claims 124 to 127, wherein the amino acid sequence of the Cas9 protein comprises the K294R, E480K, E543D, A711E, E1219V, and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

151. The Cas9 protein according to any one of claims 124 to 127, wherein the amino acid sequence of the Cas9 protein comprises the P230S, S267G, K294R, E480K, E543D, E1219V, and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

152. The Cas9 protein according to any one of claims 124 to 127, wherein the amino acid sequence of the Cas9 protein comprises the D180N, D284N, T474I, E480K, E543D, K554R, M763I, E1219V, and K1244N mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

153. The Cas9 protein according to any one of claims 124 to 127, wherein the amino acid sequence of the Cas9 protein comprises the A262T, S409I, E480K, E543D, M694I, and E1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

154. The Cas9 protein according to any one of claims 124 to 127, wherein the amino acid sequence of the Cas9 protein comprises the S267G, K294R, E480K, E543D, E1219V, and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

155. The Cas9 protein according to any one of claims 124 to 127, wherein the amino acid sequence of the Cas9 protein comprises the P230S, S267G, K294R, E480K, E543D, E1219V, and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

156. The Cas9 protein according to any one of claims 124 to 127, wherein the amino acid sequence of the Cas9 protein comprises the S267G, K294R, and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

157. The Cas9 protein according to any one of claims 124 to 127, wherein the amino acid sequence of the Cas9 protein comprises the A262T and S409I mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

158. The Cas9 protein according to any one of claims 124 to 157, wherein the amino acid sequence of the HNH domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of the HNH domain of any one of SEQ ID NOs. 9 to 262.

159. The Cas9 protein according to any one of claims 125 to 158, wherein the amino acid sequence of the HNH domain is identical to the amino acid sequence of the HNH domain of SEQ ID NO:

9.

160. The Cas9 protein according to any one of claims 125 to 159, wherein the amino acid sequence of the Ruvc domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of the Ruvc domain of any one of SEQ ID NOs. 9 to 262.

161. The Cas9 protein according to any one of claims 125 to 160, wherein the amino acid sequence of the Ruvc domain is identical to the amino acid sequence of the Ruvc domain of SEQ ID NO:

9.

162. The Cas9 protein according to any one of claims 124 to 161, wherein the Cas9 protein comprises a D10A and / or H840A mutation in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

163. The Cas9 protein has a D10X １ and / or H840X ２ mutation, or a corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10-262, wherein X １ is any amino acid other than D, and X ２ is any amino acid other than H, the Cas9 protein according to any one of claims 124 to 162.

164. The Cas9 protein according to any one of claims 124 to 163, wherein the Cas9 protein comprises a D10A mutation in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

165. The Cas9 protein according to claim 164, wherein the Cas9 protein contains H at amino acid residue 840 of the amino acid sequence shown in SEQ ID NO: 9, or at a corresponding residue in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

166. The Cas9 protein according to any one of claims 124 to 164, wherein the Cas9 protein comprises an H840A mutation in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

167. The Cas9 protein according to claim 166, wherein the Cas9 protein contains D in amino acid residue 10 of the amino acid sequence shown in SEQ ID NO: 9, or in a corresponding residue of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

168. The Cas9 protein according to any one of claims 124 to 167, wherein the Cas9 protein exhibits increased activity against a target sequence that does not contain a classical PAM (5'-NGG-3') at its 3' end, compared to Cas9 of Streptococcus pyogenes shown in SEQ ID NO:

9.

169. It contains an amino acid sequence that is at least 90 percent identical to the amino acid sequence or fragment of Cas9 of Streptococcus pyogenes shown in Sequence ID No. 9, The amino acid sequence of the Cas9 protein contains mutations in 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 ten amino acid residues selected from the group consisting of amino acid residues 23, 108, 115, 141, 180, 230, 257, 262, 267, 284, 294, 324, 409, 455, 466, 474, 480, 543, 554, 654, 694, 711, 727, 763, 1063, 1100, 1219, 1244, 1256, 1289, and 1323 of the amino acid sequence shown in Sequence ID No. 9, The amino acid sequence of the recombinant Cas9 protein is not identical to the amino acid sequence of the naturally occurring Cas9 protein. Recombinant Cas9 protein exhibits increased activity against target sequences that do not contain the classical PAM (5'-NGG-3') at the 3' end, compared to Cas9 from Streptococcus pyogenes shown in SEQ ID NO:

9.

170. The Cas9 protein according to claim 169, wherein the Cas9 protein comprises a RuvC and an HNH domain.

171. The Cas9 protein according to claim 169 or 170, wherein the Cas9 protein exhibits activity against a target sequence having a 3' end that is not directly adjacent to the classical PAM sequence (5'-NGG-3'), and the activity against the same target sequence is increased by at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 50,000-fold, at least 100,000-fold, at least 500,000-fold, or at least 1,000,000-fold compared to the activity of Cas9 of Streptococcus pyogenes shown in SEQ ID NO:

9.

172. The Cas9 protein according to claim 171, wherein the 3' end of the target sequence is directly adjacent to an AGC, GAG, TTT, GTG, or CAA sequence.

173. The Cas9 protein according to claim 171 or 172, wherein the 3' end of the target sequence is directly adjacent to a sequence selected from the group consisting of CAC, GAT, TAA, ACG, CGA, and CGT.

174. The Cas9 protein according to any one of claims 169 to 173, wherein the activity of the Cas9 protein is measured by a nuclease assay, a deamination assay, or a transcriptional activation assay.

175. The Cas9 protein according to claim 174, wherein the transcriptional activation assay is a GFP activation assay.

176. The amino acid sequence of the Cas9 protein is X23N, X108G, X115H, X141Q, X180N, X230S, X257N, X262T, X267G, X284N, X294R, X324L, X409I, X455F, X466A, X474I, X480K, X543D, X554R, X654L, X694I, X711E, X727P, X763I, X1063V, X1100I, X1 The Cas9 protein according to any one of claims 169 to 175, comprising at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations selected from the group consisting of 219V, X1244N, X1256K, X1289Q, and X1323S, or corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs. 10 to 262, wherein X represents any of the amino acids.

177. The amino acid sequence or fragment of the Cas9 protein is D23N, E108G, R115H, K141Q, D180N, P230S, D257N, A262T, S267G, D284N, K294R, R324L, S409I, L455F, T466A, T474I, E480K, E543D, K554R, R654L, M694I, A711E, L727P, M763I, X1063 The Cas9 protein according to any one of claims 169 to 176, comprising at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations selected from the group consisting of V, X1100I, E1219V, K1244N, Q1256K, K1289Q, and A1323S, or corresponding mutations in any of the amino acid sequences shown in SEQ ID NOs. 10 to 262.

