Methods and compositions for RNA-directed target DNA modification and RNA-directed transcription modulation

Abstract

To provide a DNA-targeting RNA that comprises a targeting sequence and provides, together with a modifying polypeptide, site-specific modification of a target DNA and / or a polypeptide associated with the target DNA.SOLUTION: Provided is a DNA-targeting RNA comprising: (i) a first segment comprising a nucleotide sequence complementary to a sequence in the target DNA; and (ii) a second segment that interacts with a site-directed modifying polypeptide, where, as an aspect, the first segment comprises 8 nucleotides having 100% complementarity to a sequence in the target DNA.SELECTED DRAWING: None

Description

[Technical Field]

[0001] cross reference This application is a continuation of U.S. Provisional Patent Application No. 61 / 652,086, filed May 25, 2012. No. 61 / 716,256 filed October 19, 2012, and No. 61 / 716,256 filed January 2013. No. 61 / 757,640, filed on February 28, 2013, and No. 61 / 757,640, filed on February 15, 2013. This application claims the benefit of U.S. Provisional Patent Application No. 61 / 765,576 filed in the United States and each of which is hereby incorporated by reference. No. 60 / 699,999, filed Dec. 16, 2003, the entirety of which is incorporated herein by reference.

[0002] Statement of Federally Funded Research This invention was made with funding from the National Institutes of Health, Grant No. GM081879. This invention was made with government support under the terms of the present application. The U.S. Government has certain rights in this invention. do.

[0003] Incorporation by reference of sequence listings provided as text files The sequence listing is a text file created on March 13, 2013, with a size of 7645 KB. The file "BERK-187WO-SeqList_ST25.txt" is included in this specification. The contents of the text files are incorporated herein by reference in their entirety. Be absorbed. [Background technology]

[0004] Approximately 60% of bacteria and 90% of archaea confer resistance to foreign DNA elements CRISPR (clustered regularly interspaced d short palindromic repeats) / CRISPR-related (Ca s) system. Streptococcus pyogenes The type II CRISPR system from Escherichia coli (E. pyogenes) is a novel CRISPR system that utilizes exogenous DNA induced by RNA. Only a single gene encoding the Cas9 protein is necessary and sufficient for A silencing, Two types of RNA (mature CRISPR RNA (crRNA) and a partially complementary transcript) It contains a transcription-regulating RNA (tracrRNA).

[0005] Recently, engineered nuclease enzymes designed to target specific DNA sequences have been developed. Gene deletion, gene replacement and gene repair, as well as the insertion of foreign sequences (transgenes) It is a powerful tool for genetically manipulating cells and whole organisms, allowing for insertion into the genome. Two methods for modifying site-specific DNA nucleases have been proposed. Two major techniques have emerged, both of which utilize sequence-nonspecific DNA endonuclease domains. Based on the construction of a chimeric endonuclease enzyme in which the enzyme is fused to an engineered DNA-binding domain However, targeting each new genomic locus requires new nuclei. The design of enzymes is necessary, which makes these approaches time-consuming and Furthermore, these two technologies have limited accuracy. It has drawbacks that can lead to unpredictable non-specific effects.

[0006] Systematic collation of cell genomes and gene reprogramming for expression or repression This involves targeting a set of genes. In recent years, the most popular approach has been to target any gene for regulation. The most common approach is to use RNA interference (RNAi). Techniques have limitations; for example, RNAi can exhibit significant non-specific effects and toxicity.

[0007] Nucleases can be synthesized in a way that avoids the need to design a new protein for each new target sequence. Precise targeting of enzyme activity (or other protein activity) to different locations within the target DNA There is a need in the field for techniques that allow for non-specific targeting. There is a need in the art for methods of regulating gene expression with minimal adverse effects. There are. Summary of the Invention

[0008] The present disclosure provides a method for targeting a target DNA and / or a modified polypeptide, comprising the steps of: or DNA-targeting RNAs that provide site-specific modification of polypeptides bound to target DNA. A. The present disclosure further provides a site-specifically modified polypeptide. In addition, methods for site-specific modification of target DNA and / or polypeptides bound to target DNA are also proposed. The present disclosure provides a method for cleaving a target nucleic acid with an enzymatically inactive Cas9 polypeptide and a DNA fragment. A method for modulating transcription of a target nucleic acid in a target cell generally involves contacting the target nucleic acid with a targeting RNA. Kits and compositions for carrying out the methods are also provided. and Cas9-transfected non-human multicellular organisms. Provide.

[0009] Features Features of the present disclosure include: (i) a nucleotide sequence that is complementary to a sequence within the target DNA; a first segment; and (ii) a second segment that interacts with the site-specific modifying polypeptide. In some instances, the first segment comprises a DNA-targeting RNA comprising: It contains eight nucleotides that are 100% complementary to a sequence in the target DNA. In some examples, the second segment is SEQ ID NOs: 431-682 (e.g., 431-56 2) A series of at least 8 nucleotides of any one of the nucleotide sequences described in Contains a nucleotide sequence that has at least 60% identity over consecutive nucleotides In some examples, the second segment comprises a nucleotide sequence set forth in SEQ ID NOs: 563-682. A stretch of at least 8 consecutive nucleotides for any one of the nucleotide sequences In some instances, the nucleotide sequence may be at least 60% identical to the sequence of the original. The site-specific modifying polypeptide is a Cas9 / Csn1 amino acid sequence shown in FIG. For amino acids 7 to 166 or 731 to 1003, or for SEQ ID NOs: 1 to 256 and and the corresponding portion in any of the amino acid sequences set forth as 795 to 1346. It comprises an amino acid sequence having at least about 75% amino acid sequence identity to the amino acid sequence.

[0010] A feature of the present disclosure is a DNA fragment comprising a nucleotide sequence encoding a DNA-targeting RNA. In some instances, the recombinant expression vector comprises a DNA polymerase. In some instances, the nucleotides encoding the DNA-targeting RNA include The nucleotide sequence is operably linked to a promoter. In some instances, the promoter is an inducible promoter. The loading nucleotide sequence further comprises a multiple cloning site. , and in vitro genetically modified host cells containing the DNA polynucleotide.

[0011] Features of the present disclosure include: (i) (a) a nucleotide sequence complementary to a sequence in a target DNA; (b) a first segment comprising a second segment that interacts with the site-directed modifying polypeptide; (ii) a nucleotide sequence encoding a DNA-targeting RNA comprising a ) an RNA-binding site that interacts with the DNA-targeting RNA; and (b) a site-specific enzymatic activity. wherein the site of enzymatic activity is determined by the DNA-targeting RNA. a nucleotide sequence encoding a site-specifically modified polypeptide containing a target site; Expression vectors are also included.

[0012] Features of the present disclosure include: (i) (a) a nucleotide sequence complementary to a sequence in a target DNA; (b) a first segment comprising a second segment that interacts with the site-directed modifying polypeptide; (ii) a nucleotide sequence encoding a DNA-targeting RNA comprising a ) an RNA-binding site that interacts with the DNA-targeting RNA; and (b) a transcription factor within the target DNA. The site in the target DNA where transcription is regulated is a DNA-targeted RNA. A nucleic acid encoding a site-specifically modified polypeptide containing an active site is determined by A. The present invention also includes recombinant expression vectors containing the nucleotide sequence.

[0013] Features of the present disclosure include: (i) a nucleotide sequence that is complementary to a sequence within the target DNA; an RNA-binding site that interacts with the DNA-targeting RNA; and (ii) reduced site specificity an active site that exhibits specific enzymatic activity, the site of enzymatic activity being determined by DNA-targeting RNA; Some examples include mutant site-directed modified polypeptides containing active sites that are In the present invention, the mutant site-directed modified polypeptide is a polypeptide that is capable of inhibiting S. pyogenes (S. pyogenes). s) H840A mutation of sequence (SEQ ID NO: 8), or SEQ ID NOs: 1-256 and 795-1 346. In one example, the mutant site-directed modifying polypeptide has the S. pyogenes sequence (SEQ ID NO:8) ) D10A mutation, or as set forth as SEQ ID NOs: 1-256 and 795-1346 In some instances, the variant portion contains a corresponding mutation in either of the amino acid sequences. The site-specific modified polypeptide comprises (i) the D10A mutation in the S. pyogenes sequence (SEQ ID NO: 8) or any of the amino acid sequences set forth as SEQ ID NOs: 1 to 256 and 795 to 1346 and (ii) the corresponding mutation in H8 of the S. pyogenes sequence (SEQ ID NO: 8). 40A mutation, or the amino acids set forth as SEQ ID NOS: 1-256 and 795-1346 It contains both corresponding mutations in either the amino acid sequence.

[0014] Features of the present disclosure include: (i) a nucleotide sequence that is complementary to a sequence within the target DNA; (ii) an RNA-binding site that interacts with the DNA-targeting RNA; and (ii) a site-specific enzymatic activity. wherein the site of enzymatic activity is determined by the DNA-targeting RNA. In some instances, chimeric site-directed modified polypeptides containing a chimeric site are included. The site-specific modification polypeptide is a Cas9 / Csn1 polypeptide having the amino acid sequence shown in FIG. For amino acids 7 to 166 or 731 to 1003, or for SEQ ID NOs: 1 to 256 and and the corresponding portion in any of the amino acid sequences set forth as 795 to 1346. It contains an amino acid sequence that has at least about 75% amino acid sequence identity to the In some examples, the DNA-targeting RNA is selected from the group consisting of SEQ ID NOs: 431-682 (e.g., SEQ ID NO: 5 63 to 682), a series of at least Nucleotides that share at least 60% identity over eight consecutive nucleotides In some examples, the DNA-targeting RNA further comprises the sequence of SEQ ID NOs: 431-5. 62, a series of at least eight Nucleotide sequences having at least 60% identity over consecutive nucleotides are also included. In some instances, the enzymatic activity of the chimeric site-directed modifying polypeptide is In some instances, the enzymatic activity of the chimeric site-directed modifying polypeptide modifies the DNA. The activity of the nuclease, methyltransferase, demethylase, and DNA Repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, deprotease activity Phosphorylation activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity enzyme activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photoactivity In some instances, the chimeric site-specific The enzymatic activity of the genetically modified polypeptide is a nuclease activity. The cleavage activity causes double-strand breaks in the target DNA. The enzymatic activity of the site-specific modifying polypeptide is determined by the target polypeptide bound to the target DNA. In some instances, the enzymatic activity of the chimeric site-directed modifying polypeptide 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 O-ribosylation activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity It is a cyclization activity.

[0015] Features of the disclosure include a nucleotide sequence encoding a chimeric site-directed modifying polypeptide. In some examples, the polynucleotide includes a polynucleotide comprising R In some instances, the polynucleotide is a DNA polynucleotide. A feature of the disclosure is a recombinant expression vector comprising the polynucleotide. In some examples, the polynucleotide is operably linked to a promoter. In some instances, the promoter is an inducible promoter. Features of the disclosure include in vitro genetically modified host cells containing the polynucleotides. .

[0016] Features of the present disclosure include: (i) a nucleotide sequence that is complementary to a sequence within the target DNA; (ii) an RNA-binding site that interacts with the DNA-targeting RNA; and (ii) a transcription factor within the target DNA. The site in the target DNA where transcription is regulated is a DNA-targeted RNA. A chimeric site-directed modified polypeptide comprising an active site determined by A. In some instances, the active site increases transcription within the target DNA. In this case, the active site reduces transcription within the target DNA.

[0017] Features of the present disclosure include an RNA binding site that interacts with a DNA-targeting RNA; and a site-specific The active site exhibits specific enzymatic activity, and the site of enzymatic activity is determined by the DNA-targeting RNA. a recombinant site-specifically modified polypeptide comprising an active site, In some examples, the site-directed modifying polypeptide is a Ca polypeptide shown in FIG. For amino acids 7 to 166 or 731 to 1003 of the s9 / Csn1 amino acid sequence, Or, among the amino acid sequences set forth as SEQ ID NOs: 1 to 256 and 795 to 1346 have at least about 75% amino acid sequence identity to the corresponding portion in any of In some examples, the cell is an archaeal cell, a bacterial cell, a eukaryotic cell, or a vacuole, eukaryotic unicellular organism, somatic cell, germ cell, stem cell, plant cell, algae cell, animal cell, invertebrate Animal cells, vertebrate cells, fish cells, frog cells, bird cells, mammalian cells, pig cells, female Bovine cells, goat cells, sheep cells, rodent cells, rat cells, mouse cells, non-human primates The cell is selected from the group consisting of a mammalian cell, a mammalian cell, and a human cell.

[0018] A feature of the present disclosure includes a genome comprising: (i) an RNA-binding moiety that interacts with a DNA-targeting RNA; and (ii) an active site that exhibits site-specific enzymatic activity, wherein the site of enzymatic activity is a DNA A recombinant site-specifically modified polypeptide containing an active site determined by a targeting RNA. Transgenic non-human organisms include those containing transgenes, which contain nucleotide sequences encoding the In some examples, the site-specific modifying polypeptide is a Cas polypeptide shown in FIG. 9 / Csn1 amino acid sequence, amino acids 7 to 166 or 731 to 1003, or or the amino acid sequences set forth as SEQ ID NOs: 1 to 256 and 795 to 1346 have at least about 75% amino acid sequence identity to the corresponding portion in either In some instances, the organism is an archaebacterium, a bacterium, a eukaryotic unicellular organism, or a nucleotide sequence of the amino acid sequence of the nucleotide ... , algae, plants, animals, invertebrates, flies, insects, cnidarians, vertebrates, fish, frogs, birds, mammals From the group consisting of animals, ungulates, rodents, rats, mice, and non-human primates are selected.

[0019] Features of the present disclosure include: (i) (a) a nucleotide sequence complementary to a sequence in a target DNA; (b) a first segment comprising a second segment that interacts with the site-directed modifying polypeptide; a DNA-targeting RNA comprising the fragment, or a DNA polynucleotide encoding the same; and (ii) (a) an RNA-binding site that interacts with a DNA-targeting RNA; and (b) An active site that exhibits site-specific enzymatic activity, wherein the site of enzymatic activity is determined by DNA-targeting RNA. A site-directed modified polypeptide comprising an active site determined by the method of claim 1, or a polypeptide encoding the same. In some instances, compositions comprising a DNA-targeting RNA polynucleotide are included. The first segment of A has at least 100% complementarity to a sequence in the target DNA. In some instances, the second segment of the DNA-targeting RNA comprises eight nucleotides. The components are nucleotides set forth in SEQ ID NOs: 431 to 682 (for example, SEQ ID NOs: 563 to 682). A stretch of at least 8 consecutive nucleotides for any one of the nucleotide sequences In some instances, the nucleotide sequence may be at least 60% identical to the sequence of the original. The second segment of the DNA-targeting RNA is a nucleotide sequence set forth in SEQ ID NOs: 431 to 562. A stretch of at least 8 consecutive nucleotides for any one of the nucleotide sequences In some instances, the nucleotide sequence may be at least 60% identical to the sequence of the original. The site-specific modifying polypeptide is a Cas9 / Csn1 amino acid sequence shown in FIG. For amino acids 7 to 166 or 731 to 1003, or for SEQ ID NOs: 1 to 256 and and the corresponding portion in any of the amino acid sequences set forth as 795 to 1346. It contains an amino acid sequence that has at least about 75% amino acid sequence identity to the In some instances, the enzymatic activity modifies the target DNA. , nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damaging activity, deaminating activity, dismutase activity, alkylating activity, depurination activity activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photoreactivation In some instances, the enzymatic activity is a nuclear enzyme or glycosylase activity. In some instances, nuclease activity creates double-strand breaks within the target DNA. In some instances, the enzymatic activity results in the target polypeptide binding to the target DNA. In some instances, the enzymatic activity is a methyltransferase activity, a deprotection activity, or a Methyltransferase 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 In some examples, the activity is a myristoylating activity, a myristoylating activity, or a demyristoylating activity. In this case, the target polypeptide is a histone, and the enzymatic activity is a methyltransferase activity. Demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity phospholipase activity, phosphatase activity, ubiquitin ligase activity, or deubiquitinating activity. In some instances, the DNA-targeting RNA is a double-molecule DNA-targeting RNA and is The composition contains both a targeting RNA and an activating RNA, the duplex-forming segments of which are complementary. The target sequence hybridizes to form the second segment of the DNA-targeting RNA. In this example, the duplex-forming segment of the activator RNA is A series of at least 8 consecutive nucleotides for any one of the nucleotide sequences It contains a nucleotide sequence that has at least 60% identity across the nucleotides.

[0020] Features of the present disclosure include: (i) a DNA-targeting RNA of the present disclosure, or a DNA encoding the same; and (ii) a buffer for stabilizing the nucleic acid. Features of the present disclosure include (i) a site-specifically modified polypeptide of the present disclosure, or and (ii) a polynucleotide encoding a nucleic acid and / or protein stabilizer. The features of the present disclosure include compositions comprising: (i) (a) a target DNA; (b) a first segment comprising a nucleotide sequence complementary to a sequence within a site-specific a DNA-targeting RNA comprising a second segment that interacts with the genetically modified polypeptide, or and (ii) a DNA polynucleotide encoding the targeting RNA; and (b) an RNA-binding site that interacts with the target DNA and regulates transcription. The site in the target DNA where transcription is regulated is determined by the DNA-targeting RNA. a site-directed modified polypeptide containing a functional site, or a polynucleotide encoding the same; In some instances, the active site increases transcription within the target DNA. In some instances, the active site reduces transcription within the target DNA. The features described include (i) a site-specifically modified polypeptide or a polynucleotide encoding the same; and (ii) a buffer for stabilizing nucleic acids and / or proteins. The present invention includes a composition comprising:

[0021] A feature of the disclosure includes a method for site-specifically modifying target DNA, the method comprising: (i) (a) a first sequence containing a nucleotide sequence complementary to a sequence in the target DNA; and (b) a second segment that interacts with the site-directed modifying polypeptide. a DNA-targeting RNA containing the same or a DNA polynucleotide encoding the same; and (ii) (a) an RNA-binding site that interacts with the DNA-targeting RNA; and (b) a site-specific enzyme. A site-directed modified polypeptide containing an active site exhibiting a specific activity, or a polypeptide encoding the same In some instances, the target DNA is extrachromosomal. In some instances, the target DNA has a complementary strand that is 5'-CCY-3'. The PAM sequence, where Y is any DNA nucleotide, is the target DNA The target sequence on the complementary strand is immediately 5' to the target sequence. In some instances, the target DNA is In some instances, the target DNA is part of a chromosome in vivo. In some cases, the target DNA is part of a chromosome within a cell. In some examples, the cell is an archaeal cell, a bacterial cell, a eukaryotic cell, a eukaryotic unicellular organism, a somatic cell, or a bacterial cell. cells, germ cells, stem cells, plant cells, algae cells, animal cells, invertebrate cells, vertebrate cells, Fish cells, frog cells, bird cells, mammalian cells, pig cells, cow cells, goat cells, sheep Human cells, rodent cells, rat cells, mouse cells, non-human primate cells, and human cells In some examples, the DNA-targeting RNA is selected from the group consisting of SEQ ID NO: 43 Any of the nucleotide sequences set forth in SEQ ID NOs: 1 to 682 (e.g., SEQ ID NOs: 563 to 682) For each of them, at least 60 over a stretch of at least 8 consecutive nucleotides In some instances, the DNA-targeting RNA comprises a nucleotide sequence having at least one nucleotide sequence with ... A is a series of small fragments of any of the nucleotide sequences set forth in SEQ ID NOs: 431 to 562. Nucleotides with at least 60% identity over at least 8 consecutive nucleotides In some examples, the DNA-modified polypeptide comprises a C nucleotide sequence as shown in FIG. For amino acids 7 to 166 or 731 to 1003 of the as9 / Csn1 amino acid sequence or the amino acid sequences set forth as SEQ ID NOs: 1 to 256 and 795 to 1346 have at least about 75% amino acid sequence identity to the corresponding portion in any of In some instances, the enzymatic activity modifies the target DNA. In some examples, the enzymatic activity includes a nuclease activity, a methyltransferase activity, Demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, Enzyme activity, transposase activity, recombinase activity, polymerase activity, ligase activity Some of the activities are enzyme activity, helicase activity, photolyase activity, or glycosylase activity. In some instances, the DNA modifying enzymatic activity is a nuclease activity. The cleavage activity causes double-strand breaks in the target DNA. This occurs under conditions that are permissive for non-homologous end joining or homology-directed repair. wherein the method further comprises contacting the target DNA with a donor polynucleotide; Donor polynucleotide, part of donor polynucleotide, Some copies of the donor polynucleotide are incorporated into the target DNA. In some instances, the method does not include contacting the cell with a donor polynucleotide. In some cases, the target DNA is modified such that nucleotides within the target DNA are deleted. In some instances, the enzymatic activity modifies a target polypeptide bound to the target DNA. In examples, the enzymatic activity may be a methyltransferase activity, a demethylase activity, an acetyltransferase activity, or a methyltransferase activity. Transferase 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 In some instances, the target polypeptide is a myristoylating or demyristoylating activity. Histones, and their enzymatic activities include methyltransferase activity, demethylase activity, and acetyltransferase activity. acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity In some examples, the activity is a ubiquitin ligase activity or a deubiquitinating activity. The complex further comprises an activator-RNA. In some instances, the activator-RNA is nucleotide sequences set forth in any one of 431 to 682, Nucleotides that share at least 60% identity over eight consecutive nucleotides Contains arrays.

[0022] A feature of the disclosure includes a method for modulating site-specific transcription within target DNA, the method comprising: (i) (a) a target DNA containing a nucleotide sequence complementary to a sequence within the target DNA; (b) a first segment that interacts with the site-specific modifying polypeptide; and (b) a second segment that interacts with the site-specific modifying polypeptide. DNA-targeting RNA containing the compound, or a DNA polynucleotide encoding the same; and (ii) (a) an RNA-binding site that interacts with the DNA-targeting RNA; and (b) a transcription factor. A site-directed modified polypeptide containing an active site that regulates the activity of a polypeptide or a polypeptide encoding the same. contacting the target DNA with a nucleotide, the contacting resulting in modulation of transcription in the target DNA. In some instances, transcription within the target DNA is increased. , transcription within the target DNA is reduced.