178. The Cas9 protein according to any one of claims 169 to 177, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X115H, X141Q, X267G, X294R, X480K, X543D, X1219V and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

179. The Cas9 protein according to any one of claims 169 to 177, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X267G, X294R, X480K, X543D, X1219V, and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

180. The Cas9 protein according to any one of claims 169 to 177, wherein the amino acid sequence or fragment of the Cas9 protein comprises mutations X23N, X108G, X262T, X409I, X480K, X543D, X694I, X727P, X1219V, and X1289Q of the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

181. The Cas9 protein according to any one of claims 169 to 177, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X257N, X267G, X294R, X466A, X480K, X543D, X1063V, X1219V and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

182. The Cas9 protein according to any one of claims 169 to 177, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X409I, X455F, X480K, X543D, X654L, X1100I, X1219V and X1323S mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

183. The Cas9 protein according to any one of claims 169 to 177, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X267G, X294R, X480K, X543D, X1219V, and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

184. The Cas9 protein according to any one of claims 169 to 177, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X262T, X324L, X409I, X480K, X543D, X694I and X1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

185. The Cas9 protein according to any one of claims 169 to 177, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X294R, X480K, X543D, X711E, X1219V, and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

186. The Cas9 protein according to any one of claims 169 to 177, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X230S, X267G, X294R, X480K, X543D, X1219V and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

187. The Cas9 protein according to any one of claims 169 to 177, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X180N, X284N, X474I, X480K, X543D, X554R, X763I, X1219V and X1244N mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

188. The Cas9 protein according to any one of claims 169 to 177, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X262T, X409I, X480K, X543D, X694I and X1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

189. The Cas9 protein according to any one of claims 169 to 177, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X267G, X294R, X480K, X543D, X1219V, and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

190. The Cas9 protein according to any one of claims 169 to 177, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X230S, X267G, X294R, X480K, X543D, X1219V and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

191. The Cas9 protein according to any one of claims 169 to 177, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X267G, X294R, and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

192. The Cas9 protein according to any one of claims 169 to 177, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X262T and X409I mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

193. The Cas9 protein according to any one of claims 169 to 177, wherein the amino acid sequence or fragment of the Cas9 protein comprises the R115H, K141Q, S267G, K294R, E480K, E543D, E1219V and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

194. The Cas9 protein according to any one of claims 169 to 177, wherein the amino acid sequence or fragment of the Cas9 protein comprises the S267G, K294R, E480K, E543D, E1219V and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

195. The Cas9 protein according to any one of claims 169 to 177, wherein the amino acid sequence or fragment of the Cas9 protein comprises mutations D23N, E108G, A262T, S409I, E480K, E543D, M694I, L727P, E1219V and K1289Q of the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

196. The Cas9 protein according to any one of claims 169 to 177, wherein the amino acid sequence or fragment of the Cas9 protein comprises the D257N, S267G, K294R, T466A, E480K, E543D, I1063V, E1219V and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

197. The Cas9 protein according to any one of claims 169 to 177, wherein the amino acid sequence or fragment of the Cas9 protein comprises the S409I, L455F, E480K, E543D, R654L, V1100I, E1219V and A1323S mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

198. The Cas9 protein according to any one of claims 169 to 177, wherein the amino acid sequence or fragment of the Cas9 protein comprises the S267G, K294R, E480K, E543D, E1219V and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

199. The Cas9 protein according to any one of claims 169 to 177, wherein the amino acid sequence or fragment of the Cas9 protein comprises the A262T, R324L, S409I, E480K, E543D, M694I and E1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

200. The Cas9 protein according to any one of claims 169 to 177, wherein the amino acid sequence or fragment of the Cas9 protein comprises the K294R, E480K, E543D, E543D, A711E, E1219V, and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

201. The Cas9 protein according to any one of claims 169 to 177, wherein the amino acid sequence or fragment of the Cas9 protein comprises the P230S, S267G, K294R, E480K, E543D, E1219V, and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

202. The Cas9 protein according to any one of claims 169 to 177, wherein the amino acid sequence or fragment of the Cas9 protein comprises the D180N, D284N, T474I, E480K, E543D, K554R, M763I, E1219V and K1244N mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

203. The Cas9 protein according to any one of claims 169 to 177, wherein the amino acid sequence or fragment of the Cas9 protein comprises the A262T, S409I, E480K, E543D, M694I and E1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

204. The Cas9 protein according to any one of claims 169 to 177, wherein the amino acid sequence or fragment of the Cas9 protein comprises the S267G, K294R, E480K, E543D, E1219V and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

205. The Cas9 protein according to any one of claims 169 to 177, wherein the amino acid sequence or fragment of the Cas9 protein comprises the P230S, S267G, K294R, E480K, E543D, E1219V, and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

206. The Cas9 protein according to any one of claims 169 to 177, wherein the amino acid sequence or fragment of the Cas9 protein comprises the S267G, K294R and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

207. The Cas9 protein according to any one of claims 169 to 177, wherein the amino acid sequence or fragment of the Cas9 protein comprises the A262T and S409I mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

208. The Cas9 protein according to any one of claims 170 to 207, wherein the amino acid sequence of the HNH domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of the HNH domain of any one of SEQ ID NOs. 9 to 262.

209. The Cas9 protein according to any one of claims 170 to 208, wherein the amino acid sequence of the HNH domain is identical to the amino acid sequence of the HNH domain of SEQ ID NO:

9.

210. The Cas9 protein according to any one of claims 170 to 209, wherein the amino acid sequence of the Ruvc domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of the Ruvc domain of any one of SEQ ID NOs. 9 to 262.

211. The Cas9 protein according to any one of claims 170 to 210, wherein the amino acid sequence of the Ruvc domain is identical to the amino acid sequence of the Ruvc domain of SEQ ID NO:

9.

212. The Cas9 protein according to any one of claims 169 to 211, wherein the Cas9 protein or a fragment thereof comprises a mutation D10A and / or H840A in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

213. The Cas9 protein or a fragment thereof has the D10X amino acid sequence shown in SEQ ID NO:

9. １ and / or H840X ２ A mutation, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10-262, X １ is any amino acid other than D, and X ２ The Cas9 protein according to any one of claims 169 to 211, wherein is any amino acid other than H.