[0023] A feature of the present disclosure includes a method for site-specific modification in target DNA, the method comprising: (i) (a) a nucleotide sequence complementary to a sequence in the target DNA; and (b) a second segment that interacts with the site-directed modifying polypeptide. a DNA-targeting RNA comprising the following, or a DNA polynucleotide encoding the same; and ii) (a) an RNA-binding site that interacts with the DNA-targeting RNA; and (b) a target DNA A site-specific modifying polypeptide containing an active site that regulates transcription in A, or encoding the same In some instances, the site-specific modification comprises contacting the target gene with a polynucleotide that encodes the target gene. The polypeptide increases transcription within the target DNA. In some instances, the polypeptide increases transcription within the target DNA. The modified polypeptide reduces transcription within the target DNA.

[0024] Features of the present disclosure include a method for promoting site-specific cleavage and modification of target DNA in cells. The method includes: (i) (a) injecting into a cell a nucleotide sequence complementary to a sequence within a target DNA; (b) a first segment comprising a nucleotide sequence; and (b) a first segment that interacts with the site-specific modifying polypeptide. A DNA-targeting RNA comprising the second segment, or a DNA polynucleoside encoding the same. (ii) (a) an RNA-binding site that interacts with the DNA-targeting RNA; and (b) a region containing an active site that exhibits nuclease activity, generating a double-strand break in the target DNA; This involves introducing a specifically modified polypeptide or a polynucleotide encoding the same. The site of the double-strand break is determined by the DNA-targeting RNA, and the contacts are made by non-homologous end joining or or occurs under conditions permissive for homology-directed repair, resulting in the breakage and rejoining of the target DNA. In some instances, the method comprises: The method further comprises contacting the donor polynucleotide with a donor polynucleotide. - a portion of a polynucleotide, a copy of a donor polynucleotide, or In some instances, the method involves incorporating a portion of the copy of the nucleotide into the target DNA. The method does not involve contacting a cell with a donor polynucleotide, but involves nucleotide substitution within the target DNA. In some instances, the target DNA is modified such that the target DNA is deleted. Fungal cells, bacterial cells, eukaryotic cells, eukaryotic unicellular organisms, somatic cells, germ cells, stem cells, plant cells, Algae cells, animal cells, invertebrate cells, vertebrate cells, fish cells, frog cells, bird cells, Mammalian cells, porcine cells, bovine cells, goat cells, sheep cells, rodent cells, rat cells , mouse cells, non-human primate cells, and human cells. In some instances, the cells are in vitro. It exists in Bo.

[0025] A feature of the disclosure includes a method of producing genetically modified cells in a subject, the method comprising: (I) a cell (i) containing a nucleotide sequence complementary to a sequence in (a) the target DNA; (b) a second segment that interacts with the site-specific modifying polypeptide; a DNA-targeting RNA comprising the target, or a DNA polynucleotide encoding the same; and (ii) (a) an RNA-binding site that interacts with the DNA-targeting RNA; and (b) a target D The site-specific modification polymer contains an active site that exhibits nuclease activity, generating double-strand breaks within the NA. introducing a polypeptide or a polynucleotide encoding the same (wherein The site of cleavage is determined by the DNA-targeting RNA, and contacts are made by non-homologous end joining or homologous pairing. It occurs under conditions permissive for recombination repair, and the target DNA is modified by being broken and rejoined. (II) ) transplanting the genetically modified cells into the subject. In some examples, the method comprises transplanting the cells The method further comprises contacting the donor polynucleotide with a donor polynucleotide. - a portion of a polynucleotide, a copy of a donor polynucleotide, or In some instances, the method comprises: A method for identifying a nucleotide in a target DNA, the method comprising: contacting a cell with a donor polynucleotide; In some instances, the target DNA is modified to delete the sequence. Cells, bacterial cells, eukaryotic cells, eukaryotic unicellular organisms, somatic cells, germ cells, stem cells, plant cells, algae Cells, animal cells, invertebrate cells, vertebrate cells, fish cells, amphibian cells, bird cells, mammalian cells The group consisting of mammalian cells, ungulate cells, rodent cells, non-human primate cells, and human cells is selected from.

[0026] Features of the disclosure include a nucleotide sequence encoding an exogenous site-directed modifying polypeptide. and a method for modifying target DNA in a genetically modified cell, the method comprising: By introducing DNA-targeting RNA or a DNA polynucleotide encoding it into cells, (i) the DNA-targeting RNA comprises (a) a nucleotide sequence complementary to a sequence within the target DNA; (b) a first segment containing a polypeptide sequence; and (b) a segment that interacts with the site-specific modifying polypeptide. (ii) the site-directed modifying polypeptide comprises (a) a DNA (b) an RNA-binding site that interacts with the target RNA; and (b) an activity that exhibits nuclease activity. In some examples, the site-directed modifying polypeptide comprises a C site as shown in FIG. For amino acids 7 to 166 or 731 to 1003 of the as9 / Csn1 amino acid sequence or the amino acid sequences set forth as SEQ ID NOs: 1 to 256 and 795 to 1346 have at least about 75% amino acid sequence identity to the corresponding portion in any of In some instances, the cell is an archaeal cell, a bacterial cell, a eutrophic cell, or a bacterial cell. Nuclear cells, eukaryotic unicellular organisms, somatic cells, germ cells, stem cells, plant cells, algae cells, animal cells, Vertebrate cells, vertebrate cells, fish cells, amphibian cells, bird cells, mammalian cells, ungulate cells cells, rodent cells, non-human primate cells, and human cells. In some instances, the cells are in vivo. In some instances, expression of the site-directed modified polypeptide is inducible. In some instances, the site-specific modifying polypeptide is under the control of a promoter. Expression is under the control of a cell type-specific promoter.

[0027] Features of the present disclosure include DNA-targeting RNAs or DNA polynucleotides encoding same. and kits containing reagents for reconstitution and / or dilution. In this example, the kit includes a buffer for introducing DNA-targeting RNA into cells, a wash buffer, solution, control reagent, control expression vector or RNA polynucleotide, DNA to DNA target a reagent selected from the group consisting of a reagent for transcribing modified RNA, and combinations thereof Further includes medicines.

[0028] Features of the disclosure include site-directed modified polypeptides of the disclosure, or polypeptides encoding same. and kits containing reagents for reconstitution and / or dilution. In some instances, the kit includes a method for introducing a site-specific modifying polypeptide into a cell. buffer, wash buffer, control reagent, control expression vector or RNA polynucleotide, D Reagents for in vitro production of site-specifically modified polypeptides from NAs, and their The compound further comprises a reagent selected from the group consisting of a combination of:

[0029] Features of the disclosure include site-directed modified polypeptides of the disclosure, or polypeptides encoding same. and kits containing reagents for reconstitution and / or dilution. Features of the present disclosure include: (a) a nucleotide sequence that is complementary to a sequence within a target DNA; (b) a second segment that interacts with the site-specific modifying polypeptide; a DNA-targeting RNA comprising the target, or a DNA polynucleotide encoding the same; and (ii) (a) an RNA-binding site that interacts with the DNA-targeting RNA; and (b) a site-specific The active site exhibits specific enzymatic activity, and the site of enzymatic activity is determined by the DNA-targeting RNA. A site-directed modified polypeptide containing an active site, or a polypeptide encoding the same, Kits containing nucleotides are included.

[0030] Features of the present disclosure include: (i) (a) a nucleotide sequence complementary to a sequence in a target DNA; (b) a first segment comprising a second segment that interacts with the site-directed modifying polypeptide; a DNA-targeting RNA comprising the fragment, or a DNA polynucleotide encoding the same; and (ii) (a) an RNA-binding site that interacts with a DNA-targeting RNA; and (b) An active site that regulates transcription within a target DNA, the site at which transcription within the target DNA is regulated Site-specifically modified polypeptides containing active sites whose activity is determined by DNA-targeting RNA or a kit comprising a polynucleotide encoding the same.

[0031] Features of the present disclosure include (i) any of the recombinant expression vectors described above; and (ii) a recombinant Kits containing reagents for constitution and / or dilution are included. Features of the present disclosure include: ) any of the recombinant expression vectors described above; and (ii) (a) a DNA-targeting RNA and (b) an RNA-binding site that interacts with the active site, and that exhibits site-specific enzymatic activity. , site-specific, including the active site, where the site of enzymatic activity is determined by DNA-targeting RNA a recombinant expression vector comprising a nucleotide sequence encoding a modified polypeptide; Features of the present disclosure include (i) any of the recombinant expression vectors described above; and (ii) (a) an RNA-binding site that interacts with the DNA-targeting RNA; and (b) a target an active site that regulates transcription in target DNA, and the site at which transcription in target DNA is regulated A site-specifically modified polypeptide containing an active site determined by a DNA-targeting RNA. and a recombinant expression vector containing a nucleotide sequence encoding the

[0032] Features of the disclosure include two or more DNA-targeting RNAs, or the DNAs encoding them. A kit for targeting target DNA, including a polynucleotide, is included, The first segment of at least one of the DNA-targeting RNAs may comprise two or more DNA-targeting RNAs. A first segment of at least one targeting RNA and at least one nucleic acid sequence. Only Reotide is different. [Brief explanation of the drawings]

[0033] [Figure 1] 1A-B provide schematic diagrams of two exemplary subject DNA-targeting RNAs, each associated with a site-specific modifying polypeptide and target DNA. [Figure 2] 1 depicts targeted DNA editing via double-stranded DNA breaks introduced using Cas9 / Csn1 site-specific modifying polypeptides and DNA-targeting RNA. [Figure 3A]

[0023] Figure 1 shows the amino acid sequence of the Cas9 / Csn1 protein of Streptococcus pyogenes (SEQ ID NO: 8). Cas9 has domains that are homologous to both HNH and RuvC endonucleases. Motifs 1-4 are overlined. [Figure 3B]

[0023] Figure 1 shows the amino acid sequence of the Cas9 / Csn1 protein of Streptococcus pyogenes (SEQ ID NO: 8). Cas9 has domains that are homologous to both HNH and RuvC endonucleases. Domains 1 and 2 are overlined. [Figure 4] Figures 4A-B show the percent identity between Cas9 / Csn1 proteins from multiple species. (A) Sequence identity with respect to Streptococcus pyogenes. For example, as shown in Figure 3B, domain 1 is amino acids 7-166 of Cas9 / Csn1 from Streptococcus pyogenes, and domain 2 is amino acids 731-1003 of Cas9 / Csn1 from Streptococcus pyogenes. (B) Sequence homology with respect to Neisseria meningitidis. For example, domain 1 is amino acids 13-139 of Cas9 / Csn1 from Neisseria meningitidis (SEQ ID NO: 79), and domain 2 is amino acids 475-750 of Cas9 / Csn1 from Neisseria meningitidis (SEQ ID NO: 79). [Figure 5]32 shows a multiple sequence alignment of motifs 1-4 of Cas9 / Csn1 proteins from a variety of species selected from the phylogenetic table of FIG. (See Figure 32, Figure 3A and Table 1) (Streptococcus pyogenes (SEQ ID NO: 8), Legionella pneumophila (SEQ ID NO: 17), Gamma proteobacterium (SEQ ID NO: 107), Listeria innocua (SEQ ID NO: 3), Lactobacillus gasseri (SEQ ID NO: 152), Eubacterium rectale (SEQ ID NO: 99), Staphylococcus lugdunensis (SEQ ID NO: 185), Mycoplasma synoviae (SEQ ID NO: 22), Mycoplasma mobile mobile (SEQ ID NO: 16), Wolinella succinogenes (SEQ ID NO: 10), Flavobacterium columnare (SEQ ID NO: 235), Fibrobacter succinogenes (SEQ ID NO: 121), Bacteroides fragilis (SEQ ID NO: 21), Acidothermus cellulolyticus (SEQ ID NO: 42) and Bifidobacterium dentium (SEQ ID NO: 131). [Figure 6]Figures 6A-B provide an alignment of natural tracrRNA ("activator RNA") sequences from various species (L. innocua (SEQ ID NO: 268); S. pyogenes (SEQ ID NO: 267); S. mutans (SEQ ID NO: 269); S. thermophilus 1 (SEQ ID NO: 270); M. mobile (SEQ ID NO: 274); N. meningitides (SEQ ID NO: 272); P. multocida (SEQ ID NO: 273); S. thermophilus 2 (SEQ ID NO: 271); and S. pyogenes (SEQ ID NO: 267)). (A) Multiple sequence alignment of selected tracrRNA orthologs (AlignX, VectorNTI package, Invitrogen) associated with CRISPR / Cas loci with similar structures and highly similar Cas9 / Csn1 sequences. Black boxes indicate shared nucleotides. (B) Multiple sequence alignment of selected tracrRNA orthologs (AlignX, VectorNTI package, Invitrogen) associated with CRISPR / Cas loci with different structures and unrelated Cas9 / Csn1 sequences. Note the sequence similarity of the tracrRNA orthologs of N. meningitidis and P. multocida. Black boxes indicate shared nucleotides. For more exemplary activator RNA sequences, see SEQ ID NOs: 431-562. [Figure 7]Figures 7A-B provide an alignment of natural duplex-forming segments of crRNA ("targeting RNA") sequences from various species (L. innocua (SEQ ID NO: / / ); S. pyogenes (SEQ ID NO: / / ); S. mutans (SEQ ID NO: / / ); S. thermophilus 1 (SEQ ID NO: / / ); C. jejuni (SEQ ID NO: / / ); S. pyogenes (SEQ ID NO: / / ); F. novicida (SEQ ID NO: / / ); M. mobile (SEQ ID NO: / / ); N. meningitides (SEQ ID NO: / / ); P. multocida (SEQ ID NO: / / ); and S. thermophilus 2 (SEQ ID NO: / / ). (A) Similar (B) Multiple sequence alignment (AlignX, VectorNTI package, Invitrogen) of exemplary duplex-forming segments of targeting RNA sequences associated with the structures and loci of highly similar Cas9 / Csn1 sequences. (C) Multiple sequence alignment (AlignX, VectorNTI package, Invitrogen) of exemplary duplex-forming segments of targeting RNA sequences associated with different structures and loci of a wide variety of Cas9 sequences. Black boxes indicate shared nucleotides. For more exemplary duplex-forming segment targeting RNA sequences, see SEQ ID NOs: 563-679. [Figure 8]Schematic representation of hybridization between the natural duplex-forming segment of a crRNA ("targeter RNA") and the duplex-forming segment of the corresponding tracrRNA orthologue ("activator RNA"). The upper sequence is the targeter RNA; the lower sequence is the duplex-forming segment of the corresponding activator RNA. The CRISPR locus belongs to the Type II (Nmeni / CASS4) CRISPR / Cas system. Nomenclature follows that of the CRISPR Database (CRISPR DB). S. pyogenes (SEQ ID NO: / / and / / ); S. mutans (SEQ ID NO: / / and / / ); S. thermophilus 1 (SEQ ID NO: / / and / / ); S. thermophilus 2 (SEQ ID NO: / / and / / ); L. innocua (SEQ ID NO: / / and / / ); T. denticola (SEQ ID NO: / / and / / ); N. meningitides (SEQ ID NO: / / and / / ); S. gordonii (SEQ ID NO: / / and / / ); B. bifidum (SEQ ID NO: / / and / / ); L. salivarius (SEQ ID NO: / / and / / ); F. tularensis (SEQ ID NO: / / and / / ); and L. pneumophila (SEQ ID NO: / / and / / ). Note that some species contain two Type II CRISPR loci each. For more exemplary activator RNA sequences, see SEQ ID NOs: 431-562. For more exemplary duplex-forming segment targeter RNA sequences, see SEQ ID NOs: 563-679. [Figure 9]Exemplary tracrRNA (activator RNA) and crRNA (targeter RNA) sequences from two species are shown. Some interchangeability exists; for example, the S. pyogenes Cas9 / Csn1 protein functions with tracrRNA and crRNA from L. innocua. "|" represents standard Watson-Crick base pairing, while "·" represents a GU wobble base pair. "Variable 20 nt" or "20 nt" refers to a DNA target fragment complementary to the target DNA (this region can be up to approximately 100 nt in length). Also shown is the design of a single-molecule DNA-targeting RNA incorporating features of the targeter RNA and activator RNA (Cas9 / Csn1 protein sequences from a wide variety of species are shown in Figure 3, designated SEQ ID NOS: 1-256 and 795-1346). Streptococcus pyogenes: top to bottom: (SEQ ID NOS: / / , / / , / / ); Listeria innocua: top to bottom: (SEQ ID NOS: / / , / / , / / ). The sequences provided are non-limiting examples and are intended to illustrate how single-molecule DNA-targeting RNAs and bimolecular DNA-targeting RNAs can be designed based on naturally occurring sequences from a wide variety of species. Various examples of suitable sequences from a wide variety of species are shown as follows (Cas9 protein: SEQ ID NOS: 1-259; tracrRNA: SEQ ID NOS: 431-562, or their complements; crRNA: SEQ ID NOS: 563-679, or their complements; and exemplary single-molecule DNA-targeting RNA: SEQ ID NOS: 680-682). [Figure 10A] We show that Cas9 is a DNA endonuclease guided by two RNA molecules. [Figure 10B] Same as above. [Figure 10C] Same as above. [Figure 10D] Same as above. [Figure 10E] Figure 1 shows that Cas9 is a DNA endonuclease guided by two RNA molecules (SEQ ID NOs: 278-280 and / / , from top to bottom). [Figure 11] Figures 11A-B show that Cas9 uses two nuclease domains to cleave two strands of target DNA. [Figure 12A] Illustrates that Cas9-catalyzed cleavage of target DNA requires the activation domain of tracrRNA and is governed by the seed sequence of the crRNA. [Figure 12B] Same as above. [Figure 12C] Illustrated is a diagram showing that Cas9-catalyzed cleavage of target DNA requires the activation domain of tracrRNA and is governed by the seed sequence of the crRNA (SEQ ID NOs: 278-280 and / / , from top to bottom). [Figure 12D] Illustrates that Cas9-catalyzed cleavage of target DNA requires the activation domain of tracrRNA and is governed by the seed sequence of the crRNA (SEQ ID NOs: 281-290, from top to bottom). [Figure 12E] Figure 1 shows that Cas9-catalyzed cleavage of target DNA requires the activation domain of tracrRNA and is governed by the seed sequence of the crRNA (SEQ ID NOS: 291-292, 283, 293-298, from top to bottom). [Figure 13A] We show that PAM is required to license targeted DNA cleavage by the Cas9-tracrRNA:crRNA complex. [Figure 13B] Same as above. [Figure 13C] Same as above. [Figure 14A] 1 illustrates that Cas9 can be programmed using a single engineered RNA molecule that combines features of tracrRNA and crRNA. Chimera A (SEQ ID NO: 299); Chimera B (SEQ ID NO: 300). [Figure 14B] Same as above. [Figure 14C] Same as above. [Figure 15] 1 illustrates the type II RNA-mediated CRISPR / Cas immunity pathway. [Figure 16] Figures 16A-B show the purification of Cas9 nuclease. [Figure 17A] Figure 1 shows that dual tracrRNA:crRNA-guided Cas9 cleaves the protospacer plasmid and oligonucleotide DNA. [Figure 17B]Dual tracrRNA:crRNA-guided Cas9 cleaves protospacer plasmid and oligonucleotide DNA (SEQ ID NOS: 301-303 and / / , from top to bottom). [Figure 17C] Dual tracrRNA:crRNA-guided Cas9 cleaves protospacer plasmid and oligonucleotide DNA (SEQ ID NOS: 304-306 and / / , from top to bottom). [Figure 18A] We show that Cas9 is a Mg2+-dependent endonuclease with 3'-5' exonuclease activity. [Figure 18B] Same as above. [Figure 19A] Illustrates that dual tracrRNA:crRNA-directed Cas9 cleavage of target DNA is site-specific. [Figure 19B] Same as above. [Figure 19C] Illustrates that dual tracrRNA:crRNA-directed Cas9 cleavage of target DNA is site-specific (SEQ ID NOs: 307-309, / / , 337-339, and / / , from top to bottom). [Figure 20A] We demonstrate that dual tracrRNA:crRNA-directed Cas9 cleavage of target DNA is fast and efficient. [Figure 20B] Same as above. [Figure 21] Figures 21A-B show that the HNH and RuvC-like domains of Cas9 direct cleavage of complementary and non-complementary DNA strands, respectively. [Figure 22] We show that tracrRNA is required for target DNA recognition. [Figure 23A] We show that the minimal region of tracrRNA is capable of directing dual tracrRNA:crRNA-directed cleavage of target DNA. [Figure 23B] Same as above. [Figure 24A] We demonstrate that dual tracrRNA:crRNA-guided target DNA cleavage by Cas9 is species-specific. [Figure 24B] Same as above. [Figure 24C] Same as above. [Figure 24D] Same as above. [Figure 25A] Figure 1 shows that the seed sequence of the crRNA governs dual tracrRNA:crRNA-directed cleavage of target DNA by Cas9. Target DNA probe 1 (SEQ ID NO: 310); spacer 4 crRNA(1-42) (SEQ ID NO: 311); tracrRNA(15-89) (SEQ ID NO: / / ). [Figure 25B] Panel left (SEQ ID NO: 310) shows that the seed sequence of the crRNA governs dual tracrRNA:crRNA-directed cleavage of target DNA by Cas9. [Figure 25C] We show that the seed sequence of the crRNA governs dual tracrRNA:crRNA-directed cleavage of target DNA by Cas9. [Figure 26A] We show that the PAM sequence is essential for protospacer plasmid DNA cleavage by Cas9-tracrRNA:crRNA and Cas9-mediated plasmid DNA interference in bacterial cells. [Figure 26B] Figure 1 shows that the PAM sequence is essential for protospacer plasmid DNA cleavage by Cas9-tracrRNA:crRNA and Cas9-mediated plasmid DNA interference in bacterial cells (SEQ ID NOs: 312-314, from top to bottom). [Figure 26C] Figure 1 shows that the PAM sequence is essential for protospacer plasmid DNA cleavage by Cas9-tracrRNA:crRNA and Cas9-mediated plasmid DNA interference in bacterial cells (SEQ ID NOs: 315-320, from top to bottom). [Figure 27A] We show that Cas9 guided by a single chimeric RNA mimicking the dual tracrRNA:crRNA cleaves the protospacer DNA. [Figure 27B] Same as above. [Figure 27C] Figure 1 shows that Cas9 guided by a single chimeric RNA mimicking the dual tracrRNA:crRNA cleaves the protospacer DNA (SEQ ID NOs: 321-324, from top to bottom). [Figure 28A] A novel design of chimeric RNA targeting the green fluorescent protein (GFP) gene sequence is presented. [Figure 28B] 1 shows a novel design of a chimeric RNA targeting the green fluorescent protein (GFP) gene sequence (SEQ ID NOs: 325-326, from top to bottom). [Figure 28C] 1 shows novel designs of chimeric RNAs targeting green fluorescent protein (GFP) gene sequences: GFP1 target sequence (SEQ ID NO: 327); GFP2 target sequence (SEQ ID NO: 328); GFP3 target sequence (SEQ ID NO: 329); GFP4 target sequence (SEQ ID NO: 330); GFP5 target sequence (SEQ ID NO: 331); GFP1 chimeric RNA (SEQ ID NO: 332); GFP2 chimeric RNA (SEQ ID NO: 333); GFP3 chimeric RNA (SEQ ID NO: 334); GFP4 chimeric RNA (SEQ ID NO: 335); GFP5 chimeric RNA (SEQ ID NO: 336). [Figure 28D] A novel design of chimeric RNA targeting the green fluorescent protein (GFP) gene sequence is presented. [Figure 29A] We show that co-expression of Cas9 and guide RNA in human cells results in double-stranded DNA breaks at the target locus. [Figure 29B] Same as above. [Figure 29C] Figure 1 shows that co-expression of Cas9 and guide RNA in human cells results in double-stranded DNA breaks at the target locus (SEQ ID NOS: 425-428, from top to bottom). [Figure 29D] We show that co-expression of Cas9 and guide RNA in human cells results in double-stranded DNA breaks at the target locus. [Figure 29E] Same as above. [Figure 30] Figures 30A-B show that cell lysates contain active Cas9:sgRNA and support site-specific DNA cleavage. [Figure 31A] 3A-3C show that the 3' extension of the sgRNA construct enhances site-specific NHEJ-mediated mutagenesis (SEQ ID NOs: 428-430, from top to bottom). [Figure 31B]1 shows that the 3′ extension of the sgRNA construct enhances site-specific NHEJ-mediated mutagenesis. [Figure 32A] Phylogenetic tree of representative Cas9 sequences from various organisms. [Figure 32B] The Cas9 locus structure for the major groups in a phylogenetic tree of representative Cas9 sequences from various organisms is shown. [Figure 33A] Figure 1 shows the structures of type II CRISPR-Cas from selected bacterial species. [Figure 33B] Same as above. [Figure 33C] Same as above. [Figure 33D] Same as above. [Figure 33E] Same as above. [Figure 34A] Figure 1 shows the co-processing of tracrRNA and pre-crRNA of selected Type II CRISPR Cas systems (SEQ ID NOs: / / , / / , / / , / / , / / , / / , / / , / / , / / , from top to bottom). [Figure 34B] Figure 1 shows the co-processing of tracrRNA and pre-crRNA of selected Type II CRISPR Cas systems (SEQ ID NOs: / / , / / , / / , / / , from top to bottom). [Figure 35] A sequence alignment of tracrRNA orthologs showing the diversity of tracrRNA sequences is shown. [Figure 36A] Expression of bacterial tracrRNA orthologs and crRNAs revealed by deep RNA sequencing is shown. [Figure 36B] Same as above. [Figure 36C] Same as above. [Figure 36D] Same as above. [Figure 36E] Same as above. [Figure 36F] Same as above. [Figure 37A] List all tracrRNA orthologs and mature crRNAs recovered by sequencing in the studied bacterial species, including their coordinates (regions of interest) and corresponding cDNA sequences (5' to 3'). [Figure 37B]Same as above. [Figure 37C] Same as above. [Figure 37D] Same as above. [Figure 37E] Same as above. [Figure 37F] Same as above. [Figure 37G] Same as above. [Figure 37H] Same as above. [Figure 37I] Same as above. [Figure 37J] Same as above. [Figure 37K] Same as above. [Figure 37L] Same as above. [Figure 37M] Same as above. [Figure 37N] Same as above. [Figure 37O] Same as above. [Figure 38A] Figure 1 shows a table of bacterial species containing type II CRISPR-Cas loci characterized by the presence of the signature gene cas9. These sequences were used for phylogenetic analysis. [Figure 38B] Same as above. [Figure 39] Figures 39A-B show the design of the CRISPR interference (CRISPRi) system. [Figure 40A] We show that CRISPRi effectively silences transcription elongation and initiation. [Figure 40B] Same as above. [Figure 40C] Same as above. [Figure 40D] Same as above. [Figure 40E] Same as above. [Figure 41] Figures 41A-B show that CRISPRi functions by blocking transcription elongation. [Figure 42A] 1 shows the target specificity of the CRISPRi system. [Figure 42B] Same as above. [Figure 42C] Same as above. [Figure 43A] The characteristics of factors that affect the silencing effect are shown. [Figure 43B] Same as above. [Figure 43C] Same as above. [Figure 43D] Same as above. [Figure 43E] Same as above. [Figure 43F] Same as above. [Figure 44A] 1 shows functional profiling of complex regulatory networks using CRISPRi gene knockdown. [Figure 44B] Same as above. [Figure 44C] Same as above. [Figure 45A] Gene silencing using CRISPRi in mammalian cells. [Figure 45B] Same as above. [Figure 46] Figure 1 shows the mechanism of the type II CRISPR system in S. pyogenes. [Figure 47] Figures 47A-B show growth curves of E. coli cell cultures co-transformed with dCas9 and sgRNA. [Figure 48] We demonstrate that CRISPRi can silence expression of a reporter gene on a multicopy plasmid. [Figure 49A] RNA-seq data of cells with sgRNAs targeting different genes are shown. [Figure 49B] Same as above. [Figure 49C] Same as above. [Figure 50A] 1 shows the silencing effect of sgRNAs with adjacent double mismatches. [Figure 50B] Same as above. [Figure 50C] Same as above. [Figure 50D] Same as above. [Figure 50E] Same as above. [Figure 51] Figures 51A-C show the combinatorial silencing effect of using two sgRNAs to control a single gene. [Figure 52] Figure 1 shows that sgRNA suppression depends on the target locus and its relative distance from the transcription start. [Figure 53] Figures 53A-C show experimental results demonstrating that mutant Cas9 site-directed polypeptides (dCas9) are mechanisms for the subject methods when dCas9 reduces activity in the RuvC1 domain alone (e.g., D10A), the HNH domain alone (e.g., H840A), or both domains (e.g., D10A and H840A). [Figure 54A] Examples of suitable fusion partners (or fragments thereof) for the subject mutant Cas9 site-directed polypeptides are listed below, including but not limited to those listed. [Figure 54B] Same as above. [Figure 54C] Same as above. [Figure 55A] It is shown that chimeric site-directed polypeptides can be used to activate (increase) transcription in human cells. [Figure 55B] Same as above. [Figure 55C] Same as above. [Figure 55D] Same as above. [Figure 56] It is shown that chimeric site-directed polypeptides can be used to repress (reduce) transcription in human cells. [Figure 57A] We show that artificial sequences that share approximately 50% identity with natural tracrRNA and crRNA can function together with Cas9 to cleave target DNA, as long as the structure of the protein-binding domain of the DNA-targeting RNA is preserved. [Figure 57B] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0034] Definition (Part 1) The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to any It refers to the polymeric form of nucleotides of a certain length, such as ribonucleotides or deoxyribonucleosides. Thus, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrid, or primers phosphorus and pyrimidine bases or other natural, chemically or biochemically modified, It includes polymers containing non-natural or derivatized nucleotide bases. A "nucleotide" is generally a polynucleotide of about 5 to about 100 nucleotides. It refers to single-stranded or double-stranded DNA. However, for purposes of this disclosure, the length of the oligonucleotide There is no upper limit to the number of oligonucleotides. Oligonucleotides are also known as "oligomers" or "oligos." and can be isolated from genes or chemically synthesized by methods known in the art. The terms "polynucleotide" and "nucleic acid" are used in the described embodiment. Where applicable, single-stranded polynucleotides (e.g., sense or antisense) and double-stranded polynucleotides It should be understood to include single-stranded polynucleotides.

[0035] A "stem-loop structure" is a structure that is mainly formed by a region of single-stranded nucleotides (the loop portion). Forms a double strand (step portion) that is linked at one end Nucleotides with secondary structure containing nucleotide regions known or predicted to The terms "hairpin" and "foldback" structures also refer to stem-loop structures. Such structures are well known in the art and are used herein as These terms are used consistently with their known meanings in the art. As is known in the art, stem-loop structures do not require precise base pairing. Therefore, the stem may contain one or more base mismatches. The base pairing of may be exact, i.e., not contain any mismatches.

[0036] "Hybridizable" or "complementary" or "substantially complementary" refers to a nucleic acid ( RNA) is synthesized in vitro and / or in vivo at appropriate temperatures and solution ionic strengths. under conditions that allow the nucleic acid to bind non-covalently to another nucleic acid in a sequence-specific, antiparallel manner, i.e., Watson-Crick base pairing and / or G / U base pairing, "annealing" It contains a nucleotide sequence that allows it to "bind" or "hybridize" (i.e., specifically binds to a complementary nucleic acid). As shown, canonical Watson-Crick base pairing involves adenine (A) and thymidine (T) ), adenine (A) and uracil (U), and guanine (G) and cytosine ( C) pairing [DNA, RNA]. In addition, the pairing between two RNA molecules (e.g., d In sRNA hybridization, guanine (G) and uracil (U) form a base pair. For example, G / U base pairing is known in the art. In relation to the pairing of the codons and anticodons of tRNA in A, the degeneracy of the genetic code ( In light of the present disclosure, the subject DNA-targeted R The guanine (G) in the protein-binding segment of the NA molecule (dsRNA duplex) binds to the uracil The base pair G / U is considered complementary to the base G, and vice versa. The protein-binding segment (dsRNA duplex) of the subject DNA-targeting RNA molecule is If a given nucleotide position can be formed, that position is not considered non-complementary; Instead, they are considered to be complementary.

[0037] Hybridization and washing conditions are well known and are described in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second Edition, Co. Spring Harbor Laboratory Press, Cold Spring Harbor (1989), especially Chapter 11 and and Table 11.1; and Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual. ry Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor The temperature and ionic strength conditions are as follows: Determine the "stringency".

[0038] Hybridization requires that the two nucleic acids contain complementary sequences; Mismatches between bases are possible. Conditions favorable for hybridization between two nucleic acids are the length and degree of complementarity of those nucleic acids, which are variables well known in the art. The higher the degree of complementarity between two nucleotide sequences, the more likely it is that the The melting temperature (Tm) of hybrids consisting of nucleic acids is higher. 35 or less, 30 or less, 25 or less, 22 or less, 20 or less, or 18 or less nucleotides In hybridization between nucleic acids with complementary sequences, the position of a mismatch (Sambrook et al., supra, see 11.7-11.8). Typically, hybrid The length of the hybridizable nucleic acid is at least about 10 nucleotides. Examples of minimum lengths include at least about 15 nucleotides; at least about 20 nucleotides; at least about 22 nucleotides; at least about 25 nucleotides; and at least about 30 Furthermore, the temperature and the salt concentration of the wash solution affect the length and Those skilled in the art will recognize that this may be adjusted as needed depending on factors such as the degree of complementarity. .

[0039] Polynucleotide sequences are specifically hybridizable or hybridizable. It is 100% complementary to the polynucleotide sequence of its target nucleic acid so that it can be encoded. It is understood in the art that polynucleotides need not be so that the adjacent or neighboring segments are not included in the hybridization event ( loop or hairpin structure), The polynucleotide may be hybridized to a target region within a target nucleic acid sequence. , at least 70%, at least 80%, at least 90%, at least 95%, less For example, the two sequences of an antisense compound may contain 99% or 100% sequence complementarity. 18 of the nucleotides are complementary to the target region and therefore specifically hybridize. In this example, the remaining non-complementary sequences would be 90% complementary. The complementary nucleotides are clustered or interspersed with complementary nucleotides. Nucleic acids may be present and need not be contiguous to each other or to complementary nucleotides. The percent complementarity between particular stretches of nucleic acid sequences within a BL sequence is determined using the BL method known in the art. AST program (Basic Local Alignment Search Tool) and PowerBLAS T program (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madde n, Genome Res., 1997, 7, 649-656) or by using the Gap program (Wiscon sin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park , Madison Wis.) and Smith and Waterman (Adv Appl. Math., 1981, 2, 482-489) algorithm, and It can be determined by the method.

[0040] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein. Used, coded and non-coded amino acids, chemical or biochemical Chemically modified or derivatized amino acids, as well as polypeptides having modified peptide backbones. The term "amino acid" refers to a polymeric form of amino acids of any length, which may include amino acids.

[0041] "Binding" as used herein refers to binding to an RNA-binding domain of a polypeptide (e.g., (related to the non-covalent interactions between macromolecules (e.g., between proteins and nucleic acids) In the context of non-covalent interactions, macromolecules can be "associated" or "interacted" with one another. They are said to be "bonded" or "bonded" (e.g., molecule X is said to interact with molecule Y). (In this case, molecule X binds to molecule Y non-covalently.) The components of need not be sequence-specific (e.g., contacts with phosphate residues in the DNA backbone) ), some parts of the binding interaction may be sequence-specific. , generally, 10 -6 Under M, 10 -7 Under M, 10 -8 Under M, 10 -9 Less than M, 1 0 -10 Under M, 10 -11 Under M, 10 -12 Under M, 10 -13 Under M, 10 -14 Less than M or 10 -15 Affinity is a measure of binding affinity. It refers to the strength of binding, with increased binding affinity correlated with a lower Kd.

[0042] A "binding domain" is a protein domain that is capable of non-covalently binding to another molecule. The binding domain refers to, for example, a DNA molecule (DNA binding protein), R RNA molecules (RNA-binding proteins) and / or protein molecules (protein-binding proteins) In the case of a protein domain-binding protein, it can bind to itself (forming homodimers, homotrimers, etc.), and / or one or more heterologous The protein can bind to one or more molecules comprising the protein.

[0043] The term "conservative amino acid substitution" refers to a substitution in a protein of amino acid residues having similar side chains. For example, the group of amino acids with aliphatic side chains is glycine, alanine, and bacillin. Consists of phosphorus, leucine, and isoleucine; amino with an aliphatic hydroxyl side chain The amino acid group consists of serine and threonine; the amino acid group with amide-containing side chains is asparagus amino acids with aromatic side chains are phenylalanine, thiamin, and glutamine; The amino acids with basic side chains are lysine, arginine, and tryptophan. amino acids with acidic side chains are glutamate and and aspartic acid; and the group of amino acids with sulfur-containing side chains is cysteine ​​and Exemplary conservative amino acid substitutions are valine-leucine-isoleucine phenylalanine-tyrosine, lysine-arginine, alanine-valine, and aspartate It is paragine-glutamine.

[0044] A polynucleotide or polypeptide can be linked to another polynucleotide or polypeptide. have a certain percent "sequence identity" to each other, which means that when aligned, In some cases, the percentage of bases or amino acids is identical, and the two sequences When comparing sequences, the sequence identity is the same relative position. To determine sequence identity, various methods and ncb i.nlm.nili.gov / BLAST, ebi.ac.uk / Tools / msa / tcoffee / , ebi.ac.uk / Tools / msa / muscle / , ma On sites including fft.cbrc.jp / alignment / software / Computer programs available on the World Wide Web (e.g., BLAST, Align the sequences using a tool such as T-COFFEE, MUSCLE, or MAFFT. See, e.g., Altschul et al. (1990), J. Mol. Biol. 215:403-10. stomach.

[0045] A DNA sequence that "encodes" a particular RNA is a DNA nucleic acid sequence that is transcribed into RNA. A DNA polynucleotide encodes an RNA (mRNA) that is translated into a protein. Alternatively, the DNA polynucleotide may be an RNA that is not translated into a protein. A (e.g., tRNA, rRNA, or DNA-targeting RNA; "non-coding" RNA or In some cases, they encode proteins (also called "ncRNAs").

[0046] "Protein coding sequence" or a sequence that encodes a specific protein or polypeptide The sequences described herein are capable of expressing themselves in vitro or in vivo when placed under the control of appropriate regulatory sequences. It is then transcribed into mRNA (in the case of DNA) and translated into polypeptides (in the case of mRNA). The boundaries of the coding sequence are determined by a start codon at the 5' end (N-terminus) and a 3' end. The coding sequence is determined by a translation termination nonsense codon at the end (C-terminus). cDNA derived from prokaryotic or eukaryotic mRNA, but not from prokaryotic or eukaryotic mRNA can include genomic DNA sequences derived from eukaryotic DNA, as well as synthetic nucleic acids. A termination sequence is usually located 3' to the coding sequence.

[0047] As used herein, a "promoter sequence" refers to a sequence that binds RNA polymerase. and can initiate transcription of downstream (3' direction) coding or non-coding sequences. For the purposes of defining this invention, a promoter sequence is a DNA regulatory region that It is bound at its 3' end by a transcription origin and extends upstream (5' direction) to form a background The minimum number of bases or elements required to initiate transcription at a detectable level above the threshold. The promoter sequence contains the transcription initiation site and the site for RNA polymerase binding. Eukaryotic promoters often contain protein binding domains involved in transcription. It contains a "TATA" box and a "CAT" box. A variety of promoters, including the .alpha. promoter, can be used to drive the various vectors of the present invention. It is possible.

[0048] The promoter is a constitutively active promoter (i.e., constitutively active / "ON" state) a promoter that is inactive / "ON" Or the inactive / "OFF" state may be triggered by an external stimulus, e.g., a specific temperature, compound, or temperature. It can be a promoter that is spatially restricted (a promoter controlled by the presence of a protein) (i.e., transcriptional regulatory region, enhancer) promoter promoters, etc.) (e.g., tissue-specific promoters, cell type-specific promoters, etc.) Often, a temporarily restricted promoter (i.e. That is, the promoter may be involved in the expression of a gene during a particular stage of embryonic development or at a particular stage of a biological process. For example, during the hair follicle cycle in mice, they are in an "ON" or "OFF" state. It's okay to have it.

[0049] Suitable promoters may be of viral origin and are therefore referred to as viral promoters. Alternatively, a suitable promoter may be used in any organism, including prokaryotes or eukaryotes. Any RNA polymerase can be produced by using an appropriate promoter. Expression can also be driven by pol I, pol II, pol III Examples of promoters include, but are not limited to, the SV40 early promoter. , mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter promoter (AdMLP); herpes simplex virus (HSV) promoter, cytomegalovirus a CMV promoter, e.g., a CMV immediate early promoter region (CMV IEL) , Rous sarcoma virus (RSV) promoter, human U6 small nuclear promoter (U6 ) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), highly sensitive U6 probe promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep 1;31(17)), human H1 Examples include promoter (H1).

[0050] Examples of inducible promoters include, but are not limited to, the T7 RNA polymerase promoter. promoter, T3 RNA polymerase promoter, isopropyl-β-D-thiogalactopyranoside Ionoside (IPTG)-regulated promoter, lactose-inducible promoter, heat shock promoter, tetracycline-regulated promoter, steroid-regulated promoter, Examples include metal-regulated promoters and estrogen receptor-regulated promoters. Inducible promoters include doxycycline; RNA polymerases, e.g., T7RNA polymerase; estrogen receptor; estrogen receptor fusion; etc. It can be regulated by molecules that are not determined.

[0051] In some embodiments, the promoter is a promoter that is unique in a multicellular organism. Spatially restricted promoters that are active (i.e., "ON") in certain cells (i.e., cell type-specific promoters, tissue-specific promoters, etc.) A target promoter may also be called an enhancer, a transcriptional regulatory region, a control sequence, etc. Convenient spatially restricted promoters can also be used, and suitable promoters (e.g., Brain-specific promoters, promoters that drive expression in some neurons, and promoters that drive expression in germ cells a promoter that drives expression in the cell lineage, a promoter that drives expression in the lung, A promoter that drives expression in muscle and a promoter that drives expression in pancreatic islet cells The choice of the target organism (e.g., the serotonin receptor) varies depending on the organism. For example, plants, flies, insects, mammals, and mice Various spatially restricted promoters are known in the art. Using the detector, the subject can be analyzed in a variety of different tissues and cell types, depending on the organism. The expression of a nucleic acid encoding a site-specifically modified polypeptide can be controlled. Some spatially restricted promoters are characterized by the fact that the promoter is expressed only during a specific stage of embryonic development or During specific stages of a biological process (e.g., the hair follicle cycle in mice), the "ON" state It is time-limited, like being in a powered-on or "OFF" state.