214. The Cas9 protein according to any one of claims 169 to 213, wherein the Cas9 protein or a fragment thereof comprises a D10A mutation in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

215. The Cas9 protein according to claim 214, wherein the Cas9 protein or a fragment thereof contains H at amino acid residue 840 of the amino acid sequence shown in SEQ ID NO: 9, or at a corresponding residue in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

216. The Cas9 protein according to any one of claims 169 to 214, wherein the Cas9 protein or a fragment thereof comprises an H840A mutation in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

217. The Cas9 protein according to claim 216, wherein the Cas9 protein or a fragment thereof contains D in amino acid residue 10 of the amino acid sequence shown in SEQ ID NO: 9, or in a corresponding residue of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

218. It includes an amino acid sequence that is at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identical to the amino acid sequence of Cas9 represented by any one of sequence numbers 9 to 262, The amino acid sequence of the Cas9 protein contains mutations in at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine amino acid residues selected from the group consisting of amino acid residues 170, 230, 257, 267, 294, 466, 480, 543, 711, 1063, 1207, 1219, and 1256 of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding amino acid residues of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262. Recombinant Cas9 proteins in which the amino acid sequence of the recombinant Cas9 protein is not identical to that of naturally occurring Cas9 proteins.

219. The Cas9 protein according to claim 218, wherein the Cas9 protein comprises a RuvC and an HNH domain.

220. The Cas9 protein according to claim 218 or 219, wherein the amino acid sequence of the Cas9 protein includes at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine mutations selected from the group consisting of X175T, X230F, X257N, X267G, X294R, X466A, X480K, X543D, X711E, X1207G, X1063V, X1219V, and X1256K of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, and X represents any of the amino acids.

221. The Cas9 protein according to any one of claims 218 to 220, wherein the amino acid sequence of the Cas9 protein includes at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine mutations selected from the group consisting of N175T, P230F, D257N, S267G, K294R, T466A, E480K, E543D, A711E, E1207G, I1063V, E1219V, and Q1256K of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

222. The Cas9 protein according to any one of claims 218 to 221, wherein the amino acid sequence of the Cas9 protein includes the X230F, X267G, X294R, X480K, X543D, X1219V, and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

223. The Cas9 protein according to any one of claims 218 to 221, wherein the amino acid sequence of the Cas9 protein comprises the X294R, X480K, X543D, X711E, X1207G, X1219V, and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

224. The Cas9 protein according to any one of claims 218 to 221, wherein the amino acid sequence of the Cas9 protein includes the X175T, X267G, X294R, X480K, X543D, X1219V, and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

225. The Cas9 protein according to any one of claims 218 to 221, wherein the amino acid sequence of the Cas9 protein includes the X257N, X267G, X294R, X466A, X480K, X543D, X1063V, X1219V, and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

226. The Cas9 protein according to any one of claims 218 to 221, wherein the amino acid sequence of the Cas9 protein comprises the X294R, X480K, X543D, X1219V, and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

227. The Cas9 protein according to any one of claims 218 to 221, wherein the amino acid sequence of the Cas9 protein comprises the X294R and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

228. The Cas9 protein according to any one of claims 218 to 221, wherein the amino acid sequence of the Cas9 protein comprises the P230F, S267G, K294R, E480K, E543D, E1219V, and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

229. The Cas9 protein according to any one of claims 218 to 221, wherein the amino acid sequence of the Cas9 protein comprises the K294R, E480K, E543D, A711E, E1207G, E1219V, and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

230. The Cas9 protein according to any one of claims 218 to 221, wherein the amino acid sequence of the Cas9 protein comprises the N175T, S267G, K294R, E480K, E543D, E1219V, and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

231. The Cas9 protein according to any one of claims 218 to 221, wherein the amino acid sequence of the Cas9 protein comprises the D257N, S267G, K294R, T466A, E480K, E543D, I1063V, E1219V, and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

232. The Cas9 protein according to any one of claims 218 to 221, wherein the amino acid sequence of the Cas9 protein comprises the K294R, E480K, E543D, E1219V, and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

233. The Cas9 protein according to any one of claims 218 to 221, wherein the amino acid sequence of the Cas9 protein comprises the K294R and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

234. The Cas9 protein according to any one of claims 218 to 233, wherein the amino acid sequence of the HNH domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of the HNH domain of any one of SEQ ID NOs. 9 to 262.

235. The Cas9 protein according to any one of claims 219 to 234, wherein the amino acid sequence of the HNH domain is identical to the amino acid sequence of the HNH domain of SEQ ID NO:

9.

236. The Cas9 protein according to any one of claims 219 to 235, wherein the amino acid sequence of the Ruvc domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of the Ruvc domain of any of SEQ ID NOs. 9 to 262.

237. The Cas9 protein according to any one of claims 219 to 236, wherein the amino acid sequence of the Ruvc domain is identical to the amino acid sequence of the Ruvc domain of SEQ ID NO:

9.

238. The Cas9 protein according to any one of claims 218 to 237, wherein the Cas9 protein comprises a D10A and / or H840A mutation in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

239. The Cas9 protein has the D10X amino acid sequence shown in SEQ ID NO:

9. １ and / or H840X ２ A mutation, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10-262, X １ is any amino acid other than D, and X ２ The Cas9 protein according to any one of claims 218 to 238, wherein is any amino acid other than H.

240. The Cas9 protein according to any one of claims 218 to 239, wherein the Cas9 protein comprises a D10A mutation in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

241. The Cas9 protein according to claim 240, wherein the Cas9 protein contains H at amino acid residue 840 of the amino acid sequence shown in SEQ ID NO: 9, or at a corresponding residue in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

242. The Cas9 protein according to any one of claims 218 to 240, wherein the Cas9 protein comprises an H840A mutation in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

243. The Cas9 protein according to claim 242, wherein the Cas9 protein contains D in amino acid residue 10 of the amino acid sequence shown in SEQ ID NO: 9, or in a corresponding residue of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

244. The Cas9 protein according to any one of claims 218 to 243, wherein the Cas9 protein exhibits increased activity against a target sequence that does not contain a classical PAM (5'-NGG-3') at its 3' end, compared to Cas9 of Streptococcus pyogenes shown in SEQ ID NO:

9.

245. It contains an amino acid sequence that is at least 90 percent identical to the amino acid sequence or fragment of Cas9 of Streptococcus pyogenes shown in Sequence ID No. 9, The amino acid sequence of the Cas9 protein contains at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations in amino acid residues selected from the group consisting of amino acid residues 175, 230, 257, 267, 294, 466, 480, 543, 711, 1063, 1207, 1219, and 1256 of the amino acid sequence shown in Sequence ID No.

9. The amino acid sequence of the recombinant Cas9 protein is not identical to the amino acid sequence of the naturally occurring Cas9 protein. Recombinant Cas9 protein exhibits increased activity against target sequences that do not contain the classical PAM (5'-NGG-3') at the 3' end, compared to Cas9 from Streptococcus pyogenes shown in SEQ ID NO:

9.

246. The Cas9 protein according to claim 245, wherein the Cas9 protein comprises a RuvC and an HNH domain.

247. The Cas9 protein according to claim 245 or 246, wherein the Cas9 protein exhibits activity against a target sequence having a 3' end that is not directly adjacent to the classical PAM sequence (5'-NGG-3'), and the activity against the same target sequence is increased by at least 5, at least 10, at least 50, at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, at least 50,000, at least 100,000, at least 500,000, or at least 1,000,000 compared to the activity of Cas9 of Streptococcus pyogenes shown in SEQ ID NO:

9.