[0052] For illustrative purposes, examples of spatially restricted promoters include, but are not limited to, Ion-specific promoter, adipocyte-specific promoter, cardiomyocyte-specific promoter, Examples include smooth muscle-specific promoters and photoreceptor-specific promoters. Heterogeneous spatially restricted promoters include, but are not limited to, neuron-specific enolase (NSE) promoter (see, for example, EMBL HSENO2, X51956); aromatic amino acid decarboxylase (AADC) promoter; neurofilament promoter (e.g. See, for example, GenBank HUMNFL, L04147); synapsin promoter (See, e.g., GenBank HUMSYNIB, M55301); thy-1 pro motors (e.g., Chen et al. (1987) Cell 51:7-19; and Llewellyn, et al. (2010) Nat. Med. 16(10):1161-1166); serotonin receptor promoters (e.g., Ge see nBank S62283); tyrosine hydroxylase promoter (TH) (e.g. , Oh et al. (2009) Gene Ther 16:437; Sasaoka et al. (1992) Mol. Brain Res. 16:27 4; Boundy et al. (1998) J. Neurosci. 18:9989; and Kaneda et al. (1991) Neuron 6 :583-594); the GnRH promoter (see, e.g., Radovick et al. (1991) Proc. Nat. Acad. Sci. USA 88:3402-3406); the L7 promoter (see, e.g., Oberdick et al. (1990) Science 248:223-226); DNMT promoters (see, e.g., Bartge et al. (1988) Proc. Natl. Acad. Sci. USA 85:3648-3652); enkephalin promoter myelin basic protein (see, e.g., Comb et al. (1988) EMBO J. 17:3793-3805); Ca2+-calmodulin-dependent protein kinase (MBP) promoter II-α (CamKIIα) promoter (e.g., Mayford et al. (1996) Proc. Natl Acad. Sci. USA 93:13250; and Casanova et al. (2001) Genesis 31:37); C MV enhancer / platelet-derived growth factor-β promoter (e.g., Liu et al. (2004) See Gene Therapy 11:52-60); etc.

[0053] Adipocyte-specific spatially restricted promoters include, but are not limited to, the aP2 gene promoter. promoter / enhancer, e.g., the region from -5.4 kb to +21 bp of the human aP2 gene (e.g., Tozzo et al. (1997) Endocrinol. 138:1604; Ross et al. (1990) Proc. N atl. Acad. Sci. USA 87:9590; and Pavjani et al. (2005) Nat. Med. 11:797) glucose transporter-4 (GLUT4) promoter (e.g., Knight et al. (2003) Proc. Natl. Acad. Sci. USA 100:14725); fatty acid translocase ( FAT / CD36) promoter (e.g., Kuriki et al. (2002) Biol. Pharm. Bull. 25:1476; and Sato et al. (2002) J. Biol. Chem. 277:15703); stearoyl CoA desaturase-1 (SCD1) promoter (Tabor et al. (1999) J. Biol. Chem. 274:20603); leptin promoter (e.g., Mason et al. (1998) Endocrinol. 139: 1013; and Chen et al. (1999) Biochem. Biophys. Res. Comm. 262:187); The eponectin promoter (see, e.g., Kita et al. (2005) Biochem. Biophys. Res. Comm 331:484; and Chakrabarti (2010) Endocrinol. 151:2408; Adipsymp promoter (see, e.g., Platt et al. (1989) Proc. Natl. Acad. Sci. USA 86:7490) ); resistin promoter (e.g., Seo et al. (2003) Molec. Endocrinol. 17:1522 See ); etc.

[0054] Cardiomyocyte-specific spatially restricted promoters include, but are not limited to, promoters for the following genes: These regulatory sequences include myosin light chain-2, α-myosin heavy chain, AE3, and cardiac myosin. Examples include loponin C and cardiac actin. Franz et al. (1997) Cardiovasc. Res. 35: 560-566; Robbins et al. (1995) Ann. NY Acad. Sci. 752:492-505; Linn et al. (19 95) Circ. Res. 76:584-591; Parmacek et al. (1994) Mol. Cell. Biol. 14:1870-1885; Hunter et al. (1993) Hypertension 22:608-617; and Sartorelli et al. (1992) Pro c. Natl. Acad. Sci. USA 89:4047-4051.

[0055] Smooth muscle specific spatially restricted promoters include, but are not limited to, SM22α promoter. (e.g., Akyurek et al. (2000) Mol. Med. 6:983; and U.S. Pat. No. 7,166,416. 9,874); smoothelin promoters (e.g., International Publication No. 2001 / 018048); α-smooth muscle actin promoter; etc. For example, a 0.4 kb region of the SM22α promoter contains two CArG elements. The presence of IL-14 elements has been shown to mediate vascular smooth muscle cell-specific expression ( For example, Kim, et al. (1997) Mol. Cell. Biol. 17, 2266-2278; Li, et al., (1996) J. Cell Biol. 132, 849-859; and Moessler, et al. (1996) Development 122, 2415-242 See 5).

[0056] Photoreceptor-specific spatially restricted promoters include, but are not limited to, rhodopsin promoters. Motor; rhodopsin kinase promoter (Young et al. (2003) Ophthalmol. Vis. S ci. 44:4076); β-phosphodiesterase gene promoter (Nicoud et al. (2007) J. Gene Med. 9:1015); retinitis pigmentosa gene promoter (Nicoud et al. (2007), supra) Interphotoreceptor retinoid-binding protein (IRBP) gene enhancer (Nicoud et al. t al. (2007), supra); IRBP gene promoter (Yokoyama et al. (1992) Exp Eye Res. 55:225); etc.

[0057] The terms "DNA regulatory sequence," "regulatory region," and "regulatory region" are used interchangeably herein. An "element" may be a non-coding sequence (e.g., a DNA-targeting RNA) or a coding sequence (e.g., For example, transcription of a site-specific modifying polypeptide, or a Cas9 / Csn1 polypeptide effecting and / or regulating the translation of the encoded polypeptide Regulating, promoter, enhancer, polyadenylation signal, terminator, It refers to transcriptional and translational regulatory sequences, such as protein degradation signals.

[0058] The term "naturally occurring" as used herein as applied to a nucleic acid, polypeptide, cell, or organism. "Native" or "native" refers to a nucleic acid, polypeptide, cell, or organism that is naturally occurring. For example, it can be isolated from natural sources and intentionally modified by humans in the laboratory. Polypeptides or polynucleotides present in living organisms (including viruses) that have not been identified The sequence is naturally occurring.

[0059] The term "chimeric" as used herein as applied to nucleic acids or polypeptides refers to a refers to two components defined by their structure derived from different sources. For example, "chimera" refers to a chimeric used in conjunction with chimeric polypeptides (e.g., chimeric Cas9 / Csn1 proteins) In some cases, the chimeric polypeptide contains amino acid sequences derived from different polypeptides. Chimeric polypeptides may be any of a modified or naturally occurring polypeptide sequence. (e.g., a modified or unmodified Cas9 / Csn1 protein) and a second amino acid sequence other than the Cas9 / Csn1 protein). In other words, "chimera" in reference to a polynucleotide encoding a chimeric polypeptide is a polynucleotide that encodes a different It includes nucleotide sequences derived from the coding region (e.g., modified or unmodified Cas9 / C a first nucleotide sequence encoding the sn1 protein; and a Cas9 / Csn1 protein a second nucleotide sequence encoding a polypeptide other than a protein).

[0060] The term "chimeric polypeptide" refers to a chimeric polypeptide that is a mixture of two uncombined amino acid sequences. by combining (i.e., "fusion") segments, usually through human intervention. A polypeptide comprising a chimeric amino acid sequence is called a chimeric polypeptide. Some chimeric polypeptides can be referred to as "fusion variants."

[0061] "Heterologous," as used herein, refers to a molecule that is not present in the native nucleic acid or protein, respectively. For example, chimeric Ca The s9 / Csn1 protein is a naturally occurring bacterial Cas9 / Csn1 polypeptide ( or variants thereof) can be coupled to a heterologous polypeptide sequence (i.e., Cas Polypeptide sequences derived from proteins other than 9 / Csn1 or polypeptides from other organisms The heterologous polypeptide sequence may be fused to a chimeric Cas9 / may exhibit an activity (e.g., an enzymatic activity) also exhibited by the Csn1 protein (e.g., Methyltransferase activity, acetyltransferase activity, kinase activity, ubiquitin chitinization activity, etc.) The heterologous nucleic acid sequence may be a naturally occurring nucleic acid sequence (or a variant thereof) (e.g., For example, by genetic engineering) to form a chimeric polypeptide encoding a chimeric polypeptide. As another example, a nucleotide sequence can be generated to generate a fusion mutant Cas9 site-specific polypeptide. In this case, the mutant Cas9 site-directed polypeptide is a heterologous polypeptide (i.e., C The fusion mutant Cas9 site-specific The heterologous nucleic acid sequence exhibits an activity also exhibited by the mutant Cas9 site-specific polypeptide. The fusion mutant Cas is linked (e.g., by genetic engineering) to a target polypeptide. A nucleotide sequence encoding a site-specific polypeptide can be generated.

[0062] "Recombinant," as used herein, refers to a form of a particular nucleic acid (DNA or RNA) and having structural coding or non-coding sequences that are distinguishable from endogenous nucleic acids present in natural systems. cloning, restriction, polymerase chain reaction (PCR) and The products of various recombinations of the PCR and / or ligation steps It means that the DNA sequence encoding the polypeptide is obtained from a cDNA fragment or Constructed from a series of synthetic oligonucleotides, they can be used in intracellular or cell-free transcription systems and It is possible to generate synthetic nucleic acids that can be expressed from recombinant transcription units contained within a translation system. Genomic DNA containing relevant sequences can also be used to form recombinant genes or transcription units. The sequence of non-translated DNA may be located 5' or 5' of the open reading frame. 3' to the coding region, where such sequences do not interfere with manipulation or expression of the coding region and, in fact, The DNases can act to regulate the production of desired products by a variety of mechanisms (see "DNases" below). A regulatory sequence). Alternatively, non-translated RNA (e.g., DNA-targeting RNA) A DNA sequence encoding a recombinant vector may also be considered recombinant. Nucleic acids are not naturally occurring, e.g., they are not two combinations of sequences, usually by human intervention. nucleic acids, created by the artificial combination of otherwise separate segments This artificial combination is often achieved by chemical synthesis or by the synthesis of a single nucleic acid. This can be achieved by artificial manipulation of isolated segments, for example, by genetic engineering techniques. Artificial recombination usually involves alternating codons with identical amino acids, conservative amino acids, or non-conservative amino acids. Alternatively, a codon encoding a desired amino acid may be substituted for a codon encoding a desired amino acid. This is done to join nucleic acid segments to create a desired functional combination. An artificial combination is often achieved by chemical synthesis means, or by synthesis of isolated segments of nucleic acids. This is achieved by the artificial manipulation of the gene, for example by genetic engineering techniques. If the code encodes a polypeptide, the sequence of the encoded polypeptide is the same as that of a naturally occurring ( "wild-type") or may be a variant (e.g., mutant) of a naturally occurring sequence. Thus, the term "recombinant" polypeptide does not necessarily mean that the sequence is not naturally occurring. In fact, a "recombinant" polypeptide does not refer to a polypeptide that is not recombinant D. Although the polypeptide sequence is encoded by a naturally occurring ("wild-type") ) or may be non-naturally occurring (e.g., variants, mutants, etc.) Thus, a "recombinant" polypeptide is the product of human intervention, but does not contain naturally occurring amino acids. It may be an array.

[0063] A "vector" or "expression vector" is a vector that contains another DNA segment, i.e., an "insertion vector." The binding of the "segment" to the target protein causes replication of the bound segment within the cell. The replicon is a plasmid, phage, virus, or cosmid that can

[0064] An "expression cassette" comprises a DNA coding sequence operably linked to a promoter. "Operably linked" means that the components so described can function in their intended manner. For example, a promoter is placed in a coding sequence. A promoter is operably linked to a coding sequence if it affects the transcription or expression of the sequence. It is tied.

[0065] The terms "recombinant expression vector" or "DNA construct" are used interchangeably herein. It is commonly used to refer to a DNA molecule containing a vector and at least one insert. Expression vectors typically contain a vector for expression and / or propagation of the insert(s). nucleotide sequences, or for the construction of other recombinant nucleotide sequences. The insert(s) may or may not be operably linked to a promoter sequence. It may or may not be operably linked to DNA regulatory sequences.

[0066] The cells may be transformed if exogenous DNA (e.g., a recombinant expression vector) has been introduced into the cells. If so, it has been "genetically modified" or "transformed" or "transfected" with such DNA. The presence of foreign DNA results in permanent or transient genetic changes. A may or may not be integrated into the genome of the cell (covalently linked). For example, in prokaryotes, yeast, and mammalian cells, transformation The DNA may be maintained on an episomal element such as a plasmid. Stably transformed cells have the gene(s) inherited by daughter cells through chromosome replication. Transforming: A cell in which the transforming DNA has become integrated into a chromosome. This stability allows the eukaryotic cell to It is explained by the ability to establish cell lines or clones containing a population of daughter cells containing the transformed DNA. A "clone" is a set of cells derived by mitosis from a single cell or common ancestor. A "cell line" is a population of cells capable of stable growth in vitro over many generations. It is a clone of a viable primary cell.

[0067] Suitable methods of genetic modification (also called "transformation") include, for example, viral infection. Infection or bacteriophage infection, transfection, conjugation n), protoplast fusion, lipofection, electroporation, calcium phosphate precipitation , polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gas Nucleic acid delivery using nanoparticles, such as the phospholipid technique, calcium phosphate precipitation, direct microinjection, and nanoparticle-mediated nucleic acid delivery (e.g., Panyam et al. et., al Adv Drug Deliv Rev. 2012 Sep 13. pii: S0169-409X(12)00283-9. doi: 10.10 16 / j.addr.2012.09.023)

[0068] The choice of genetic modification method generally depends on the type of cell to be transformed and the nature of the transformation. The type of reaction depends on the environment (e.g., in vitro, ex vivo, or in vivo). For a comprehensive discussion of the method, see Ausubel, et al., Short Protocols in Molecular Biology, 3rd edition. d., Wiley & Sons, 1995.

[0069] As used herein, "target DNA" refers to a DNA that contains a "target site" or "target sequence." A polynucleotide. The terms "target site" or "target sequence" or "target protoss" are used interchangeably. "Protospacer DNA" is used interchangeably herein and refers to a binding site of a The DNA-targeting segment of a subject DNA-targeting RNA is bound when conditions sufficient for the This refers to a nucleic acid sequence present in the target DNA that binds to the target DNA (see Figure 1 and Figure 39). Target site (or target sequence) within target DNA: 5'-GAGCATATC-3' (SEQ ID NO: / ) is targeted by the RNA sequence 5'-GAUAUGCUC-3' (SEQ ID NO: / / ) or binds to, hybridizes to, or is complementary to Suitable DNA / RNA binding conditions include physiological conditions normally present within cells. Other suitable DNA / RNA binding conditions (e.g., conditions in a cell-free system) are known in the art. Complementary to DNA-targeting RNAs, as known in the art (e.g., Sambrook, supra). The target DNA strand that hybridizes with it is called the "complementary strand" and the opposite The target DNA strand that is complementary (and therefore not complementary to the DNA-targeting RNA) is called the "non-complementary strand (n "non-complementary strand" or "non-complementary strand" This is called the "supplementary strand" (see Figure 12).

[0070] "site-specific modifying polypeptide" or "RNA-binding site-specific polypeptide" or "RNA-binding site-specific modified polypeptide" or "site-specific polypeptide" means a polypeptide that is It refers to a polypeptide that binds to NA and is targeted to a specific DNA sequence. The site-specifically modified polypeptide is bound to a specific D The RNA molecule is targeted to a target sequence within the target DNA. Therefore, a sequence that targets the bound polypeptide to a specific location within the target DNA (target sequence) is Contains columns.

[0071] "Cleavage" refers to the breaking of the covalent backbone of a DNA molecule. Cleavage refers to the enzymatic or chemical hydrolysis of a phosphodiester bond. Initiation can be achieved by a variety of methods, including but not limited to, single-strand breaks and double-strand breaks. Both strand breaks are possible, and double-strand breaks occur as a result of two separate single-strand break events DNA cleavage can result in the generation of blunt or cohesive ends. In certain embodiments, a complex comprising a DNA-targeting RNA and a site-specific modifying polypeptide is The ligation is used to cleave the target double-stranded DNA.

[0072] "Nuclease" and "endonuclease" are used interchangeably herein and are intended to mean a It refers to an enzyme that has catalytic activity for NA cleavage.

[0073] The "cleavage domain" or "activity domain" or "nuclease domain" of a nuclease "A" refers to a polypeptide sequence or polypeptide within a nuclease that has catalytic activity for DNA cleavage. It means a peptide domain. When the cleavage domain is contained within a single polypeptide chain, Alternatively, the cleavage activity may be due to the binding of two (or more) polypeptides. A single nuclease domain may be generated by two or more nucleotides within a given polypeptide. It may consist of one or more isolated amino acid chains.

[0074] A method for binding a site-specific modifying polypeptide and targeting the polypeptide to a specific location within the target DNA. The RNA molecule that targets the target is called a "DNA-targeting RNA" or "DNA-targeting RNA polynucleotide." The DNA of the subject is called a "guide" (also called a "guide RNA" or "gRNA"). The targeting RNA consists of two segments: a DNA-targeting segment and a protein-binding segment. A "segment" refers to a segment / section / region of a molecule (e.g. A segment refers to a continuous chain of nucleotides in an RNA. It can also refer to a region / section of a complex that may contain regions of more than one molecule. For example, in some instances, the protein-binding segment of the DNA-targeting RNA ( (see below) is an RNA molecule, so the protein-binding segment is located in that RNA. In other cases, the protein-binding segment of a DNA-targeting RNA A hybridization (see below) involves two separate molecules that hybridize along complementary regions. Examples include, but are not limited to, DNA-targeting RNA proteins comprising two separate molecules. The binding segment is (i) base pairs 40 to 75 of a 100 base pair long first RNA molecule; and (ii) a "segment" that may comprise 10 to 25 base pairs of a second RNA molecule that is 50 base pairs long. The definition of is limited to a specific number of total base pairs unless otherwise defined in a particular context. It is not limited to any particular number of base pairs from any particular, given RNA molecule, but rather to multiple base pairs. It is not limited to a specific number of separate molecules within a complex, but includes regions of RNA molecules of any length. may or may not contain regions of complementarity to other molecules. .

[0075] A DNA targeting segment (or "DNA targeting sequence") is a sequence that targets a specific sequence within the target DNA. It contains a nucleotide sequence complementary to the target DNA (the complementary strand). The cleavage region (or "protein binding sequence") interacts with the site-specifically modified polypeptide. The site-specific modifying polypeptide is a Cas9 or a Cas9-related polypeptide (see below). (described in more detail below), site-specific cleavage of the target DNA can be achieved by (i) a DNA-targeting RNA and (ii) base pairing complementarity between the target DNA and the short motif within the target DNA. short motif (called protospacer adjacent motif (PAM)) The ion beam occurs at a position determined by both the

[0076] The protein-binding segments of the subject DNA-targeting RNAs hybridize to each other to form double stranded RNA duplex (ds It contains two complementary strands of nucleotides that form an RNA duplex.

[0077] In some embodiments, the subject nucleic acids (e.g., DNA-targeting RNA, DNA-targeting a nucleic acid comprising a nucleotide sequence encoding a targeting RNA; a nucleic acid comprising a nucleotide sequence encoding a site-specific polypeptide; nucleic acids; etc.) may be modified to have additional desired characteristics (e.g., modified or controlled stability; intracellular targeting); labeling; tracking (e.g., fluorescent labeling); binding sites for proteins or protein complexes; etc.) Examples include, but are not limited to, modifications or sequences that provide a 5' cap (e.g., For example, a 7-methylguanylic acid cap (m7G); a 3' polyadenylated tail (i.e. , 3' poly(A) tail); riboswitch sequence (e.g., for regulating stability and / or transcription) allowing for modulation of accessibility by proteins and / or protein complexes); stability control sequences; sequences that form dsRNA duplexes (i.e., hairpins); RNA Modifications or sequences that target a subcellular location (e.g., nucleus, mitochondria, chloroplasts, etc.); traces (e.g., direct conjugation to fluorescent molecules, moieties that facilitate fluorescent detection, sequences that allow for fluorescent detection) modifications or sequences that result in proteins (e.g., transcriptional activators, transcription factors, etc.); Repressor, DNA methyltransferase, DNA demethylase, histone acetyl proteins that act on DNA, including transferases, histone deacetylases, etc. and combinations thereof.

[0078] In some embodiments, the DNA-targeting RNA has any of the above characteristics. For example, the appropriate nucleotide sequence may be included at either the 5' or 3' end. Three segments include a 5' cap (e.g., a 7-methylguanylic acid cap (m7G)); a 3' polyadenylation tail (i.e., a 3' poly(A) tail); a riboswitch sequence (e.g., , modulation of stability, and / or accessibility by proteins and protein complexes stability control sequences; sequences that form dsRNA duplexes (i.e., hairpin); targeting RNA to subcellular locations (e.g., nucleus, mitochondria, chloroplasts, etc.) tracking (e.g., direct conjugation to a fluorescent molecule, a moiety that facilitates fluorescent detection, a sequence that modifications or sequences that result in proteins (e.g., transcriptional activity); transcription factor, transcription repressor, DNA methyltransferase, DNA demethylase, His Acts on DNA containing histone acetyltransferase, histone deacetylase, etc. Modifications or sequences that provide binding sites for the target protein; and combinations thereof. It can be seen.

[0079] The subject DNA-targeting RNAs and the subject site-specific modifying polypeptides (i.e., site specific polypeptides) form a complex (i.e., by non-covalent interactions) DNA-targeting RNA binds to a target DNA with a nucleotide sequence complementary to the target DNA. The inclusion of the site-specifically modified polypeptide of the complex confers target specificity to the complex. In other words, the site-specifically modified polypeptide itself binds to the protein-binding segment of the DNA-targeting RNA, thereby sequences (e.g., target sequences within chromosomal nucleic acids; extrachromosomal nucleic acids (e.g., episomal nucleic acids, mini- target sequences within nucleic acids (e.g., mitochondrial nucleic acids); target sequences within chloroplast nucleic acids ; a target sequence within a plasmid; etc.

[0080] In some embodiments, a subject DNA-targeting RNA comprises two separate RNA molecules (RNA polynucleotides: "activator RNA" and "targeter RNA", see below) and is referred to as a "double-molecule DNA-targeting RNA" or "two-molecule DNA-targeting RNA." In other embodiments, the subject DNA-targeting RNA is a single RNA molecule (a single RNA polynucleotide). leotide), and are referred to as "single molecule DNA-targeting RNA," "single guide RNA," or "single molecule DNA-targeting RNA." The term "DNA-targeting RNA" or "gRNA" refers to a double molecule DNA Both DNA-targeting RNA and single-molecule DNA-targeting RNA (i.e., sgRNA) Refers to something comprehensively.

[0081] Exemplary bimolecular DNA-targeting RNAs are crRNA-like ("CRISPR RNA" or is a "targeting RNA" or "crRNA" or "crRNA repeat") molecule and a targeting RNA. tracrRNA-like ("trans-acting CRISPR RNA" or "activating R crRNA-like molecules (targeting RNAs) include , the DNA-targeting segment (single strand) of the DNA-targeting RNA and the DNA-targeting RNA The nucleotide strand that forms one half of the dsRNA duplex of the protein-binding segment (the "duplex") The corresponding tracrRNA-like molecule (activator RNA) contains both the forms the other half of the dsRNA duplex of the protein-binding segment of the DNA-targeting RNA. In other words, it contains a crRNA-like molecule. The nucleotide chain of the molecule is complementary to the nucleotide chain of the tracrRNA-like molecule. Hybridizes to the dsRNA duplex of the protein-binding domain of the DNA-targeting RNA Thus, each crRNA-like molecule forms a corresponding tracrRNA-like molecule. The crRNA-like molecule can further comprise a single-stranded DNA targeting segment. Therefore, the crRNA-like and tracrRNA-like molecules are (corresponding pairs) and hybridize to form a DNA-targeting RNA. The exact sequence of a tracrRNA molecule is specific to the type of RNA molecule present. The crRNA and tracrRNA are shown in corresponding complementary pairs in Figure 8. The double-molecule DNA-targeting RNA can be used with any corresponding crRNA and tracrRNA pair. The subject double-molecule DNA-targeting RNA may also comprise any corresponding crRN. A and tracrRNA pair.

[0082] The term "activator RNA" refers to a double-molecule DNA-targeting RNA tracrRNA-like molecule. As used herein, the term "targeted RNA" refers to a double-molecule DNA-targeted RNA. The term "duplex-forming segment" is used herein to refer to the crRNA-like molecule of A. The "antigen" hybridizes to the nucleotide sequence of the corresponding activator or targeter RNA molecule. activator or targeter RNAs that, when activated, contribute to the formation of dsRNA duplexes In other words, activator RNA is a duplex-forming segment complementary to a duplex-forming segment of the corresponding target RNA, Thus, activator RNAs contain duplex-forming segments, while targeter RNAs contain duplex-forming segments. It includes both the strand-forming segment and the DNA-targeting segment of the DNA-targeting RNA. Thus, the subject dual-molecule DNA-targeting RNA can be used in combination with any corresponding activating RNA and targeting RNA. The nucleic acid sequence may consist of a pair of RNA molecules.

[0083] "Host cell," as used herein, refers to a eukaryotic cell, either in vivo or in vitro. cells, prokaryotic cells (e.g., bacterial or archaeal cells), or unicellular organisms. refers to cells (e.g., cell lines) derived from multicellular organisms that are cultivated; eukaryotic or prokaryotic cells may contain nuclei. can be or has been used as a recipient of nucleic acid and transformed by nucleic acid The progeny of a single cell may vary depending on natural, accidental, or intentional mutations. Due to the inherent variation in morphology or in the genome or total DNA complement (com It is understood that the parent does not necessarily have to be completely identical to the original parent. A "recombinant host cell" (also called a "genetically modified host cell") is a cell that contains heterologous nucleic acid (e.g., A subject bacterial host cell is a host cell into which a suitable vector (e.g., expression vector) has been introduced. Introduction of exogenous nucleic acid (e.g., a plasmid or recombinant expression vector) into a bacterial host cell and the subject eukaryotic host cell is a suitable eukaryotic host cell. Genetically modified eukaryotic host cells (e.g., (e.g., mammalian germ cells).

[0084] The term "stem cell" refers to a cell that has the ability to self-renew and give rise to differentiated cell types (e.g., , plant stem cells, vertebrate stem cells) (Morrison et al. (1999) 7) See Cell 88:287-298. The adjective "differentiated," or "Differentiating" is a relative term. A "differentiating cell" is one that is further along a developmental pathway than the cell it is being compared to. Thus, pluripotent stem cells (see below) are lineage-restricted progenitor cells. These lineage-restricted progenitor cells can then differentiate into further lineage-restricted progenitor cells (e.g., mesodermal stem cells). can differentiate into more restricted cells (e.g., neuronal progenitor cells) and The cells are end-stage cells (i.e., well-differentiated cells, e.g., neurons, cardiomyocytes, etc.). The final stage cells play a characteristic role in a particular tissue type, They may or may not retain the ability to further proliferate. Stem cells are those that express specific markers. characterize both the presence of specific markers (e.g., proteins, RNA, etc.) and the absence of specific markers Stem cells may also be used in functional analyses both in vitro and in vivo, particularly in stem cell The cells can be identified by assays relating to their ability to give rise to multiple differentiated progeny.

[0085] Stem cells of interest include pluripotent stem cells (PSCs). "PSC" refers to stem cells that can give rise to all cell types of that organism. As used herein, PSCs are defined as any germ layer of an organism (e.g., a vertebrate). Pluripotent cells can give rise to cells of various germ layers (endodermal, mesodermal, and ectodermal). They can form teratomas and contribute to ectodermal, mesodermal, or endodermal tissue. Plant pluripotent stem cells can give rise to all cell types of the plant (e.g., roots, stems, leaves, etc.). can produce cells of the

[0086] Animal PSCs can be obtained in several different ways, for example, from embryonic stem cells (ESCs). C) is obtained from the inner cell mass of the embryo (Thomson et al., Science. 1998 Nov 6;282(5391):11 45-7), whereas induced pluripotent stem cells (iPSCs) are derived from somatic cells (Takahashi et. al , Cell. 2007 Nov 30;131(5):861-72; Takahashi et. al, Nat Protoc. 2007;2(12):3081 -9; Yu et. al, Science. 2007 Dec 21;318(5858):1917-20. Epub 2007 Nov 20). term The term PSC refers to pluripotent stem cells regardless of their origin, and therefore the term PSC is interchangeable with the terms ESC and and the term iPSC, as well as the term embryonic germ stem cell (EGSC), which is another example of a PSC. PSCs may be in the form of established cell lines, may be derived directly from primary embryonic tissue, or Alternatively, they may be derived from somatic cells. PSCs are the target cells for the methods described herein. obtain.

[0087] "Embryonic stem cells" (ESCs) are cells isolated from embryos, typically from the inner cell mass of blastocysts. ESCs are the NIH Human Embryonic Stem Cells. Cell Registry, e.g., hESBGN-01, hESB GN-02, hESBGN-03, hESBGN-04 (Bresagen) en, Inc.);HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International) nal); Miz-hES1(MizMedi Hospital-Seoul National University(MizMedi Ho spital-Seoul National University));HSF-1 , HSF-6 (University of California, San Francisco) California at San Francisco)); and H1, H7, H 9, H13, H14 (Wisconsin Alumni Research Fund) Wissel Research Foundation WiCell Research Institute, etc. Stem cells that can be used include embryonic stem cells derived from other primates, such as rhesus monkey stem cells and marmoset stem cells. The stem cells may be derived from any mammalian species, including humans, horses, cows, pigs, and cattle. obtained from dogs, cats, rodents (e.g., mice, rats, hamsters), primates, etc. (Thomson et al. (1998) Science 282:1145; Thomson et al. (1995) Proc. Na tl. Acad. Sci USA 92:7844; Thomson et al. (1996) Biol. Reprod. 55:254; Shamblott (E. et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). In culture, ESCs typically Generally, they appear as flat colonies with a large nucleo-cytoplasmic ratio, clear borders and prominent nucleoli. Furthermore, ESCs proliferate by SSEA-3, SSEA-4, TRA-1-60, and TR A-1-81 and alkaline phosphatase, but not SSEA-1 Examples of methods for generating and characterizing ESCs are described, for example, in U.S. Patent No. 7,029,913. Nos. 5,843,780, and 6,200,806; The disclosures of which are incorporated herein by reference. Methods for this purpose are described in WO 99 / 20741, WO 01 / 51616, and WO 01 / 51616. It is described in issue 3 / 020920.

[0088] "Embryonic germ stem cells" (EGSCs) or "embryonic germ cells" or "EG cells" refer to cells that are derived from living Germ cells and / or germ cell precursors, such as primordial germ cells (i.e., sperm and eggs) Embryonic germ cells (EG cells) refer to PSCs derived from the above-mentioned EG cells are thought to have similar properties to embryonic stem cells. Examples of methods are described, for example, in U.S. Pat. No. 7,153,684; Matsui, Y., et al., (1992 ) Cell 70:841; Shamblott, M., et al. (2001) Proc. Natl. Acad. Sci. USA 98: 113; Shamblott, M., et al. (1998) Proc. Natl. Acad. Sci. USA, 95:13726; and Koshimiz U., et al. (1996) Development, 122:1235, the disclosures of which are incorporated herein by reference. and is incorporated herein by reference.

[0089] "Induced pluripotent stem cells" or "iPSCs" refer to cells that are not PSCs (i.e., PS iPSCs are PSCs derived from cells that are more differentiated than iPSCs. iPSCs have an ES cell-like morphology and can be obtained from various types of cells (e.g., well-differentiated cells). They grow as flat colonies with a large nucleus-to-cytoplasm ratio, clear borders and prominent nuclei. Additionally, iPSCs express one or more important pluripotency markers known to those skilled in the art. For example, but not limited to, alkaline phosphatase, SSEA3, SSEA4, So x2, Oct3 / 4, Nanog, TRA160, TRA181, TDGF1, Dnmt 3b, FoxD3, GDF3, Cyp26a1, TERT, and zfp42. Examples of methods for generating and characterizing iPSCs are found in, for example, U.S. Patent Application Publication No. US200 90047263, US20090068742, US200901911 59, US20090227032, US20090246875, and and US20090304646, the disclosures of which are incorporated by reference. Generally, to generate iPSCs, cells are cultured to become pluripotent stem cells. Reprogramming factors known in the art for reprogramming somatic cells such as For example, Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc.) are expressed in somatic cells is given to.

[0090] "Somatic cells" refer to cells that do not normally give rise to all cell types in an organism in the absence of experimental manipulation. It refers to all cells in an organism. In other words, somatic cells are cells of all three germ layers ( cells that are sufficiently differentiated that they do not naturally give rise to cells of the germ layer (i.e., ectoderm, mesoderm, and endoderm). For example, somatic cells include both neurons and neural progenitor cells, the latter of which may be capable of spontaneously giving rise to all or some cell types of the central nervous system, but mesoderm or cannot give rise to cells of the endodermal lineage.

[0091] "Mitotic cell" means a cell undergoing mitosis. Mitosis is a process that occurs in eukaryotic cells. is the process by which the chromosomes contained in that nucleus are separated into two identical sets in two separate nuclei Usually, cytokinesis occurs immediately afterwards, and the nucleus, cytoplasm, organelles, and cell membrane are separated. It divides into two cells containing these cellular components in roughly equal proportions.

[0092] "Postmitotic cells" are cells that have exited mitosis, i.e., are in a "quiescent state"; This state of quiescence is temporary, i.e., reversible. It may be temporary or permanent.

[0093] "Meiotic cell" means a cell undergoing meiosis. It is the process of dividing the nuclear material in order to produce a germ or spore. Unlike mitosis, meiosis involves a recombination step in which chromosomes recombine to reassemble genetic material between chromosomes. Furthermore, for the two (genetically identical) diploid cells produced by mitosis, Thus, meiosis results in four (genetically unique) haploid cells.

[0094] "Recombination" refers to the process of exchange of genetic information between two polynucleotides. As used herein, "homologous recombination repair (HDR)" refers to the repair of a double stranded gene in a cell, e.g. This refers to a specialized form of DNA repair that occurs during repair of heavy strand breaks. It requires sequence homology between the "donor" molecule and the "target" molecule (i.e., the molecule that has undergone the double-strand break). Homologous recombination (HRM) is used to repair the template of the target molecule, resulting in the transfer of genetic information from the donor to the target. Repair occurs when the donor polynucleotide differs from the target molecule and a partial deletion of the donor polynucleotide sequence occurs. When part or all of the target molecule is integrated into the target DNA, it causes a change in the target molecule sequence (e.g., an insertion). In some embodiments, the donor polynucleotide , a portion of a donor polynucleotide, a copy of a donor polynucleotide, or a donor polynucleotide Some of the nucleotide copies are incorporated into the target DNA.

[0095] "Non-homologous end joining (NHEJ)" refers to the process of repair without the need for a homologous template (to guide repair). (In contrast to homology-directed repair, which requires homologous sequences, the cut ends are directly ligated together.) NHEJ refers to the repair of double-strand breaks in DNA by cleaving the DNA. This results in the loss (deletion) of nucleotide sequences near the heavy chain cleavage site.

[0096] The terms "treatment," "treating," and the like refer to the process of achieving a desired pharmacological and / or physiological effect. As used herein, the term "effective" generally means to achieve a therapeutic effect. may be prophylactic in that it prevents, in whole or in part, is a partial or complete cure of the disease and / or adverse effects caused by the disease. As used herein, "treatment" refers to the treatment of a mammal. (a) includes any treatment of a disease or condition in a person who is prone to contracting the disease or condition; The development of a disease or condition in a subject who has not yet been diagnosed with it. (b) inhibiting a disease or condition, i.e., arresting its development; or (c) to alleviate the disease, i.e., to cause regression of the disease. Therapeutic agents may be administered prior to the onset of disease or injury or during the onset of disease or injury. Treatment of ongoing disease may be initiated or administered after onset. Treatments that stabilize or reduce undesirable clinical symptoms are of particular importance. It is desirable to treat the disease before complete loss of function in the affected tissue. It is desirable to administer the compound during the symptomatic stage, and optionally after the symptomatic stage of the disease.

[0097] The terms "individual," "subject," "host," and "patient" are used interchangeably herein. "Antibody" refers to any mammalian subject, particularly humans, for whom diagnosis, treatment, or therapy is desired.

[0098] General methods in molecular and cellular biochemistry are described in Molecular Cloning: A Laboratory Guide. ory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001); Short Proto cols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999) ; Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Bio technology (Doyle & Griffiths, John Wiley & Sons 1998) and other standard textbooks. and the disclosures of which are incorporated herein by reference.

[0099] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, and therefore It should be understood that variations may, of course, be made. It is intended that the terminology be used for the purpose of describing particular embodiments only, and the scope of the present invention will be limited only by the appended claims. It is understood that the scope of the claims is limited only by the scope of the claims, and is not intended to be limiting. .

[0100] When a range of values ​​is given, the difference between the upper and lower limits of the range is not always clear, as the context may dictate. Each intervening value and its description to the tenth of the unit of the lower limit unless otherwise indicated Any other stated or intervening values ​​within the range are intended to be encompassed by the present invention. It should be understood that the upper and lower limits of these smaller ranges are not necessarily construed as limiting the scope of the smaller ranges. The ranges may be independently included within the ranges, and are also encompassed by the present invention, but the specific ranges described may not be included within the ranges. Exclusionary limits apply. If the stated range includes one or both limits, Ranges excluding either or both of the inclusive limits are also included in the invention.

[0101] Certain ranges are provided herein using numerical values ​​preceded by the term "about." As used herein, the term "about" refers to the exact numerical value that follows it, as well as the value of the term itself. Used to provide literal support for numbers that are close to or approximate the number that follows. Determine whether a value is close to or approximates a specifically stated value In such cases, any unstated figures that are close or approximate will be used to specifically address the issue in the context in which they are presented. It may be a numerical value that provides a substantial equivalent of the numerical value stated.

[0102] Unless otherwise defined, all technical and scientific terms used herein are defined by the principles of the present invention. The term "term" has the same meaning as commonly understood by a person skilled in the art. Any methods and materials similar or equivalent to those described herein may be used to practice the present invention. Although any method and material can be used for the implementation or testing of the present invention, the preferred methods and materials are described herein. All publications mentioned in the specification are incorporated by reference in their entirety for purposes of illustration only and are not intended to be limiting unless expressly stated otherwise. No. 6,299,333, filed Dec. 1, 2003, which is incorporated herein by reference to disclose and describe the materials.

[0103] All publications and patents cited herein are to be construed as though individually incorporated by reference. Each such disclosure is specifically and individually indicated to be incorporated by reference herein. As such, the methods and methods for which publications are cited in connection with which they are incorporated by reference herein are also incorporated by reference. and / or materials are incorporated herein by reference to disclose and describe the same. The citation of any publication is for its disclosure prior to the filing date and does not constitute a substitute for prior invention. Nothing contained herein should be construed as an admission that the reader is not entitled to antedate such publication. Additionally, any dates of publication provided may be different from the actual publication dates, which may be independently confirmed. There may be a need.

[0104] As used in this specification and the appended claims, the singular forms "a," "an," and "an" are used interchangeably. Note that "and the" include plural referents unless the context clearly indicates otherwise. Thus, for example, a reference to a "polynucleotide" includes a plurality of such polynucleotides. Reference to a "polypeptide" includes reference to one or more polypeptides. and the equivalents thereof known to those skilled in the art. Note that ranges can be drafted to exclude any optional element. Thus, this statement should not be construed as limiting the scope of the invention to "solely," "or" in connection with the recitation of claim elements. The use of exclusive language such as "only" or "negative" limitations is subject to the existence of a condition precedent. It is intended to serve as a benchmark.

[0105] Certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be incorporated into a single It will be appreciated that in an embodiment, they may be provided in combination. Various features of the invention that are, for purposes of illustration, described in the context of a single embodiment may also be provided separately. or may be provided in any suitable subcombination. Combinations of such aspects are treated as if each and every combination were individually and expressly disclosed. As such, these are specifically embraced by the present invention and disclosed herein. The embodiments and all subcombinations of elements thereof are also included herein, each and every such subcombination. are specifically embraced by the present invention as if each such compound were individually and explicitly disclosed herein. and disclosed herein.

[0106] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. All disclosures herein are to be construed as a lien on any such publication by virtue of prior invention. Nothing in this document should be construed as an admission that no prior right exists. Dates of publication may be different from the actual publication dates which may need to be independently confirmed.

[0107] MODE FOR CARRYING OUT THE INVENTION (Part 1) The present disclosure provides a method for targeting a target DNA and / or a modified polypeptide, comprising the steps of: or DNA-targeting RNAs that provide site-specific modification of polypeptides bound to target DNA. A. The present disclosure further provides a site-specifically modified polypeptide. In addition, methods for site-specific modification of target DNA and / or polypeptides bound to target DNA are also proposed. The present disclosure provides a method for cleaving a target nucleic acid with an enzymatically inactive Cas9 polypeptide and a DNA fragment. A method for modulating transcription of a target nucleic acid in a target cell generally involves contacting the target nucleic acid with a targeting RNA. Kits and compositions for carrying out the methods are also provided. and Cas9-transgenic non-human multicellular organisms. Provide.

[0108] nucleic acid DNA targeting RNA The present disclosure provides a method for the production of polypeptides (e.g., fragments) that bind to specific target sequences within target DNA. The present invention provides a DNA-targeting RNA that directs the activity of a site-specific modifying polypeptide. The DNA-targeting RNA comprises a first segment (referred to herein as a "DNA-targeting segment" or a second segment (also referred to herein as a "protein The nucleotide sequence comprises a nucleotide sequence (also referred to as a "binding segment" or "protein binding sequence").

[0109] DNA-targeting segment of DNA-targeting RNA The DNA-targeting segment of the subject DNA-targeting RNA is complementary to a sequence within the target DNA. In other words, the DNA target of the subject DNA-targeting RNA comprises a complementary nucleotide sequence. The hybridization segments are sequence-specifically linked through hybridization (i.e., base pairing). Therefore, the nucleotide sequence of the DNA targeting segment is variable. The position within the target DNA where the DNA-targeting RNA and the target DNA interact may be moved. The DNA-targeting segment of the subject DNA-targeting RNA determines the modified (e.g., by genetic engineering) to hybridize to any desired sequence; It is possible.

[0110] The DNA targeting segment has a length of about 12 nucleotides to about 100 nucleotides. For example, the DNA targeting segment can be from about 12 nucleotides (nt) to about 80 nt, Approximately 12nt to approximately 50nt, approximately 12nt to approximately 40nt, approximately 12nt to approximately 30nt, approximately 12n and having a length of about 1 to about 25 nt, about 12 nt to about 20 nt, or about 12 nt to about 19 nt. For example, the DNA targeting segment may be about 19 nt to about 20 nt, about 19 nt to about 2 5nt, about 19nt to about 30nt, about 19nt to about 35nt, about 19nt to about 40nt, Approximately 19nt to approximately 45nt, approximately 19nt to approximately 50nt, approximately 19nt to approximately 60nt, approximately 19nt t ~ approx. 70nt, approx. 19nt ~ approx. 80nt, approx. 19nt ~ approx. 90nt, approx. 19nt ~ approx. 1 00nt, about 20nt to about 25nt, about 20nt to about 30nt, about 20nt to about 35nt , about 20nt to about 40nt, about 20nt to about 45nt, about 20nt to about 50nt, about 20 nt ~ approx. 60nt, approx. 20nt ~ approx. 70nt, approx. 20nt ~ approx. 80nt, approx. 20nt ~ approx. The length of the target DNA may be 90 nt, or about 20 nt to about 100 nt. The nucleotide sequence of the DNA targeting segment (DNA) that is complementary to the target sequence The targeting sequence may have a length of at least about 12 nt. For example, the target sequence of the target DNA may The DNA targeting sequence of the DNA targeting segment complementary to the sequence is at least about 12 nt , at least about 15 nt, at least about 18 nt, at least about 19 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 35 nt, or It may have a length of at least about 40 nt. For example, it may be complementary to a target sequence of a target DNA. The DNA targeting sequence of the DNA targeting segment can be from about 12 nucleotides (nt) to about 80 nt. t, about 12nt to about 50nt, about 12nt to about 45nt, about 12nt to about 40nt, about 1 2nt ~ approx. 35nt, approx. 12nt ~ approx. 30nt, approx. 12nt ~ approx. 25nt, approx. 12nt ~ Approximately 20nt, approximately 12nt~approximately 19nt, approximately 19nt~approximately 20nt, approximately 19nt~approximately 25n t, about 19nt to about 30nt, about 19nt to about 35nt, about 19nt to about 40nt, about 1 9nt ~ approx. 45nt, approx. 19nt ~ approx. 50nt, approx. 19nt ~ approx. 60nt, approx. 20nt ~ Approx. 25nt, approx. 20nt ~ approx. 30nt, approx. 20nt ~ approx. 35nt, approx. 20nt ~ approx. 40n t, about 20 nt to about 45 nt, about 20 nt to about 50 nt, or about 20 nt to about 60 nt The length of the DNA target is complementary to the nucleotide sequence of the target DNA (target sequence). The nucleotide sequence of the targeting segment (DNA targeting sequence) is at least about 12 nt in length. It may have a certain quality.