248. The Cas9 protein according to claim 247, wherein the 3' end of the target sequence is directly adjacent to an AGC, GAG, TTT, GTG, or CAA sequence.

249. The Cas9 protein according to claim 247 or 248, wherein the 3' end of the target sequence is directly adjacent to a sequence selected from the group consisting of CAC, GAT, TAA, ACG, CGA, and CGT.

250. The Cas9 protein according to any one of claims 246 to 249, wherein the activity of the Cas9 protein is measured by a nuclease assay, a deamination assay, or a transcriptional activation assay.

251. The Cas9 protein according to claim 250, wherein the transcriptional activation assay is a GFP activation assay.

252. The Cas9 protein according to any one of claims 245 to 251, wherein the amino acid sequence of the Cas9 protein includes at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine mutations selected from the group consisting of X175T, X230F, X257N, X267G, X294R, X466A, X480K, X543D, X711E, X1207G, X1063V, X1219V, and X1256K of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, and X represents any of the amino acids.

253. The Cas9 protein according to any one of claims 245 to 252, wherein the amino acid sequence or fragment of the Cas9 protein includes at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine mutations selected from the group consisting of N175T, P230F, D257N, S267G, K294R, T466A, E480K, E543D, A711E, E1207G, I1063V, E1219V, and Q1256K of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

254. The Cas9 protein according to any one of claims 245 to 253, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X230F, X267G, X294R, X480K, X543D, X1219V and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

255. The Cas9 protein according to any one of claims 245 to 253, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X294R, X480K, X543D, X711E, X1207G, X1219V and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

256. The Cas9 protein according to any one of claims 245 to 253, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X175T, X267G, X294R, X480K, X543D, X1219V and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

257. The Cas9 protein according to any one of claims 245 to 253, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X257N, X267G, X294R, X466A, X480K, X543D, X1063V, X1219V and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

258. The Cas9 protein according to any one of claims 245 to 253, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X294R, X480K, X543D, X1219V, and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

259. The Cas9 protein according to any one of claims 245 to 253, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X294R and X1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

260. The Cas9 protein according to any one of claims 245 to 253, wherein the amino acid sequence or fragment of the Cas9 protein comprises the P230F, S267G, K294R, E480K, E543D, E1219V and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

261. The Cas9 protein according to any one of claims 245 to 253, wherein the amino acid sequence or fragment of the Cas9 protein comprises the K294R, E480K, E543D, A711E, E1207G, E1219V and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

262. The Cas9 protein according to any one of claims 245 to 253, wherein the amino acid sequence or fragment of the Cas9 protein comprises the N175T, S267G, K294R, E480K, E543D, E1219V and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

263. The Cas9 protein according to any one of claims 245 to 253, wherein the amino acid sequence or fragment of the Cas9 protein comprises the D257N, S267G, K294R, T466A, E480K, E543D, I1063V, E1219V and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

264. The Cas9 protein according to any one of claims 245 to 253, wherein the amino acid sequence or fragment of the Cas9 protein comprises the K294R, E480K, E543D, E1219V and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

265. The Cas9 protein according to any one of claims 245 to 253, wherein the amino acid sequence or fragment of the Cas9 protein comprises the K294R and Q1256K mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

266. The Cas9 protein according to any one of claims 246 to 265, wherein the amino acid sequence of the HNH domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of the HNH domain of any one of SEQ ID NOs. 9 to 262.

267. The Cas9 protein according to any one of claims 246 to 266, wherein the amino acid sequence of the HNH domain is identical to the amino acid sequence of the HNH domain of SEQ ID NO:

9.

268. The Cas9 protein according to any one of claims 246 to 267, wherein the amino acid sequence of the Ruvc domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of the Ruvc domain of any one of SEQ ID NOs. 9 to 262.

269. The Cas9 protein according to any one of claims 247 to 268, wherein the amino acid sequence of the Ruvc domain is identical to the amino acid sequence of the Ruvc domain of SEQ ID NO:

9.

270. The Cas9 protein according to any one of claims 245 to 269, wherein the Cas9 protein or a fragment thereof comprises a D10A and / or H840A mutation in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

271. The Cas9 protein or a fragment thereof has the D10X amino acid sequence shown in SEQ ID NO:

9. １ and / or H840X ２ A mutation, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10-262, X １ is any amino acid other than D, and X ２ The Cas9 protein according to any one of claims 245 to 269, wherein is any amino acid other than H.

272. The Cas9 protein or fragment thereof comprises a D10A mutation in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, according to any one of claims 245 to 271.

273. The Cas9 protein according to claim 272, wherein the Cas9 protein or a fragment thereof contains H at amino acid residue 840 of the amino acid sequence shown in SEQ ID NO: 9, or at a corresponding residue in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

274. The Cas9 protein according to any one of claims 245 to 272, wherein the Cas9 protein or a fragment thereof comprises an H840A mutation in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

275. The Cas9 protein according to claim 274, wherein the Cas9 protein or a fragment thereof contains D in amino acid residue 10 of the amino acid sequence shown in SEQ ID NO: 9, or in a corresponding residue of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

276. It includes an amino acid sequence that is at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identical to the amino acid sequence of Cas9 represented by any one of sequence numbers 9 to 262, The amino acid sequence of the Cas9 protein contains at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations in amino acid residues selected from the group consisting of amino acid residues 108, 217, 262, 324, 409, 480, 543, 673, 694, 1219, 1264, and 1365 of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding amino acid residues of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262. Recombinant Cas9 proteins in which the amino acid sequence of the recombinant Cas9 protein is not identical to that of naturally occurring Cas9 proteins.

277. The Cas9 protein according to claim 276, wherein the Cas9 protein comprises a RuvC and an HNH domain.

278. The Cas9 protein according to claim 276 or 277, wherein the amino acid sequence of the Cas9 protein includes at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine mutations selected from the group consisting of X108G, X217A, X262T, X324L, X409I, X480K, X543D, X673E, X694I, X1219V, X1264Y, and X1365I of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, and X represents any of the amino acids.