[0111] In some instances, a DNA targeting segment that is complementary to a target sequence of the target DNA. The DNA targeting sequence is 20 nucleotides in length. The DNA targeting sequence of the DNA targeting segment complementary to the target sequence of He is a chid leader.

[0112] Complementarity between the DNA targeting sequence of the DNA targeting segment and the target sequence of the target DNA The percentage should be at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 9 5%, at least 97%, at least 98%, at least 99%, or 100%) In some instances, the DNA targeting sequence and target of the DNA targeting segment The percent complementarity between target sequences of DNA is determined by dividing the seven nucleotides of the target sequence on the complementary strand of the target DNA. In some instances, the DNA is 100% over the 5'-terminal nucleotides following the The percentage of complementarity between the DNA targeting sequence of the targeting segment and the target sequence of the target DNA The percentage is at least 60% over about 20 consecutive nucleotides. between the DNA targeting sequence of the DNA targeting segment and the target sequence of the target DNA Percent complementarity is calculated by the number of consecutive 5'-terminal nucleotides of the target sequence on the complementary strand of the target DNA. It is 100% across the octides and as low as 0% across the rest of the sequence. In such cases, the DNA targeting sequence can be considered to be 14 nucleotides in length (Figure 1). 12D-E). In some instances, the DNA-targeting sequence of the DNA-targeting segment The percent complementarity between a target sequence on the complementary strand of the target DNA and a target sequence on the target DNA is 100% across the seven consecutive 5'-terminal nucleotides of the string and 100% across the remainder of the sequence. In such cases, the DNA targeting sequence is 7 nucleotides long. It can be considered that there is.

[0113] Protein-binding segments of DNA-targeting RNA The protein-binding segment of the subject DNA-targeting RNA comprises a site-specifically modified polypeptide. The subject DNA-targeting RNAs target the bound polypeptide to the DNA. The targeting segment directs the target to a specific nucleotide sequence within the target DNA. The protein-binding segment of the targeting RNA contains two complementary nucleotide strands. The complementary nucleotides of the protein-binding segment hybridize to form double-stranded RNA. A double-stranded (dsRNA) is formed (see Figure 1A and Figure 1B).

[0114] A subject double-molecule DNA-targeting RNA comprises two separate RNA molecules. Each of the two RNA molecules of the DNA-targeting RNA is a complementary nucleotide sequence of the two RNA molecules. The proteoides hybridize to form a double-stranded RNA duplex of the protein-binding segment. They contain complementary nucleotide chains, such as

[0115] In some embodiments, the duplex-forming segment of the activator-RNA is SEQ ID NO: 43 1-562, or its complement. at least about 60% identical over a stretch of at least 8 consecutive nucleotides to For example, the double-stranded segment of the activator RNA (or the double-stranded segment of the activator RNA) The DNA encoding the tracrRNA segment is represented by SEQ ID NOs: 431 to 562. A stretch of at least 8 consecutive nucleotides to one of the sequences or its complement. at least about 60% identical, at least about 65% identical, at least about 70% identical, At least about 75% identical, at least about 80% identical, at least about 85% identical, at least Also about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical % identical, or 100% identical.

[0116] In some embodiments, the duplex-forming segment of the targeting RNA is SEQ ID NO: 56 3-679, or its complement. , are at least about 60% identical over a stretch of at least 8 contiguous nucleotides. For example, the duplex-forming segment of the targeting RNA (or the duplex-forming segment of the targeting RNA) The DNA encoding the crRNA is one of the crRNA sequences set forth in SEQ ID NOs: 563 to 679, or its complement over a stretch of at least 8 contiguous nucleotides at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical 1. At least about 98% identical, at least about 99% identical, or 100% identical.

[0117] The bimolecular DNA-targeting RNA is regulated by the interaction of the targeting RNA with the activating RNA (i.e. The bimolecular DNA-targeting RNA can be designed to allow for conditional (or conditional) binding. Both the activating RNA and the targeting RNA bind to dCas9 in the form of a functional complex. It is inducible to bind activating and targeting RNAs because it is not functional unless By doing so, the bimolecular DNA-targeting RNA can be made inducible (e.g., drug-inducible). As one non-limiting example, RNA aptamers can be used to target activated RNAs. Therefore, binding of the activating RNA to the targeting RNA can be controlled (i.e., regulated). And / or the targeting RNA may comprise an RNA aptamer sequence.

[0118] RNA aptamers are known in the art and generally represent synthetic versions of riboswitches. The terms "RNA aptamer" and "riboswitch" are used interchangeably herein. used to identify synthetic and naturally occurring nucleic acid sequences, Synthetic nuclei that provide inducible regulation of the structure (and therefore availability of specific sequences) of RNA molecules. RNA aptamers typically contain specific structures (e.g., The sequence contains a sequence that folds into a small molecule (e.g., a hairpin), and the sequence binds to a specific drug (e.g., a small molecule). The binding of the drug induces a structural change in the folding of the RNA, which alters the characteristics of the nucleic acid of which the aptamer is a part. (i) Activated RNA with an aptamer will not bind to the appropriate drug unless the aptamer binds to the appropriate drug. (ii) unable to bind to the cognate target RNA; The targeting RNA with an aptamer will not bind to the cognate activating RNA unless the aptamer binds to the appropriate drug. (iii) different aptamers that bind to different drugs, The targeting RNA and activating RNA bind to each other unless both agents are present. As illustrated by these examples, bimolecular DNA-targeting RNA is inducible. It can be designed to be functional.

[0119] Examples of aptamers and riboswitches are described in, for example, Nakamura et al., Genes Cells. 2012 May;17(5):344-64; Vavalle et al., Future Cardiol. 2012 May;8(3):371-82; Citarta n et al., Biosens Bioelectron. 2012 Apr 15;34(1):1-11; and Liberman et al., Wil ey Interdiscip Rev RNA. 2012 May-Jun;3(3):369-84, all of which No. 6,299,333, filed Dec. 1, 2003, all of which are incorporated herein by reference in their entirety.

[0120] Non-limiting examples of nucleotide sequences that can be included in the bimolecular DNA-targeting RNA include SEQ ID NO: Any of the sequences set forth in Nos. 431 to 562, or their complements, hybridize. The protein-binding segment can be formed by the The term "antibody" refers to a nucleic acid sequence that is a nucleic acid sequence of a nucleic acid molecule, such as a nucleotide sequence of a nucleic acid molecule, ... or a complement thereof.

[0121] The subject single-molecule DNA-targeting RNAs are complementary to each other and have an intervening nucleotide ("ligand"). The hybridization is performed by covalently linking the nucleotides with one another via a "linker" or "linker nucleotide" ("linker" or "linker nucleotide"). It is then purified to form a double-stranded RNA duplex (dsRNA duplex) of the protein-binding segment. The target RNA is then inserted into the targeting region of the target RNA, forming a two-nucleotide strand (TRNA) that forms a stem-loop structure. and activator RNAs) (Figure 1B). Targeter RNAs and activator RNAs are Alternatively, the activator RNA may be covalently linked via the 3' end of the NA and the 5' end of the activator RNA. The targeter RNA and the activator RNA are located at the 5' end of the targeter RNA and the 5' end of the activator RNA. It may be covalently linked via the 3' end.

[0122] The linker of the single-molecule DNA-targeting RNA is about 3 nucleotides to about 100 nucleotides. For example, the linker may have a length of about 3 nucleotides (nt) to about 90 nt, about 3 Nucleotide (nt) to about 80 nt, about 3 nucleotides (nt) to about 70 nt, about 3 nucleotides nucleotide (nt) to about 60 nt, about 3 nucleotides (nt) to about 50 nt, about 3 nucleotides nucleotide (nt) to about 40 nt, about 3 nucleotides (nt) to about 30 nt, about 3 nucleotides (nt) to about 20 nt or from about 3 nucleotides (nt) to about 10 nt in length. For example, the linker may be about 3 nt to about 5 nt, about 5 nt to about 10 nt, or about 10 nt to about 15 nt. nt, about 15nt to about 20nt, about 20nt to about 25nt, about 25nt to about 30nt, about 30nt ~ approx. 35nt, approx. 35nt ~ approx. 40nt, approx. 40nt ~ approx. 50nt, approx. 50nt ~about 60nt, about 60nt to about 70nt, about 70nt to about 80nt, about 80nt to about 90 In some embodiments, the length of the nucleotide sequence may be about 90 nt to about 100 nt. , the linker of the single-molecule DNA-targeting RNA is 4 nt.

[0123] Exemplary single-molecule DNA-targeting RNA hybridizes to form a dsRNA duplex In some embodiments, the single molecule DNA comprises two complementary nucleotide strands. A. One of the two complementary nucleotide strands of the targeting RNA (or the D NA) is an activating RNA (tracrRNA) molecule set forth in SEQ ID NOs: 431 to 562. or its complement to a series of at least 8 consecutive nucleotides For example, the two strands of a single-molecule DNA-targeting RNA are at least about 60% identical across the entire DNA sequence. One of the complementary nucleotide strands (or the DNA encoding that sequence) is SEQ ID NO: 43 1-562, or its complement. , at least about 65% identical over a stretch of at least 8 consecutive nucleotides, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical 1. At least about 99% identical or 100% identical.

[0124] In some embodiments, the two complementary nucleosides of the single-molecule DNA-targeting RNA One of the peptide chains (or the DNA encoding the sequence) is set forth in SEQ ID NOS: 563 to 679. A series of targeting RNA (crRNA) sequences are generated against one of the targeting RNA (crRNA) sequences, or its complement. are at least about 60% identical over at least 8 consecutive nucleotides of , one of the two complementary nucleotide strands of the single-molecule DNA-targeting RNA (or its sequence The DNA encoding the crRNA sequence is a sequence of the crRNA sequence set forth in SEQ ID NOs: 563 to 679. or its complement over a stretch of at least 8 consecutive nucleotides , at least about 65% identical, at least about 70% identical, at least about 75% identical, At least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 9 5% identical, at least about 98% identical, at least about 99% identical, or 100% identical .

[0125] Proper naturally occurring cognate pairing of crRNA and tracrRNA determines proper cognate pairing. When specifying the target gene, please provide the species name and base pairing (protein binding domain of dsRNA duplex) By taking into consideration the above, SEQ ID NOs: 431 to 679 can be obtained by the usual method. can be determined (see FIG. 8 for a non-limiting example).

[0126] About the subject single-molecule DNA-targeting RNA and the subject double-molecule DNA-targeting RNA , Figure 57 has little in common with naturally occurring tracrRNA and crRNA Even artificial sequences (approximately 50% identity) can be used to identify protein-binding domains of DNA-targeting RNA. As long as the main structure is preserved, it can function together with Cas9 to cut the target DNA. Therefore, naturally occurring protein binding of DNA-targeting RNA can be demonstrated. The RNA folding structure of the domain is then used to construct an artificial protein-binding domain (either bimolecular or monomolecular). As a non-limiting example, the function of FIG. Functional artificial DNA-targeting RNAs target the protein-binding segments of naturally occurring DNA targets. designed based on structure (e.g., containing the same number of base pairs along the RNA duplex, and (Contains the same "bulge" region as present in nascent RNA.) Structure can be identified by one skilled in the art as any naturally occurring crRNA from any species: tracrR Because crRNA pairs can be easily generated (crRNA sequences from a wide variety of species and and tracrRNA sequences (see SEQ ID NOS: 431-679), artificial DNA targets The targeting RNA is a Cas9 (or related Cas9, see Figure 32A) derived from a given species. When used, they can be designed to mimic the natural structure of the species (Figure 24D and (See relevant details in Example 1.) Therefore, suitable DNA-targeting RNAs may be derived from naturally occurring A target protein designed to mimic the structure of the protein-binding domain of a specific DNA-targeting RNA Even artificially designed (non-naturally occurring) RNA containing protein-binding domains (See SEQ ID NOS: 431-679, consider species names when determining appropriate conspecific pairs.) ).

[0127] The protein-binding segment has a length of about 10 nucleotides to about 100 nucleotides. For example, the protein-binding segment may be from about 15 nucleotides (nt) to about 80 nt. t, about 15 nt to about 50 nt, about 15 nt to about 40 nt, about 15 nt to about 30 nt, or It may have a length of about 15 nt to about 25 nt.

[0128] Also included are the subject single-molecule DNA-targeting RNAs and the subject double-molecule DNA-targeting RNAs. Regarding the protein-binding segment, the dsRNA duplex is about 6 base pairs (bp) to about 50 For example, the dsRNA duplex of the protein-binding segment may have a length of about 6 bp. bp ~ approx. 40bp, approx. 6bp ~ approx. 30bp, approx. 6bp ~ approx. 25bp, approx. 6bp ~ approx. 20b p, about 6bp~about 15bp, about 8bp~about 40bp, about 8bp~about 30bp, about 8bp~ It may have a length of about 25 bp, about 8 bp to about 20 bp, or about 8 bp to about 15 bp. For example, the dsRNA duplex of the protein-binding segment may be about 8 bp to about 10 bp, about 10 bp, or p ~ about 15bp, about 15bp - about 18bp, about 18bp - about 20bp, about 20bp - about 2 5bp, about 25bp to about 30bp, about 30bp to about 35bp, about 35bp to about 40bp, Alternatively, the length may be about 40 bp to about 50 bp. The dsRNA duplex of the protein-binding segment is 36 base pairs in length. Complementarity between the nucleotide sequences that form the dsRNA duplex of the protein-binding segment The percent identity can be at least about 60%. For example, the percent identity can be at least about 60%. The percent complementarity between the nucleotide sequences forming the dsRNA duplex of the binding segment is , at least about 65%, at least about 70%, at least about 75%, at least about 80% , at least about 85%, at least about 90%, at least about 95%, at least about 98% In some instances, the hybridization may be at least about 99%. Complementarity between the nucleotide sequences forming the dsRNA duplex of the protein-binding segment The cent is 100%.

[0129] Site-specifically modified polypeptides A subject DNA-targeting RNA and a subject site-specific modifying polypeptide form a complex. DNA-targeting RNA is a molecule that is complementary to the sequence of the target DNA (as mentioned above). The inclusion of a nucleotide sequence confers target specificity to the complex. The modified polypeptide confers site-specific activity. In other words, the site-specific modified polypeptide that it binds to at least the protein-binding segment of the DNA-targeting RNA. DNA sequences (e.g., chromosomal sequences or extrachromosomal sequences, e.g., episomal sequences, Minicircle sequences, mitochondrial sequences, chloroplast sequences, etc.) (see above).

[0130] The subject site-specific modifying polypeptides modify target DNA (e.g., induce cleavage of target DNA). cleavage or methylation), and / or modify the polypeptide bound to the target DNA (e.g. For example, methylation or acetylation of histone tails). In the specification, the term "site-specific polypeptide" or "RNA-binding site-specific modified polypeptide" refers to Also known as "Chido".

[0131] In some instances, the site-directed modified polypeptide is a naturally occurring modified polypeptide. In other cases, the site-directed modified polypeptide is a naturally occurring polypeptide. The polypeptides are not chimeric polypeptides (e.g., chimeric polypeptides described below) or modified (e.g., mutations, deletions, insertions) ) naturally occurring polypeptides).

[0132] Exemplary naturally occurring site-specific modifying polypeptides include naturally occurring Cas9 / Csn A non-limiting and non-exhaustive list of endonucleases is set forth in SEQ ID NOs: 1 to 255. These naturally occurring polypeptides described herein are capable of binding to DNA-targeting RNAs. binds to the target DNA, thereby directing it to a specific sequence within the target DNA and cleaving the target DNA to form a The subject site-directed modified polypeptides comprise two portions: an RNA-binding domain; and an active site. In some embodiments, the subject site-specifically modified polypeptides The target DNA is (i) a DNA-targeting R&D gene that contains a nucleotide sequence complementary to a sequence within the target DNA. (ii) an RNA-binding site that interacts with NA; and (ii) an active site that exhibits site-specific enzymatic activity. (e.g., DNA methylation activity, DNA cleavage activity, histone acetylation activity, histone metabolite activity) The site of enzymatic activity is determined by the DNA-targeting RNA.

[0133] In other embodiments, the subject site-directed modifying polypeptides are those that: (i) target a sequence within the target DNA; RNA binding moieties that interact with DNA-targeting RNAs containing nucleotide sequences complementary to and (ii) active sites that regulate transcription within the target DNA (e.g., increase or decrease transcription). (for reducing transcription), and the site of regulated transcription within the target DNA is a DNA-targeting RNA is determined by.

[0134] In some instances, the subject site-specific modifying polypeptides modify target DNA. Has enzymatic activity (e.g., nuclease activity, methyltransferase activity, demethylase activity) deamination activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, Alkylating activity, depurinating activity, oxidizing activity, pyrimidine dimer forming activity, integrating activity enzyme activity, transposase activity, recombinase activity, polymerase activity, ligase activity , helicase activity, photolyase activity or glycosylase activity).

[0135] In other cases, the subject site-directed modifying polypeptides are polypeptides bound to target DNA. have enzymatic activity that modifies polypeptides (e.g., histones) (e.g., methyltransferases) ferase activity, demethylase activity, acetyltransferase activity, deacetylation Enzyme activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, sumoylation activity, desumoylation activity, ribozylation activity silating activity, deribosylating activity, myristoylating activity or demyristoylating activity).

[0136] Exemplary Site-Directly Modified Polypeptides In some instances, the site-directed modifying polypeptide is a Cas9 / C polypeptide, as shown in FIG. to amino acids 7 to 166 or 731 to 1003 of the sn1 amino acid sequence, or Any of the amino acid sequences set forth as numbers 1 to 256 and 795 to 1346 At least about 75%, at least about 80%, at least about 8% of the corresponding portion in 5%, at least about 90%, at least about 95%, at least about 99%, or 100% and amino acid sequences having an amino acid sequence identity of

[0137] Nucleic acid modification In some embodiments, the subject nucleic acids (e.g., DNA-targeting RNAs) are novel or to provide nucleic acids with enhanced characteristics (e.g., improved stability), or multiple modifications (e.g., base modifications, backbone modifications, etc.). As such, a nucleoside is a base-sugar combination. The two most common classes of such heterocyclic bases are: Purines and pyrimidines. Nucleotides are covalently linked to the sugar moiety of a nucleoside. Nucleosides that further contain a linked phosphate group. In nucleosides, the phosphate group is attached to the 2', 3', or 5' hydroxyl moiety of the sugar. In oligonucleotide formation, phosphate groups may link adjacent nucleotides. The cleosides are covalently linked to each other to form a linear polymeric compound. The ends of the linear polymer compound may be further linked to form a cyclic compound. Linear compounds are generally suitable. Furthermore, linear compounds have an internal nucleotide base phase. may have complementary sequences and thus be folded to form fully or partially double-stranded compounds. Within an oligonucleotide, the phosphate group acts as a These are commonly referred to as internucleotide backbones. Alternatively, the backbone is 3' to 5' phosphodiester linkages.

[0138] Modified backbones and modified internucleoside linkages Examples of suitable nucleic acids containing modifications include those containing modified backbones or non-natural internucleoside linkages. Nucleic acids with modified backbones include nucleic acids containing a phosphorus atom in the backbone. These include nucleic acids that have a phosphorus atom and nucleic acids that do not have a phosphorus atom in their backbone.

[0139] Suitable modified oligonucleotide backbones containing an internal phosphorus atom include, for example, , phosphorothioates, chiral phosphorothioates, phosphorothioates with normal 3'-5' linkages Phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphonates, e.g., 3'-alkylene phosphonates, 5 '-Alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates 3'-aminophosphoramidates and aminoalkylphosphoramidates phosphates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates , thionoalkylphosphotriesters, selenophosphates and boranophosphates, These 2'-5' linked analogs, as well as analogs of one or more internucleotide linkages 3' to Included are those with reverse polarity, which are 3', 5' to 5' or 2' to 2' linkages. Suitable oligonucleotides with reverse polarity have a single nucleotide at the 3'-terminal internucleotide linkage. a single 3' to 3' linkage, i.e., a single reverse nucleoside residue, The cytosine residue is abasic (either the nucleobase is missing or it has a hydroxyl group in that position) Various salts (e.g., potassium or sodium, etc.), mixed salts and free salts may also be used. The acid form is also included.

[0140] In some embodiments, the subject nucleic acids comprise one or more phosphorothioates and and / or heteroatom internucleoside linkages, specifically, -CH2-NH-O-CH2 -, -CH2-N(CH3)-O-CH2-(methylene(methylimino) or MMI bone known as -CH2-ON(CH3)-CH2-, -CH2-N(CH 3) -N(CH3)-CH2- and -ON(CH3)-CH2-CH2- (wherein The original phosphodiester internucleotide linkage is -OP(=O)(OH)-O-CH2- MMI-type internucleoside linkages include those described in the above-referenced U.S. Pat. Suitable amide internucleoside linkages are disclosed in U.S. Pat. This is disclosed in No. 02,240.