279. The Cas9 protein according to any one of claims 276 to 278, wherein the amino acid sequence of the Cas9 protein includes at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine mutations selected from the group consisting of E108G, S217A, A262T, R324L, S409I, E480K, E543D, K673E, M694I, E1219V, H1264Y, and L1365I of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

280. The Cas9 protein according to any one of claims 276 to 279, wherein the amino acid sequence of the Cas9 protein includes the X108G, X262T, X409I, X480K, X543D, X673E, X694I, X1219V, and X1365I mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

281. The Cas9 protein according to any one of claims 276 to 279, wherein the amino acid sequence of the Cas9 protein includes the X108G, X217A, X262T, X409I, X480K, X543D, X694I, X1219V, and X1365I mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

281. The Cas9 protein according to any one of claims 276 to 279, wherein the amino acid sequence of the Cas9 protein comprises the X262T, X324L, X409I, X480K, X543D, X694I, and X1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

283. The Cas9 protein according to any one of claims 276 to 279, wherein the amino acid sequence of the Cas9 protein comprises the X262T, X409I, X480K, X543D, X694I, X1219V, and X1264Y mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

284. The Cas9 protein according to any one of claims 276 to 279, wherein the amino acid sequence of the Cas9 protein includes the X262T, X409I, X480K, X543D, X694I, and X1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

285. The Cas9 protein according to any one of claims 276 to 279, wherein the amino acid sequence of the Cas9 protein comprises the X262T, X409I, X480K, X543D, X694I, X1219V, and X1365I mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

286. The Cas9 protein according to any one of claims 276 to 279, wherein the amino acid sequence of the Cas9 protein comprises the X108G, X262T, X409I, X480K, X543D, X673E, X694I, and X1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

287. The Cas9 protein according to any one of claims 276 to 279, wherein the amino acid sequence of the Cas9 protein includes the X108G, X262T, X409I, X480K, X543D, X694I, X1219V, and X1365I mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

288. The Cas9 protein according to any one of claims 276 to 279, wherein the amino acid sequence of the Cas9 protein comprises the X262T and X409I mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

289. The Cas9 protein according to any one of claims 276 to 279, wherein the amino acid sequence of the Cas9 protein comprises mutations E108G, A262T, S409I, E480K, E543D, K673E, M694I, E1219V, and L1365I of the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

290. The Cas9 protein according to any one of claims 276 to 279, wherein the amino acid sequence of the Cas9 protein includes mutations E108G, S217A, A262T, S409I, E480K, E543D, M694I, E1219V, and L1365I of the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

291. The Cas9 protein according to any one of claims 276 to 279, wherein the amino acid sequence of the Cas9 protein comprises the A262T, R324L, S409I, E480K, E543D, M694I, and E1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

292. The Cas9 protein according to any one of claims 276 to 279, wherein the amino acid sequence of the Cas9 protein comprises the A262T, S409I, E480K, E543D, M694I, E1219V, and H1264Y mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

293. The Cas9 protein according to any one of claims 276 to 279, wherein the amino acid sequence of the Cas9 protein comprises the A262T, S409I, E480K, E543D, M694I, and E1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

294. The Cas9 protein according to any one of claims 276 to 279, wherein the amino acid sequence of the Cas9 protein comprises the A262T, S409I, E480K, E543D, M694I, E1219V, and L1365I mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

295. The Cas9 protein according to any one of claims 276 to 279, wherein the amino acid sequence of the Cas9 protein comprises the E108G, A262T, S409I, E480K, E543D, K673E, M694I, and E1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

296. The Cas9 protein according to any one of claims 276 to 279, wherein the amino acid sequence of the Cas9 protein comprises the E108G, A262T, S409I, E480K, E543D, M694I, E1219V, and L1365I mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

297. The Cas9 protein according to any one of claims 276 to 279, wherein the amino acid sequence of the Cas9 protein comprises the A262T and S409I mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

298. The Cas9 protein according to any one of claims 276 to 297, wherein the amino acid sequence of the HNH domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of the HNH domain of any one of SEQ ID NOs. 9 to 262.

299. The Cas9 protein according to any one of claims 277 to 298, wherein the amino acid sequence of the HNH domain is identical to the amino acid sequence of the HNH domain of SEQ ID NO:

9.

300. The Cas9 protein according to any one of claims 277 to 299, wherein the amino acid sequence of the Ruvc domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of the Ruvc domain of any one of SEQ ID NOs. 9 to 262.

301. The Cas9 protein according to any one of claims 277 to 300, wherein the amino acid sequence of the Ruvc domain is identical to the amino acid sequence of the Ruvc domain of SEQ ID NO:

9.

302. The Cas9 protein according to any one of claims 276 to 301, wherein the Cas9 protein comprises a D10A and / or H840A mutation in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

303. The Cas9 protein has the D10X amino acid sequence shown in SEQ ID NO:

9. １ and / or H840X ２ A mutation, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10-262, X １ is any amino acid other than D, and X ２ The Cas9 protein according to any one of claims 276 to 302, wherein is any amino acid other than H.

304. The Cas9 protein according to any one of claims 276 to 303, wherein the Cas9 protein comprises a D10A mutation in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

305. The Cas9 protein according to claim 304, wherein the Cas9 protein contains H at amino acid residue 840 of the amino acid sequence shown in SEQ ID NO: 9, or at a corresponding residue in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

306. The Cas9 protein according to any one of claims 276 to 304, wherein the Cas9 protein comprises an H840A mutation in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

307. The Cas9 protein according to claim 306, wherein the Cas9 protein contains D in amino acid residue 10 of the amino acid sequence shown in SEQ ID NO: 9, or in a corresponding residue of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

308. The Cas9 protein according to any one of claims 276 to 307, wherein the Cas9 protein exhibits increased activity against a target sequence that does not contain a classical PAM (5'-NGG-3') at its 3' end, compared to Cas9 of Streptococcus pyogenes shown in SEQ ID NO:

9.

309. It contains an amino acid sequence that is at least 90 percent identical to the amino acid sequence or fragment of Cas9 of Streptococcus pyogenes shown in Sequence ID No. 9, The amino acid sequence of the Cas9 protein contains at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations in amino acid residues selected from the group consisting of amino acid residues 108, 217, 262, 324, 409, 480, 543, 673, 694, 1219, 1264, and 1365 of the amino acid sequence shown in Sequence ID No.

9. The amino acid sequence of the recombinant Cas9 protein is not identical to the amino acid sequence of the naturally occurring Cas9 protein. Recombinant Cas9 protein exhibits increased activity against target sequences that do not contain the classical PAM (5'-NGG-3') at the 3' end, compared to Cas9 from Streptococcus pyogenes shown in SEQ ID NO:

9.

310. The Cas9 protein according to claim 309, wherein the Cas9 protein comprises a RuvC and an HNH domain.

311. The Cas9 protein according to claim 309 or 310, wherein the Cas9 protein exhibits activity against a target sequence having a 3' end that is not directly adjacent to the classical PAM sequence (5'-NGG-3'), and the activity against the same target sequence is increased by at least 5, at least 10, at least 50, at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, at least 50,000, at least 100,000, at least 500,000, or at least 1,000,000 compared to the activity of Cas9 of Streptococcus pyogenes shown in SEQ ID NO:

9.

312. The Cas9 protein according to claim 311, wherein the 3' end of the target sequence is directly adjacent to an AGC, GAG, TTT, GTG, or CAA sequence.

313. The Cas9 protein according to claim 311 or 312, wherein the 3' end of the target sequence is directly adjacent to a sequence selected from the group consisting of CAC, GAT, TAA, ACG, CGA, and CGT.

314. The Cas9 protein according to any one of claims 310 to 313, wherein the activity of the Cas9 protein is measured by a nuclease assay, a deamination assay, or a transcriptional activation assay.

315. The Cas9 protein according to claim 314, wherein the transcriptional activation assay is a GFP activation assay.

316. The Cas9 protein according to any one of claims 309 to 315, wherein the amino acid sequence of the Cas9 protein includes at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine mutations selected from the group consisting of X108G, X217A, X262T, X324L, X409I, X480K, X543D, X673E, X694I, X1219V, X1264Y, and X1365I of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, and X represents any of the amino acids.

317. The Cas9 protein according to any one of claims 309 to 316, wherein the amino acid sequence or fragment of the Cas9 protein includes at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine mutations selected from the group consisting of E108G, S217A, A262T, R324L, S409I, E480K, E543D, K673E, M694I, E1219V, H1264Y, and L1365I of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

318. The Cas9 protein according to any one of claims 309 to 317, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X108G, X262T, X409I, X480K, X543D, X673E, X694I, X1219V, and X1365I mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

319. The Cas9 protein according to any one of claims 309 to 317, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X108G, X217A, X262T, X409I, X480K, X543D, X694I, X1219V and X1365I mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

320. The Cas9 protein according to any one of claims 309 to 317, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X262T, X324L, X409I, X480K, X543D, X694I and X1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

321. The Cas9 protein according to any one of claims 309 to 317, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X262T, X409I, X480K, X543D, X694I, X1219V, and X1264Y mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

322. The Cas9 protein according to any one of claims 309 to 317, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X262T, X409I, X480K, X543D, X694I and X1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

323. The Cas9 protein according to any one of claims 309 to 317, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X262T, X409I, X480K, X543D, X694I, X1219V, and X1365I mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

324. The Cas9 protein according to any one of claims 309 to 317, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X108G, X262T, X409I, X480K, X543D, X673E, X694I and X1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

325. The Cas9 protein according to any one of claims 309 to 317, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X108G, X262T, X409I, X480K, X543D, X694I, X1219V and X1365I mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

326. The Cas9 protein according to any one of claims 309 to 317, wherein the amino acid sequence or fragment of the Cas9 protein comprises the X262T and X409I mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262, where X represents any of the amino acids.

327. The Cas9 protein according to any one of claims 309 to 317, wherein the amino acid sequence or fragment of the Cas9 protein comprises mutations E108G, A262T, S409I, E480K, E543D, K673E, M694I, E1219V, and L1365I of the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

328. The Cas9 protein according to any one of claims 309 to 317, wherein the amino acid sequence or fragment of the Cas9 protein comprises mutations E108G, S217A, A262T, S409I, E480K, E543D, M694I, E1219V, and L1365I of the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

329. The Cas9 protein according to any one of claims 309 to 317, wherein the amino acid sequence or fragment of the Cas9 protein comprises the A262T, R324L, S409I, E480K, E543D, M694I and E1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

330. The Cas9 protein according to any one of claims 309 to 317, wherein the amino acid sequence or fragment of the Cas9 protein comprises the A262T, S409I, E480K, E543D, M694I, E1219V and H1264Y mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

331. The Cas9 protein according to any one of claims 309 to 317, wherein the amino acid sequence or fragment of the Cas9 protein comprises the A262T, S409I, E480K, E543D, M694I and E1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutations of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

332. The Cas9 protein according to any one of claims 309 to 317, wherein the amino acid sequence or fragment of the Cas9 protein comprises the A262T, S409I, E480K, E543D, M694I, E1219V and L1365I mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

333. The Cas9 protein according to any one of claims 309 to 317, wherein the amino acid sequence or fragment of the Cas9 protein comprises the E108G, A262T, S409I, E480K, E543D, K673E, M694I and E1219V mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

334. The Cas9 protein according to any one of claims 309 to 317, wherein the amino acid sequence or fragment of the Cas9 protein comprises the E108G, A262T, S409I, E480K, E543D, M694I, E1219V and L1365I mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

335. The Cas9 protein according to any one of claims 309 to 317, wherein the amino acid sequence or fragment of the Cas9 protein comprises the A262T and S409I mutations of the amino acid sequence shown in SEQ ID NO: 9, or the corresponding mutation of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

336. The Cas9 protein according to any one of claims 310 to 335, wherein the amino acid sequence of the HNH domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of the HNH domain of any one of SEQ ID NOs: 9 to 262.

337. The Cas9 protein according to any one of claims 310 to 330, wherein the amino acid sequence of the HNH domain is identical to the amino acid sequence of the HNH domain of SEQ ID NO:

9.

338. The Cas9 protein according to any one of claims 310 to 337, wherein the amino acid sequence of the Ruvc domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the amino acid sequence of the Ruvc domain of any of SEQ ID NOs. 9 to 262.

339. The Cas9 protein according to any one of claims 310 to 338, wherein the amino acid sequence of the Ruvc domain is identical to the amino acid sequence of the Ruvc domain of SEQ ID NO:

9.

340. The Cas9 protein according to any one of claims 309 to 339, wherein the Cas9 protein or a fragment thereof comprises a mutation D10A and / or H840A in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

341. The Cas9 protein or a fragment thereof has the D10X amino acid sequence shown in SEQ ID NO:

9. １ and / or H840X ２ A mutation, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10-262, X １ is any amino acid other than D, and X ２ The Cas9 protein according to any one of claims 309 to 339, wherein is any amino acid other than H.

342. The Cas9 protein according to any one of claims 309 to 341, wherein the Cas9 protein or a fragment thereof comprises a D10A mutation in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

343. The Cas9 protein according to claim 342, wherein the Cas9 protein or a fragment thereof contains H at amino acid residue 840 of the amino acid sequence shown in SEQ ID NO: 9, or at a corresponding residue in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

344. The Cas9 protein according to any one of claims 309 to 342, wherein the Cas9 protein or a fragment thereof comprises an H840A mutation in the amino acid sequence shown in SEQ ID NO: 9, or a corresponding mutation in any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

345. The Cas9 protein according to claim 344, wherein the Cas9 protein or a fragment thereof contains D in amino acid residue 10 of the amino acid sequence shown in SEQ ID NO: 9, or in a corresponding residue of any of the amino acid sequences shown in SEQ ID NOs: 10 to 262.

346. A fusion protein comprising the Cas9 protein according to any one of claims 1 to 345, wherein the Cas9 protein or a fragment thereof fuses with an effector domain to form a fusion protein.