[0141] For example, the nuclei having morpholino backbone structures described in U.S. Pat. No. 5,034,506 Acids are also suitable. For example, in some embodiments, the subject nucleic acids are In some of these embodiments, the phosphorodiamine comprises a 6-membered morpholino ring. Amidates or other non-phosphodiester internucleoside linkages are not phosphodiester bonds. Replace with.

[0142] Suitable modified polynucleotide backbones that do not contain internal phosphorus atoms include short chain alkyl or Cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkenyl internucleoside linkage, or one or more short chain heteroatoms or heterocyclic They have a backbone formed by internucleoside linkages. Partially formed from the sugar moiety of the nucleoside; siloxane backbone; sulfide, sulfoxide and and sulfone skeletons; formacetyl and thioformacetyl skeletons; methyleneformacetyl acetyl and thioformacetyl backbones; riboacetyl backbones; alkene-containing backbones; sulfamethoxazole carboxylate backbone; methyleneimino and methylenehydrazino backbone; sulfonate and sulfone Amide backbone; Amide backbone; and other compounds with mixed N, O, S and CH2 moieties These include those having a skeleton of the formula:

[0143] Mimics The subject nucleic acids can be nucleic acid mimetics. The term "mimetics" applies to polynucleotides. When the furanose ring alone or both the furanose ring and the internucleotide linkage are non-furanose, It is intended to include polynucleotides in which the furanose ring is substituted with a furanose group. Substitutions of only the heterocyclic base moiety or The modified heterocyclic base moiety is suitable for hybridization with an appropriate target nucleic acid. One such nucleic acid has excellent hybridization properties. The polynucleotide mimics shown here are called peptide nucleic acids (PNAs). In the present invention, the sugar backbone of the polynucleotide is an amide-containing backbone, specifically an aminoethylglycol. The nucleotides are retained and the aza-nitrogens in the amide portion of the backbone are replaced by It is directly or indirectly bound to an elementary atom.

[0144] One polynucleotide that has been reported to have excellent hybridization properties is The peptide mimetic is a peptide nucleic acid (PNA). The backbone in a PNA compound is an amide Two or more linked aminoethylglycine units give a heterocyclic backbone. The base moiety is directly or indirectly bound to an aza nitrogen atom of the amide portion of the backbone. Representative U.S. patents describing the preparation of Compound A include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262 Examples include:

[0145] Another class of polynucleotide mimetics being investigated is heterodimers linked to a morpholino ring. It is based on linked morpholino units (morpholino nucleic acids) with a cyclic base. Several linking groups have been reported to link morpholino monomer units within morpholino nucleic acids. One class of groups is selected to provide non-ionic oligomeric compounds. The morpholino-based oligomeric compounds have undesirable interactions with cellular proteins. Morpholino-based polynucleotides are less likely to interact with cellular proteins in an undesirable manner. It is a non-ionic mimic of oligonucleotides that is less likely to form interactions (Dwai ne A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510). Mol Homologous polynucleotides are disclosed in U.S. Patent No. 5,034,506. Within the morpholino class of thiolamide, a variety of different linking groups are used to link the monomeric subunits. A variety of compounds have been prepared.

[0146] A further class of polynucleotide mimics is called cyclohexenyl nucleic acids (CeNA). The furanose ring normally present in DNA / RNA molecules is replaced with a cyclohexenyl ring. CeNA DMT-protected phosphoramidite monomers have been prepared and synthesized using classical phosphoramidites. It has been used in the synthesis of oligomeric compounds following pyramidite chemistry. eNA oligomeric compounds and oligonucleosides with specific moieties modified with CeNA The thiol amides have been prepared and studied (Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-86 02). Generally, the incorporation of CeNA monomers into DNA strands is essential for DNA / RNA hybridization. The CeNA oligoadenylates increase the stability of the lid, similar to the original complex. It forms stable complexes with RNA and DNA complements. Studies incorporating eNA structures have been performed using NMR and circular dichroism to initiate facile conformational adaptation. was shown by.

[0147] Further modifications include the attachment of a 2'-hydroxyl group to the 4' carbon atom of the sugar ring. A 2'-C,4'-C-oxymethylene bond is formed between the 2'-C,4'-C-hydroxymethylene bond to form a bicyclic sugar moiety. The linkage is between the 2' oxygen atom and the 2' nucleotide, where n is 1 or 2. and methylene (-CH2-), a group bridging the 4' carbon atom (Singh et al. , Chem. Commun., 1998, 4, 455-456). LNA and LNA analogs bind to complementary DNA and Highly stable double stranded structure with RNA (Tm = +3 to +10°C), 3'-exonuclease activity It exhibits stability against enzyme degradation and good solubility properties. Toxic antisense oligonucleotides have been described (Wahlestedt et al., Proc. N atl. Acad. Sci. USA, 2000, 97, 5633-5638).

[0148] LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and guanine The synthesis and preparation of uracils are described, along with their oligomerization and nucleic acid recognition properties. (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNA and its preparation is also described in WO 98 / 39352 and WO 99 / 14226.

[0149] Modified sugar moiety The subject nucleic acids may also contain one or more substituted sugar moieties. The alkyl, alkenyl and alkynyl are substituted or unsubstituted C1-C 10 Archi C2~C 10 may be alkenyl and alkynyl, OH; F; O-, S -, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl. and O((CH2)) where m is 1 to about 10. n O) m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2, and O(C H2) n ON((CH2) n CH3)2 is particularly suitable. Other suitable polynucleotides C is C1 to C 10 Lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkane Aryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino , polyalkylamino, substituted silyl, RNA cleaving group, reporter group, interfering substance, oligonucleic acid Groups for improving the pharmacokinetic properties of nucleotides or the pharmacodynamic properties of oligonucleotides Sugar substitutions selected from groups for enhancing properties and other substituents with similar properties Suitable modifications include 2'-methoxyethoxy (2'-O-CH2CH2OCH3 , also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Mart in et al., Helv. Chim. Acta, 1995, 78, 486-504), i.e., alkoxyalkoxy Further suitable modifications include the 2'-DMAOE group, as described in the Examples below. Also known as 2'-dimethylaminooxyethoxy, i.e., O(CH2) 2ON(CH3)2 group, and 2'-dimethylaminoethoxyethoxy (known in the art) as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE Also known as 2'-O-CH2-O-CH2-N(CH3)2. do.

[0150] Other suitable sugar substituents include methoxy (-O-CH), aminopropoxy (-OCH 2CH2CH2NH2), allyl (-CH2-CH=CH2), -O-allyl (--O- 2'-sugar substituents include arabino (-CH2-CH=CH2) and fluoro (F). It can be in the 'no' (arabino) (upper) position or the 'ribo' (lower) position. A suitable 2'-arabino modification is 2'-F. Similar modifications can be made to other oligomeric compounds. of sugars, particularly on the 3'-terminal nucleoside or within 2'-5' linked oligonucleotides. It can occur at the 3' position and also at the 5' position of the 5' terminal nucleotide. , sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0151] Base modifications and substitutions The subject nucleic acids are composed of nucleobases (often simply referred to in the art as "bases") As used herein, "unmodified" or "Natural" nucleobases include the purine bases adenine (A) and guanine (G), as well as the pi It contains the imidine bases thymine (T), cytosine (C) and uracil (U). The nucleobases identified include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine (5-me-C), adenine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and adenine and and other alkyl derivatives of guanine, 2-propyl and other alkyl derivatives of adenine and guanine alkyl derivatives, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halogen uracil and cytosine, 5-propynyl (-C=C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine , 5-uracil (pseudouracil), 4-thiouracil, 8- 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted substituted adenine and guanine, 5-halo, especially 5-bromo, 5-trifluoromethyl and Other 5-substituted uracils and cytosines, 7-methylguanines and 7-methyladenines, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-Deazaguanine and 7-Deazaadenine and 3-Deazaguanine and 3-Dea Other synthetic and natural nucleobases are included, such as zaadenine. Nucleobases include phenoxazine cytidine (1H-pyrimido(5,4-b)(1,4)benzoxazole). Zoxadin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4- b) (1,4) benzothiazin-2(3H)-one), G-shaped clamps, e.g., substituted phenanthroitin Noxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimidin (5,4-( b) (1,4) benzoxazin-2(3H)-one), carbazole cytidine (2H-pyridinone) Pyridoindole cytidine (H-pyridindole) (3',2':4,5)pyrrolo(2,3-d)pyrimidin-2-one, etc. Includes gin.

[0152] The heterocyclic base moiety is a heterocyclic base in which the purine or pyrimidine base is attached to another heterocycle, e.g., a 7-dea substituted with 7-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone Further nucleobases may include those described in U.S. Pat. Nucleic acid bases listed in issue 8, The Concise Encyclopedia Of Polymer Science And Engine ering, pages 858-859, Kroschwitz, JI, ed. John Wiley & Sons, 1990 Nucleobases, Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 6 13, and Sanghvi, YS, Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleobases are oligomeric compounds. These include 5-substituted pyrimidines, 6-substituted pyrimidines, pyrimidines and N-2, N-6 and O-6 substituted purines, such as 2-aminopropyl These include 5-methyl-5-propynyl-2-methyl-1-propynyl-2-methyl-1-propynyl-2-methyl-1-propynyl-1-methyl-1-propynyl-2-methyl-1 ... The cytosine substitution increases the duplex stability of nucleic acids by 0.6–1.2°C (Sanghvi et al. , eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 27 6-278), for example, when combining 2'-O-methoxyethyl sugar modifications, a suitable base It is shown to be a substitution.

[0153] combination Other possible modifications of the subject nucleic acids include those that alter the activity, cellular distribution or cellular uptake of the oligonucleotides. Chemical linkage of one or more moieties or conjugates to a polynucleotide that enhances incorporation These moieties or conjugates include functional groups such as primary or secondary hydroxyl groups. Binding groups include, but are not limited to, interfering substances, receptors, and the like. Super molecules, polyamines, polyamides, polyethylene glycols, polyethers, oligomers Examples of suitable groups include groups that enhance the pharmacodynamic properties of the dimer and groups that enhance the pharmacokinetic properties of the oligomer. Suitable binding groups include, but are not limited to, cholesterol, lipids, phospholipids, Biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluo These include methicone, rhodamine, coumarin, and dyes. Groups that enhance the pharmacodynamic properties The nucleotides may include groups that enhance uptake, groups that enhance resistance to degradation, and / or groups that enhance binding to the target nucleic acid. Groups that enhance sequence-specific hybridization are included. Groups include those that improve uptake, distribution, metabolism or excretion of the subject nucleic acids.

[0154] Binding moieties include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan e al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), thioethers, e.g., hexyl Sil-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 3 06-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), Chioko Lesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), aliphatic chain , e.g., dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; huk et al., Biochimie, 1993, 75, 49-54), phospholipids, e.g., di-hexadecyl-r ac-Glycerol or Triethylammonium 1,2-di-O-hexadecyl-r ac-Glycero-3-H-phosphonic acid (Manoharan et al., Tetrahedron Lett., 1995, 3 6, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), and polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 199 5, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1 995, 36, 3651-3654), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 199 5, 1264, 229-237), or octadecylamine or hexylamino-carbonyl- The oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923 -937).

[0155] The conjugates are "protein transduction domains" or PTDs (CPPs: cell-penetrating peptides). (also known as lipid bilayers, micelles, cell membranes, cells, etc.) Polypeptides, polynucleotides, and carbohydrates that facilitate crossing of the organelle membrane or vesicle membrane It can refer to compounds, or organic or inorganic compounds. PTDs bound to another molecule, which may range from a nanoparticle to a membrane, allow the molecule to cross the membrane. and facilitates migration, for example, from the extracellular space to the intracellular space or from the cytosol into organelles. In some embodiments, the PTD is a foreign polypeptide (e.g., a site-specific In some embodiments, the amino acid sequence is covalently linked to the amino terminus of the target polypeptide. In this case, the PTD is a protein that binds to the carbohydrate of a foreign polypeptide (e.g., a site-directed modified polypeptide). In some embodiments, the PTD is covalently linked to the xyl terminus of a nucleic acid ( For example, a DNA-targeting RNA, a polynucleotide encoding the DNA-targeting RNA, a site The modified polypeptide is covalently linked to a polynucleotide (e.g., a polynucleotide encoding the specific modified polypeptide). Examples of PTDs include, but are not limited to, minimal undecapeptide protein transduction domains. Residue 47 of HIV-1 TAT containing YGRKKRRQRRR (SEQ ID NO: 264) ~57); several arginines sufficient to direct entry into cells (e.g., Polyalcohols containing 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines ginine sequence; VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96 );Drosophila Antennapedia a) Protein transduction domain (Noguchi et al. (2003) Diabetes 52(7):1732-1737) ; Truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21:1248-1 256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97:13003-1300 8); RRQRRTSKLMKR (SEQ ID NO: 265); Transportan (Transp ortan) GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 26 6);KALAWEAKLAKALAKALAKHLAKALAKALKCEA(Sequence No. No. 267); and RQIKIWFQNRRMKWKK (SEQ ID NO: 268). Examples of PTDs include, but are not limited to, YGRKKRRQRRR (SEQ ID NO: 264) , RKKRRQRRR (SEQ ID NO: 269); 3 arginine residues to 50 arginines and arginine homopolymers of residues; examples of PTD domain amino acid sequences include: , including but not limited to: YGRKKRRQRRR (SEQ ID NO: 264); RKKRRQ RR (SEQ ID NO: 270); YARAAARQARA (SEQ ID NO: 271); THRLPRR RRRR (SEQ ID NO: 272); and GGRRARRRRRR (SEQ ID NO: 273) In some embodiments, the PTD is an activatable CPP (ACP P) (Aguilera et al. (2009) Integr Biol (Camb) June; 1(5-6): 371-381). ACPPs can be attached to suitable polyanions (e.g., Glu9 or and polycationic CPPs (e.g., Arg9 or "R9") linked to a CPP (e.g., Arg9 or "E9"). This reduces the net charge to nearly zero, preventing adhesion and uptake by cells. Cleavage of the linker releases the polyanion, which then binds to polyarginine and its anchoring agent. The membrane-crossing of ACPP is "activated" by the localized exposure of the adhesive properties of the target cells.

[0156] Exemplary DNA-targeting RNAs In some embodiments, a suitable DNA-targeting RNA comprises two separate RNA polymerases. The first of two separate RNA polynucleotide molecules (activated RNA) NA) is any one of the nucleotide sequences set forth in SEQ ID NOs: 431 to 562. or its complement over a stretch of at least 8 contiguous nucleotides at least about 60%, at least about 65%, at least about 70%, at least about 75%, At least about 80%, at least about 85%, at least about 90%, at least about 95%, have at least about 98%, at least about 99%, or 100% nucleotide sequence identity The second of the two separate RNA polynucleotide molecules (target The modified RNA may be any of the nucleotide sequences set forth in SEQ ID NOs: 563 to 679. or its complement over a stretch of at least 8 consecutive nucleotides , at least about 60%, at least about 65%, at least about 70%, at least about 75% , at least about 80%, at least about 85%, at least about 90%, at least about 95% , at least about 98%, at least about 99%, or 100% nucleotide sequence identity The nucleotide sequence comprises:

[0157] In some embodiments, a suitable DNA-targeting RNA is a single RNA polynucleotide. nucleotide sequence of any of SEQ ID NOs: 431 to 562. for at least about 60 over a stretch of at least 8 contiguous nucleotides %, at least about 65%, at least about 70%, at least about 75%, at least about 80 %, at least about 85%, at least about 90%, at least about 95%, at least about 98% %, at least about 99%, or 100% nucleotide sequence identity with a first nucleotide and any one of the nucleotide sequences set forth in SEQ ID NOs: 463 to 679. at least about 60% over a stretch of at least 8 consecutive nucleotides , at least about 65%, at least about 70%, at least about 75%, at least about 80% , at least about 85%, at least about 90%, at least about 95%, at least about 98% , a second nucleotide sequence having at least about 99%, or 100% nucleotide sequence identity It contains a nucleotide sequence.

[0158] In some embodiments, the DNA-targeting RNA is a double-molecule DNA-targeting RNA. The targeting RNA is linked at its 5' end to a nucleotide sequence complementary to the target DNA. The sequence 5'GUUUUAGAGCUA-3' (SEQ ID NO: 679) is included in some experiments. In an embodiment, the DNA-targeting RNA is a double-molecule DNA-targeting RNA, and the activated RNA A is the sequence 5'UAGCAAGUUAAAAUAAGGCUAGUCCG-3' (SEQ ID NO: Includes the number / / .

[0159] In some embodiments, the DNA-targeting RNA is a single-molecule DNA-targeting RNA. and a sequence 5'-G linked at its 5' end to a nucleotide strand complementary to the target DNA. UUUUAGAGCUA-LINKER-UAGCAAGUUAAAAUAAGGCUAGU CCG-3' (wherein "linker" can comprise any nucleotide sequence). (SEQ ID NO: / / ) Other exemplary single molecule D NA-targeting RNAs include those set forth in SEQ ID NOs: 680-682.

[0160] A subject DNA-targeting RNA and / or a subject site-specific modifying polypeptide are encoded by the subject DNA-targeting RNA and / or the subject site-specific modifying polypeptide. Nucleic acid The present disclosure provides a method for producing a subject DNA-targeting RNA and / or a subject site-specifically modified polypeptide. In some embodiments, a nucleic acid comprising a nucleotide sequence encoding the Nucleic acids encoding the subject DNA-targeting RNAs can be expressed in expression vectors, e.g., recombinant expression vectors. He is a ctor.

[0161] In some embodiments, the subject methods include converting the target DNA into a DNA-targeting RNA and and / or one or more nucleotide sequences encoding the site-directed modifying polypeptide. contacting a cell (or a population of cells) with a nucleic acid or nucleic acids, or administering a DNA-targeting RNA to the cell (or a population of cells) and / or one containing a nucleotide sequence encoding a site-directed modifying polypeptide. In some embodiments, the target D The cells containing the DNA are in vitro. In some embodiments, the cells contain target DNA. The cells containing the DNA-targeting RNA and / or site-specifically modified polypeptides are present in vivo. Suitable nucleic acids comprising a nucleotide sequence encoding a peptide include expression vectors, and a nucleic acid encoding a DNA-targeting RNA and / or a site-specific modifying polypeptide. An expression vector containing an octide sequence is a "recombinant expression vector."

[0162] In some embodiments, the recombinant expression vector is a viral construct, e.g., recombinant adeno-associated virus constructs (see, e.g., U.S. Patent No. 7,078,387); Recombinant adenovirus constructs, recombinant lentivirus constructs, recombinant retrovirus constructs Things etc.

[0163] Suitable expression vectors include, but are not limited to, viral vectors (e.g., vaccinia vectors). Viral vectors based on rhesus monkey viruses; poliovirus; adenovirus (e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6 :515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gen e Ther 5:1088 1097, 1999; WO 94 / 12649, WO 93 / 03769; No. 93 / 19191; No. 94 / 28938; No. 95 / 11984 and No. 95 / 00655); adeno-associated viruses (see, e.g., Ali et al., Hum Gene Ther r 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Inves t Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997 , Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:59 1 594, 1996; Srivastava, Samulski et al., J. Vir. in WO 93 / 09239. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus Viruses that cause epidemic deficiency (e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al. , J Virol 73:7812 7816, 1999); retroviral vectors (e.g., mouse leukemia vectors) Disease virus, spleen necrosis virus, and Rous sarcoma virus, Harvey sarcoma virus, avian Leukemia viruses, lentiviruses, human immunodeficiency viruses, myeloproliferative sarcoma viruses (m erythroproliferative sarcoma virus, and breast cancer virus vectors derived from retroviruses such as rabies);

[0164] Large numbers of suitable expression vectors are known to those of skill in the art, and many are commercially available. The following vectors are described: eukaryotic host cells: pXT1, pSG5 (Stratagene) (Stratagene), pSVK3, pBPV, pMSG, and pSVLSV4 0 (Pharmacia). However, as long as it is compatible with the host cell, Any other vector may be used.

[0165] Depending on the host / vector system used, several appropriate transcriptional and translational regulatory regions may be used. Any of the regions, e.g., constitutive and inducible promoters, transcription enhancers, Ser elements, transcription terminators, etc. may be used in the expression vector (e.g., Bitter (See et al. (1987) Methods in Enzymology, 153:516-544).

[0166] In some embodiments, the DNA-targeting RNA and / or the site-specifically modified polynucleotide The nucleotide sequence encoding the peptide may be linked to a transcriptional regulatory region, such as a promoter. The transcriptional regulatory region is operably linked to a transcriptional regulatory region. or prokaryotic cells (e.g., bacterial or archaeal cells). In some embodiments, the DNA-targeting RNA and / or the site-specific modifying polypeptide The nucleotide sequence encoding the nucleotide sequence is a DNA targeting sequence in both prokaryotic and eukaryotic cells. Expression of Nucleotide Sequences Encoding RNA and / or Site-Directly Modified Polypeptides The gene is operably linked to a plurality of regulatory regions that enable

[0167] Examples of suitable eukaryotic promoters (promoters functional in eukaryotic cells) include: , including but not limited to, cytomegalovirus (CMV) immediate early stage, herpes simplex virus (HSV) V) Thymidine kinase, early and late SV40, retrovirus-derived long terminal repeat sequences ( LTR), and eukaryotic promoters derived from mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the skill of the art. Expression vectors also contain a ribosome binding site for translation initiation and a The expression vector may contain a transcription terminator. The expression vector may contain an appropriate sequence. Conjugated protein tags (e.g., 6xHis tag, hemagglutinin tag, green fluorescent protein and a nucleotide sequence encoding a chimeric polypeptide, such as a polypeptide of the present invention. That's fine.

[0168] In some embodiments, the DNA-targeting RNA and / or the site-specifically modified polynucleotide The nucleotide sequence encoding the peptide is operably linked to an inducible promoter. In some embodiments, the DNA-targeting RNA and / or the site-specific modifying polymerase The nucleotide sequence encoding the polypeptide is operably linked to a constitutive promoter. There are.