347. The fusion protein according to claim 346, wherein the effector domain is fused to the N-terminus of the Cas9 protein.

348. The fusion protein according to claim 346, wherein the effector domain is fused to the C-terminus of the Cas9 protein.

349. A fusion protein according to any one of claims 346 to 348, wherein the Cas9 protein and the effector domain are fused via a linker.

350. The fusion protein according to any one of claims 346 to 349, wherein the effector domain is a nucleic acid editing domain.

351. Linker, (GGGGS) ｎ (Sequence No. 5), (G) ｎ (EAAAAK) ｎ (Sequence No. 6), (GGS) ｎ , SGSETPGTSEATPES (Sequence ID 7) or (XP) ｎ The fusion protein according to claim 349 or 350, comprising a motif or any combination thereof, wherein n is independently an integer from 1 to 30.

352. Linker, (GGS) ３ A fusion protein according to any one of claims 349 to 351, comprising a motif.

353. The fusion protein according to any one of claims 346 to 352, wherein the effector domain includes an enzyme domain.

354. The fusion protein according to any one of claims 346 to 353, wherein the effector domain comprises a nuclease domain, a nickasase domain, a recombinase domain, a deaminase domain, a methyltransferase domain, a methylase domain, an acetylase domain, an acetyltransferase domain, a transcriptional activator domain, or a transcriptional repressor domain.

355. The fusion protein according to claim 354, wherein the effector domain is a domain comprising nuclease activity, nickas activity, recombinase activity, deaminase activity, methyltransferase activity, methylase activity, acetylase activity, acetyltransferase activity, transcriptional activation activity, or transcriptional repression activity.

356. The fusion protein according to claim 350, wherein the effector domain is a deaminase domain.

357. The fusion protein according to claim 356, wherein the deaminase is cytosine deaminase or cytidine deaminase.

358. The fusion protein according to claim 357, wherein the deaminase is a deaminase of the apolipoprotein B messenger RNA editing complex (APOBEC) family.

359. The fusion protein according to claim 357, wherein the deaminase is ABOBEC1 deaminase.

360. The fusion protein according to claim 357, wherein the deaminase is ABOBEC2 deaminase.

361. The fusion protein according to claim 357, wherein the deaminase is ABOBEC3 deaminase.

362. The fusion protein according to claim 357, wherein the deaminase is ABOBEC3A deaminase.

363. The fusion protein according to claim 357, wherein the deaminase is ABOBEC3D deaminase.

364. The fusion protein according to claim 357, wherein the deaminase is ABOBEC3E deaminase.

365. The fusion protein according to claim 357, wherein the deaminase is ABOBEC3F deaminase.

366. The fusion protein according to claim 357, wherein the deaminase is ABOBEC3G deaminase.

367. The fusion protein according to claim 357, wherein the deaminase is ABOBEC3H deaminase.

368. The fusion protein according to claim 357, wherein the deaminase is ABOBEC4 deaminase.

369. The fusion protein according to claim 357, wherein the deaminase is deaminase induced by activation (AID).

370. The fusion protein according to any one of claims 350 to 369, wherein the effector domain has at least 80 percent, at least 85 percent, at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, at least 99 percent, or at least 99.5 percent identity with the deaminase domain of any one of sequence numbers 263 to 281.

371. The fusion protein according to claim 353, wherein the enzyme domain is a nuclease domain.

372. The fusion protein according to claim 371, wherein the nuclease domain is a FokI DNA cleavage domain.

373. A dimer of the fusion protein according to claim 371 or 372.

374. A fusion protein according to any one of claims 346 to 373, wherein the fusion protein fuses with a second Cas9 protein.

375. The fusion protein according to claim 374, wherein the second Cas9 protein is Cas9.

376. The fusion protein according to claim 374 or 375, wherein the second Cas9 protein is the Cas9 protein according to any one of claims 1 to 345.

377. The fusion protein according to claim 374 or 376, wherein a second Cas9 protein is fused to the N-terminus of the fusion protein.

378. The fusion protein according to claim 374 or 376, wherein a second Cas9 protein is fused to the C-terminus of the fusion protein.

379. A fusion protein according to any one of claims 375 to 378, wherein a Cas9 protein and a second Cas9 protein are fused via a second linker.

380. Linker, (GGGGS) ｎ (Sequence No. 5), (G) ｎ (EAAAAK) ｎ (Sequence No. 6), (GGS) ｎ , SGSETPGTSEATPES (Sequence ID 7) or (XP) ｎ The fusion protein according to claim 379, comprising a motif or any combination thereof, wherein n is an independent integer from 1 to 30.

381. The second linker, (GGS) ３ A fusion protein according to claim 379, comprising a motif.

382. A fusion protein comprising a Cas9 protein according to any one of claims 22 to 345, fused to a second Cas9 protein.

383. The fusion protein according to claim 382, ​​wherein the second Cas9 protein is the Cas9 protein described in any one of claims 22 to 345.

384. The fusion protein according to claim 382, ​​wherein the second Cas9 protein is the fusion protein according to any one of claims 346 to 373.

385. The fusion protein according to claim 383 or 384, wherein a second Cas9 protein is fused to the N-terminus of a Cas9 protein.

386. The fusion protein according to claim 383 or 384, wherein a second Cas9 protein is fused to the C-terminus of a Cas9 protein.

387. A fusion protein according to any one of claims 382 to 386, wherein a second Cas9 protein and a Cas9 protein are fused via a linker.

388. Linker, (GGGGS) ｎ (Sequence No. 5), (G) ｎ , (EAAAAK)n (Sequence ID 6), (GGS) ｎ , SGSETPGTSEATPES (Sequence ID 7) or (XP) ｎ The fusion protein according to claim 387, comprising a motif or any combination thereof, wherein n is an independent integer from 1 to 30.

389. Linker, (GGS) ３ A fusion protein according to claim 387, comprising a motif.

390. The fusion protein according to claim 387, wherein the linker is the sequence SGSETPGTSEATPES (SEQ ID NO: 7).

391. A complex comprising a Cas9 protein according to any one of claims 22 to 345 or a fusion protein according to any one of claims 346 to 390, and a guide RNA that binds to the Cas9 protein or the Cas9 fusion protein.

392. The complex according to claim 391, wherein the guide RNA is about 15 to 100 nucleotides long and contains at least 10 consecutive nucleotides complementary to the target sequence.

393. The complex according to claim 392, wherein the guide RNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long.

394. The complex according to any one of claims 391 to 393, wherein the guide RNA comprises 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 consecutive nucleotides complementary to the target sequence.

395. The complex according to any one of claims 391 to 394, wherein the target sequence is a DNA base sequence.

396. The complex according to claim 395, wherein the target sequence is a sequence in the genome of a mammal.

397. The complex according to claim 396, wherein the target sequence is a sequence in the human genome.

398. The complex according to any one of claims 392 to 397, wherein the 3' end of the target sequence is not directly adjacent to the classical PAM sequence (5'-NGG-3').

399. A complex comprising a fusion protein according to any one of claims 374 to 390, a first guide RNA that binds to the Cas9 protein of the fusion protein, and a second guide RNA that binds to the second Cas9 protein of the fusion protein.

400. The complex according to claim 399, wherein the first guide RNA is about 15 to 100 nucleotides long and contains at least 10 consecutive nucleotides complementary to the first target sequence, and the second guide RNA is about 15 to 100 nucleotides long and contains at least 10 consecutive nucleotides complementary to the second target sequence.

401. The complex according to claim 400, wherein the first guide RNA and / or the second guide RNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long.

402. The complex according to any one of claims 399 to 401, wherein the first guide RNA and the second guide RNA are different.

403. The complex according to any one of claims 399 to 402, wherein the first guide RNA comprises a sequence of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides complementary to the first target sequence, and the second guide RNA comprises a sequence of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides complementary to the second target sequence.

404. The complex according to any one of claims 400 to 403, wherein the first target sequence and the second target sequence are different.

405. The complex according to any one of claims 400 to 404, wherein the first target sequence and the second target sequence are DNA base sequences.

406. The complex according to claim 405, wherein the first target sequence and the second target sequence are present in the genome of a mammal.

407. The complex according to claim 406, wherein the first target sequence and the second target sequence are present in the human genome.

408. The complex according to any one of claims 405 to 407, wherein the first target sequence is located within 30 nucleotides of the second target sequence.

409. The complex according to any one of claims 405 to 408, wherein the 3' end of the first target sequence is not directly adjacent to the classical PAM sequence (5'-NGG-3').

410. The complex according to any one of claims 405 to 409, wherein the 3' end of the second target sequence is not directly adjacent to the classical PAM sequence (5'-NGG-3').

411. To DNA molecules: (a) Contacting a Cas9 protein according to any one of claims 22 to 123 or a fusion protein according to any one of claims 346 to 373 with a guide RNA having a length of about 15 to 100 nucleotides and containing at least 10 consecutive nucleotides complementary to the target sequence, or (b) A method comprising contacting the composite according to any one of claims 391 to 410.

412. The method according to claim 411, wherein the target sequence is a DNA base sequence.

413. The method according to claim 411 or 412, wherein the 3' end of the target sequence is not directly adjacent to the classical PAM sequence (5'-NGG-3').

414. The complex according to any one of claims 411, wherein the 3' end of the target sequence is directly adjacent to an AGC, GAG, TTT, GTG, or CAA sequence.

415. The method according to any one of claims 411 to 414, wherein the target sequence includes a sequence associated with a disease or abnormality.

416. The method according to claim 415, wherein the target sequence includes a point mutation associated with a disease or abnormality.

417. The method according to claim 415, wherein the activity of the Cas9 protein, Cas9 fusion protein, or complex results in the modification of a point mutation.

418. The method according to any one of claims 411 to 417, wherein the target DNA sequence includes a T→C point mutation associated with a disease or abnormality, and deamination of the mutant C base results in a sequence not associated with a disease or abnormality.

419. The method according to claim 418, wherein the target DNA sequence encodes a protein, and the point mutation is located in a codon and results in a change in the amino acid encoded by the mutant codon compared to the wild-type codon.

420. The method according to claim 419, wherein deamination of mutant C results in a change in the amino acid encoded by the mutant codon.

421. The method according to claim 420, wherein deamination of mutant C results in a codon encoding a wild-type amino acid.

422. The method according to any one of claims 411 to 421, wherein the contact is made in vivo with respect to the object.

423. The method according to claim 422, wherein the subject has been diagnosed with a disease or abnormality.

424. The method according to any one of claims 418 to 423, wherein the disease or abnormality is a neoplasm associated with cystic fibrosis, phenylketonuria, exfoliative hyperkeratosis (EHK), Charcot-Marie-Tooth disease type 4J, neuroblastoma (NB), von Willebrand disease (vWD), congenital myotonia, hereditary renal amyloidosis, dilated cardiomyopathy (DCM), hereditary lymphedema, familial Alzheimer's disease, HIV, prion disease, chronic infantile neurocutaneous arthral syndrome (CINCA), desmin-related myopathy (DRM), mutant PI3KCA protein, mutant CTNNB1 protein, mutant HRAS protein, or mutant p53 protein.

425. The method according to any one of claims 415 to 423, wherein the disease or abnormality is related to a T>C or A>G mutation in a gene selected from the genes disclosed in Tables 6 and 7, respectively.

426. The method according to any one of claims 415 to 423, wherein the disease or abnormality is related to a T>C or A>G mutation in a gene selected from the genes disclosed in Tables 6 and 7, respectively.

427. (a) a nucleic acid sequence encoding the Cas9 protein according to any one of claims 22 to 123 or the fusion protein according to any one of claims 246 to 373; and (b) Heterogeneous promoter that drives the expression of the sequence in (a) A kit containing nucleic acid constructs.

428. The kit according to claim 427, further comprising an expression construct encoding a guide RNA backbone, wherein the construct includes a cloning site positioned to allow cloning of a nucleic acid sequence identical or complementary to a target sequence into the guide RNA backbone.

429. A polynucleotide encoding the Cas9 protein according to any one of claims 22 to 345 or the fusion protein according to any one of claims 346 to 373.

430. A vector comprising the polynucleotide described in claim 429.

431. The vector according to claim 430, wherein the vector comprises a heterologous promoter that drives the expression of a polynucleotide.

432. A cell comprising a nucleic acid molecule encoding the Cas9 protein according to any one of claims 22 to 345 or the fusion protein according to any one of claims 346 to 373, or the Cas9 protein according to any one of claims 22 to 345 or the fusion protein according to any one of claims 346 to 373.

433. AAA, AAC, AAG, AAT, CAA, CAC, CAG, CAT, GAA, GAC, GAG, GAT, TAA, TAC, TAG, TAT, ACA, A CC, ACG, ACT, CCA, CCC, CCG, CCT, GCA, GCC, GCG, GCT, TCA, TCC, TCG, TCT, AGA, AGC, AGT , CGA, CGC, CGT, GGA, GGC, GGT, TGA, TGC, TGT, ATA, ATC, ATG, ATT, CTA, CTC, CTG, CTT, A mutant Cas9 protein that recognizes a non-classical PAM sequence selected from the group consisting of GTA, GTC, GTG, GTT, TTA, TTC, TTG and TTT.

434. The mutant Cas9 protein according to claim 433, wherein the mutant Cas9 protein comprises the Cas9 protein according to any one of claims 1 to 345.

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