[0169] Methods for introducing nucleic acids into host cells are known in the art and may be any known method. Any method can be used to introduce a nucleic acid (e.g., an expression construct) into a cell. Examples of such methods include viral or bacteriophage infection, transfection, , conjugation, protoplast fusion, lipofection, electroporation ion, calcium phosphate precipitation, and polyethyleneimine (PEI)-mediated transfection transfection, DEAE-dextran mediated transfection, liposome-mediated transfection Infection, particle gun method, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated conventional nucleic acid delivery (e.g. Panyam et., al Adv Drug Deliv Rev. 2012 Sep 13. pii: S0169 -409X(12)00283-9. doi: 10.1016 / j.addr.2012.09.023).

[0170] Chimeric Polypeptides The present disclosure provides chimeric site-directed modified polypeptides. The polypeptide interacts with (eg, binds to) a subject DNA-targeting RNA (described above). The DNA-targeting RNA targets the chimeric site-specific modified polypeptide to a target DNA (e.g., a dye). Chromosomal sequences or extrachromosomal sequences, e.g., episomal sequences, minicircle sequences, mitochondrial sequences The subject chimeric site-specific modified polypeptides are directed to target sequences within the target sequence (e.g., chromatin sequences, chloroplast sequences, etc.). The peptide modifies the target DNA (e.g., cleavage or methylation of the target DNA), and / or or modifying polypeptides bound to target DNA (e.g., methylation of histone tails) or acetylation).

[0171] The subject chimeric site-directed modifying polypeptides modify target DNA (e.g., target DNA cleavage or methylation of target DNA) and / or modifying polypeptides bound to target DNA (e.g., methylation or acetylation of histone tails). Chimeric site-specific modified polypeptides The peptide is referred to as a "chimeric site-specific polypeptide" or a "chimeric RNA-binding site-specific modification polypeptide." It is also called a "lipeptide."

[0172] The subject chimeric site-directed modified polypeptides comprise two portions, an RNA binding site and an activity The subject chimeric site-directed modified polypeptides comprise at least two different polypeptides. The subject chimeric site-directed modified polypeptides comprise an amino acid sequence derived from a peptide comprising: Modified and / or naturally occurring polypeptide sequences (e.g., modified or unmodified) a first amino acid sequence derived from the modified Cas9 / Csn1 protein; and a Cas9 / Csn The second amino acid sequence may be a second amino acid sequence other than the first protein.

[0173] RNA binding site In some instances, the RNA-binding site of a subject chimeric site-directed modified polypeptide is In another example, the subject chimeric site-directed modified polypeptides The RNA binding site of the polypeptide is not a naturally occurring molecule (it is modified, e.g. Naturally occurring RNA binding sites of interest can be identified by any of the methods known in the art. For example, SEQ ID NOs: 1 to 256 and 795 to 1000 are derived from site-directed modified polypeptides. 1346 is a naturally occurring Cas polypeptide that can be used as a site-specific modifying polypeptide. This provides a non-limiting and non-exhaustive list of 9 / Csn1 endonucleases. In an example, the RNA binding site of a subject chimeric site-directed modifying polypeptide is SEQ ID NO:1 A polynucleotide having either of the amino acid sequences described as 795-1346 and 795-256. At least about 75%, at least about 80%, or at least about 100% of the peptide's RNA binding site At least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least Both contain amino acid sequences with approximately 99% or 100% amino acid sequence identity.

[0174] In some instances, the site-directed modifying polypeptide is a Cas9 / C polypeptide, as shown in FIG. to amino acids 7 to 166 or 731 to 1003 of the sn1 amino acid sequence, or Any of the amino acid sequences set forth as numbers 1 to 256 and 795 to 1346 At least about 75%, at least about 80%, at least about 8% of the corresponding portion in 5%, at least about 90%, at least about 95%, at least about 99%, or 100% and amino acid sequences having an amino acid sequence identity of

[0175] active site In addition to the RNA binding site, the chimeric site-directed modifying polypeptide contains an "active site." In some embodiments, the active site of a subject chimeric site-directed modifying polypeptide comprises: Natural site-specific modification of polypeptides (e.g., Cas9 / Csn1 endonucleases) In another embodiment, the subject chimeric site-directed modifying polypeptide comprises a generative active site. The active site of the site-directed modified polypeptide is a modified amino acid of the naturally occurring active site of the site-directed modified polypeptide. The naturally occurring active site of interest may be any of the amino acid sequences (e.g., substitutions, deletions, insertions) that are known in the art. It is derived from site-directed modified polypeptides known in the art, for example, SEQ ID NOs: 1 to 25 6 and 795-1346 are autologous polypeptides that can be used as site-directed modifying polypeptides. Provides a non-exhaustive and non-limiting list of naturally occurring Cas9 / Csn1 endonucleases The active sites of the subject chimeric site-directed modified polypeptides are variable and can be modified as described herein. It may contain any heterologous polypeptide sequence that may be useful in the indicated methods.

[0176] In some embodiments, a subject chimeric site-directed modifying polypeptide comprises (i) a target interacts with DNA-targeting RNA containing a nucleotide sequence complementary to a sequence within the target DNA and (ii) an active site that exhibits site-specific enzymatic activity (e.g., a DNA A methylation activity, DNA cleavage activity, histone acetylation activity, histone methylation activity, etc.) and the site of enzymatic activity is determined by the DNA-targeting RNA.

[0177] In other embodiments, the subject chimeric site-directed modifying polypeptides are capable of (i) targeting the target DNA. RNA that interacts with a DNA-targeting RNA containing a nucleotide sequence complementary to a sequence of (ii) an active site that regulates transcription within the target DNA (e.g., increases transcription) or to reduce), and the site of regulated transcription within the target DNA is a DNA targeting Determined by RNA.

[0178] In some instances, the active site of a subject chimeric site-directed modifying polypeptide is a target Enzymatic activity that modifies DNA (e.g., nuclease activity, methyltransferase activity) , demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase Enzyme activity, alkylating activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, enzyme activity, Tegulases, transposases, recombinases, polymerases, ligases The enzyme has the activity of glycans (glycosylase, helicase, photolyase or glycosylase).

[0179] In other examples, the active site of a subject chimeric site-directed modifying polypeptide binds to a target DNA Enzymatic activity that modifies polypeptides (e.g., histones) bound to the ATP (e.g., methyltransferases) acetyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity ubiquitin ligase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity Desinolation activity, adenylation activity, deadenylation activity, sumoylation activity, desumoylation activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity) It has.

[0180] In some instances, the active site of a subject chimeric site-directed modifying polypeptide comprises an enzyme In another example, the activity of a subject chimeric site-directed modified polypeptide is The site regulates transcription of the target DNA (see above). The active site of the polypeptide is variable and can be any heterologous polypeptide that may be useful in the methods disclosed herein. It may also contain a polypeptide sequence.

[0181] Exemplary Chimeric Site-Directly Modified Polypeptides In some embodiments, the active site of the chimeric site-directed modifying polypeptide is Ca In some instances, the Cas9 / Csn1 protein may be a modified form of the Cas9 / Csn1 protein. Modified forms of the Cas9 / Csn1 protein mimic the naturally occurring nuclease activity of the Cas9 / Csn1 protein. These include amino acid changes (e.g., deletions, insertions, or substitutions) that reduce activity. In some instances, modified forms of the Cas9 / Csn1 protein are capable of activating the corresponding wild-type Ca Less than 50%, less than 40%, less than 30%, less than 20%, and less than 10% of the s9 / Csn1 polypeptide %, less than 5%, or less than 1% of the nuclease activity. The modified form of the Cas9 / Csn1 polypeptide does not have substantial nuclease activity. .

[0182] In some embodiments, the modified form of the Cas9 / Csn1 polypeptide is capable of targeting a target DNA. A can cleave the complementary strand of A, but has a reduced ability to cleave the non-complementary strand of the target DNA. D10A (aspartic acid to alanine at amino acid position 10 of SEQ ID NO:8) Mutations (or proteins shown in SEQ ID NOS: 1 to 256 and 795 to 1346) or any corresponding mutation) (see Figure 11). Modified forms of the 9 / Csn1 polypeptide are capable of cleaving the non-complementary strand of target DNA However, H840A (amino acid position 84) has a reduced ability to cleave the complementary strand of the target DNA. 0) mutation from histidine to alanine (or SEQ ID NOS: 1-256 and 795-1 346) (see Figure 11). In some embodiments, the modified form of the Cas9 / Csn1 polypeptide is The ability of the polypeptide to cleave both complementary and non-complementary strands of target DNA is reduced. The D10A and H840A mutations (or SEQ ID NOs: 1-256 and 795-134) 6) and the corresponding mutation in any of the proteins described as other Mutating residues can be used to achieve the above effects (i.e., to enhance the activity of one or other nuclease moieties). Non-limiting examples include residues D10, G12, G17, E 762, H840, N854, N863, H982, H983, A984, D986, and and / or A987 (or as set forth as SEQ ID NOs: 1-256 and 795-1346) The corresponding mutation in one of the proteins is then altered (i.e., substituted) (For more information on the conservation of Cas9 amino acid residues, see Figure 3, Figure 5, (See Figure 11A, and Table 1.) Mutations other than alanine substitutions are also suitable.

[0183] For more information on the purpose Table 1. Table 1 lists four motifs present in Cas9 sequences from various species. (See also Figures 3 and 5.) The amino acids listed in the table are from C as9 (SEQ ID NO: 8). [Table 1]

[0184] In some instances, the chimeric site-directed modifying polypeptide is a Cas polypeptide shown in FIG. 9 / Csn1 amino acid sequence to amino acids 7-166 or 731-1003, and Any of the amino acid sequences set forth as SEQ ID NOs: 1 to 256 and 795 to 1346 at least about 75%, at least about 80%, at least at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 10% In some instances, chimeric amino acids include amino acid sequences that share 0% amino acid sequence identity. The site-specifically modified polypeptides were constructed using four motifs (listed in Table 4 and shown in Figures 3A and 5). Each of the four motifs listed in Table 1 (SEQ ID NOs: 260-262) 63) or as SEQ ID NOs: 1 to 256 and 795 to 1346 At least about 75%, at least about 75% of the corresponding portion of any of the amino acid sequences described At least about 80%, at least about 85%, at least about 90%, at least about 95%, They have amino acid sequences that have at least about 99% or 100% amino acid sequence identity. In some instances, the chimeric site-directed modifying polypeptide is a Cas9 polypeptide shown in FIG. / to amino acids 7 to 166 or 731 to 1003 of the Csn1 amino acid sequence, or Any of the amino acid sequences set forth as SEQ ID NOs: 1 to 256 and 795 to 1346 at least about 75%, at least about 80%, at least About 85%, at least about 90%, at least about 95%, at least about 99% or 100 % amino acid sequence identity.

[0185] In some embodiments, the active site of the site-directed modifying polypeptide is a DNA modification and / or transcription factor activity and / or DNA-binding polypeptide modifying activity. In some instances, the heterologous polypeptide comprises a nuclease. In another embodiment, the Cas9 / Csn1 polypeptide is substituted at a position that confers activity. The subject site-specific modifying polypeptides are Cas9 / Cas9 polypeptides that normally confer nuclease activity. A portion of a Csn1 polypeptide (and a fully active, or alternatively, a corresponding wild-type active, of a Cas9 / Csn1 polypeptide, which may be modified to have less than 100% of the nucleotide sequence. In other words, in some instances, The subject chimeric site-directed modifying polypeptides normally confer nuclease activity to Cas9. / a fusion polypeptide comprising both a portion of a Csn1 polypeptide and a heterologous polypeptide In other examples, the subject chimeric site-directed modifying polypeptides are Cas9 / Csn 1. Modified variants (e.g., amino acid changes, deletions, insertions) and variants of the active site of a polypeptide. In yet another example, the subject chimeric portion is a fusion polypeptide comprising a species polypeptide. Heterologous modified polypeptides include heterologous polypeptides and naturally occurring or modified site-specific polypeptides. It is a fusion polypeptide containing an RNA-binding site of a heterologously modified polypeptide.

[0186] For example, in a chimeric Cas9 / Csn1 protein, naturally occurring (or modified ( For example, a bacterial Cas9 / Csn1 polypeptide that has been mutated, deleted, or inserted can be used in combination with a heterologous polypeptide. Peptide sequences (i.e., polypeptide sequences derived from proteins other than Cas9 / Csn1) or a polypeptide sequence derived from another organism). Columns indicate activities also exhibited by the chimeric Cas9 / Csn1 protein (e.g., enzymatic activity). ) (e.g., methyltransferase activity, acetyltransferase activity) activity, kinase activity, ubiquitination activity, etc.) by linking the heterologous nucleic acid sequence to another nucleic acid sequence (e.g. a chimeric nucleotide sequence encoding the chimeric polypeptide (e.g., by genetic engineering) In some embodiments, chimeric Cas9 / Csn1 polypeptides may be generated. The promoter may be a Cas9 / Csn1 polypeptide (e.g., wild-type Cas9 or a Cas9 mutant). For example, Cas9 with reduced or inactivated nuclease activity can be used to localize the cells intracellularly. Heterologous sequences that can be used in the targeting of the nucleus (e.g., nuclear localization signals (NLS) for targeting to the nucleus; mitochondrial sequences Mitochondrial localization signal for targeting to the mitochondrion; chloroplast for targeting to the chloroplast These are produced by fusing them with a target signal (ER localization signal; ER retention signal; etc.). In this embodiment, the heterologous sequence may provide a tag to facilitate tracking or purification ( For example, fluorescent proteins, such as green fluorescent protein (GFP), YFP, RFP, C FP, mCherry, tdTomato, etc.; His tag, e.g., 6XHis tag; Red Hemagglutinin (HA) tag; FLAG tag; Myc tag; etc. In some embodiments, Thus, the heterologous sequence may confer increased or decreased stability. The seed sequence may be used to identify a target protein (e.g., a target protein) that is likely to be present in the chimeric Cas9 polypeptide. A or histone modifying proteins, transcription factors or transcription repressors, recruitment ing) a binding domain (to confer the ability to bind to proteins, etc.) may be provided.

[0187] A variety of additional suitable fusion partners for the subject mutant Cas9 site-directed polypeptides. Examples of (or fragments thereof) include, but are not limited to, those listed in Figure 54. .

[0188] Nucleic acids encoding the subject chimeric site-directed modified polypeptides The present disclosure provides nucleotide sequences encoding the subject chimeric site-directed modifying polypeptides. In some embodiments, the subject chimeric site-directed modified polypeptides are provided. The nucleic acid comprising a nucleotide sequence encoding the peptide can be incorporated into an expression vector, e.g., a recombinant expression vector. This is the current vector.

[0189] In some embodiments, the subject method comprises converting the target DNA into a chimeric site-specific modified polynucleotide. contacting the cell (or cell population) with one or more nucleic acids containing the peptide; introducing into the host one or more nucleic acids comprising a chimeric site-directed modifying polypeptide. Suitable nucleic acids comprising nucleotide sequences encoding chimeric site-directed modifying polypeptides. The chimeric site-directed modifying polypeptide is encoded by an expression vector. An expression vector containing the nucleotide sequence is a "recombinant expression vector."

[0190] In some embodiments, the recombinant expression vector is a viral construct, e.g., Recombinant adeno-associated virus constructs (see, e.g., U.S. Patent No. 7,078,387) , recombinant adenoviral constructs, recombinant lentiviral constructs, etc.

[0191] Suitable expression vectors include, but are not limited to, viral vectors (e.g., vaccinia vectors). Viral vectors based on rhesus monkey viruses; poliovirus; adenovirus (e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6 :515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gen e Ther 5:1088 1097, 1999; WO 94 / 12649, WO 93 / 03769; No. 93 / 19191; No. 94 / 28938; No. 95 / 11984 and No. 95 / 00655); adeno-associated viruses (see, e.g., Ali et al., Hum Gene Ther r 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Inves t Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997 , Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:59 1 594, 1996; Srivastava, Samulski et al., J. Vir. in WO 93 / 09239. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus Viruses that cause epidemic deficiency (e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al. , J Virol 73:7812 7816, 1999); retroviral vectors (e.g., mouse leukemia vectors) Disease virus, spleen necrosis virus, and Rous sarcoma virus, Harvey sarcoma virus, avian Leukemia viruses, lentiviruses, human immunodeficiency viruses, myeloproliferative sarcoma viruses (m erythroproliferative sarcoma virus, and breast cancer virus vectors derived from retroviruses such as rabies);

[0192] Large numbers of suitable expression vectors are known to those of skill in the art, and many are commercially available. The following vectors are described: eukaryotic host cells: pXT1, pSG5 (Stratagene) ), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). However, any other vector may be used so long as it is compatible with the host cell.

[0193] Depending on the host / vector system used, several appropriate transcriptional and translational regulatory regions may be used. Any of the regions, e.g., constitutive and inducible promoters, transcription enhancers, Ser elements, transcription terminators, etc. may be used in the expression vector (e.g., Bitter (See et al. (1987) Methods in Enzymology, 153:516-544).

[0194] In some embodiments, the nucleic acid encoding the chimeric site-directed modifying polypeptide The nucleotide sequence is operably linked to a regulatory region, for example, a transcription regulatory region such as a promoter. The transcriptional regulatory region may be present in eukaryotic cells, e.g., mammalian cells; or in prokaryotic cells, e.g., bacterial cells. In some embodiments, the chimeric antigen receptor may be functional in a bacterial cell (e.g., a bacterial cell or an archaeal cell). Nucleotide sequences encoding site-specifically modified polypeptides can be used in prokaryotic and eukaryotic cells. Expression of nucleotide sequences encoding chimeric site-directed modified polypeptides in both cells. The gene is operably linked to multiple regulatory regions that enable expression of the gene.

[0195] Examples of suitable eukaryotic promoters (promoters functional in eukaryotic cells) include: , including but not limited to, cytomegalovirus (CMV) immediate early stage, herpes simplex virus (HSV) V) Thymidine kinase, early and late SV40, retrovirus-derived long terminal repeat sequences ( LTR), and eukaryotic promoters derived from mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the skill of the art. Expression vectors also contain a ribosome binding site for translation initiation and a The expression vector may contain a transcription terminator. The expression vector may contain an appropriate sequence. Protein tags fused to the nucleotides (e.g., 6xHis tag, hemagglutinin (HA) tag, fluorescent Nucleotides encoding proteins (e.g., green fluorescent protein, yellow fluorescent protein, etc.) It may also contain a nucleotide sequence.

[0196] In some embodiments, the nucleic acid encoding the chimeric site-directed modifying polypeptide The nucleotide sequence may be an inducible promoter (e.g., a heat shock promoter, a tetracycline promoter, etc.). steroid-regulated promoters, metal-regulated promoters, estrogen-regulated promoters In some embodiments, the promoter is operably linked to a promoter (e.g., a promoter regulated by an agonist receptor). The nucleotide sequence encoding the chimeric site-directed modifying polypeptide is spatially restricted and and / or time-restricted promoters (e.g., tissue-specific promoters, cell type-specific promoters) In some embodiments, the chimeric site is operably linked to a promoter. The nucleotide sequence encoding the heterologously modified polypeptide is operable with a constitutive promoter. is linked to.

[0197] Methods for introducing nucleic acids into host cells are known in the art and may be any known method. The method can also be used to introduce nucleic acids (e.g., expression constructs) into stem or progenitor cells. Suitable methods include, for example, viral or bacteriophage infection. , transfection, conjugation, protoplast fusion, lipofection ion, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI) DEAE-dextran mediated transfection, transfection, particle gun method, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery (e.g., Panyam et., al Adv Drug Deliv Rev. 2 012 Sep 13. pii: S0169-409X(12)00283-9. doi: 10.1016 / j.addr.2012.09.023) etc.

[0198] method The present disclosure provides methods for modifying target DNA and / or target DNA-binding polypeptides. Generally, the subject method involves combining a target DNA with a DNA-targeting RNA and a site-specific with a complex (a "targeting complex") that includes the modified polypeptide.

[0199] As previously described, the subject DNA-targeting RNAs and subject site-specific modifying polypeptides can be prepared by: The DNA-targeting RNA binds to a nucleotide sequence complementary to the target DNA. The inclusion of a sequence confers target specificity to the conjugate. In some embodiments, the subject conjugates are targeted It modifies DNA, for example, DNA breakage, DNA methylation, DNA damage, and DNA repair. In other embodiments, the subject complexes comprise a target polypeptide (e.g., a target polypeptide) bound to a target DNA. For example, histones, DNA-binding proteins, etc.) are modified, e.g., histone methylation, histone This results in histone acetylation, histone ubiquitination, etc. The target DNA is then naked DNA in vitro, chromosomal DNA in cells in vitro, and cellular DNA in vivo It may be chromosomal DNA within a cell.

[0200] In some instances, the site-specific modifying polypeptide is a DNA-targeting RNA and a target Cleave the target DNA at the target DNA sequence determined by the complementary region between the target DNA In some instances, the site-specifically modified polypeptide exhibits nuclease activity. In the case of as9 or a Cas9-related polypeptide, site-specific cleavage of the target DNA is (i) base-pairing complementarity between the DNA-targeting RNA and the target DNA; and (ii) target D Both short motifs within the NA (called protospacer adjacent motifs (PAMs)) In some embodiments (e.g., S. piogeoides When Cas9 derived from Ness or a related Cas9 is used (SEQ ID NOs: 1 to 256 and 795-1346), and the PAM sequence of the non-complementary strand is 5'-XGG-3', where X is any DNA nucleotide and X is any target sequence on the non-complementary strand of the target DNA. The PAM sequence of the complementary strand is 5'-CCY-3'. where Y is any DNA nucleotide and Y is the target sequence of the complementary strand of the target DNA. The PAM on the non-complementary strand is 5'-GGG-3' and the PAM on the complementary strand is 5'-GGG-3'. 5'-CCC-3' (see Figure 10). In some such embodiments, X and Y can be complementary, and the XY base pair can be any base pair (e.g., X =C and Y=G;X=G and Y=C;X=A and Y=T, X=T and Y=A).

[0201] In some instances, different Cas9 proteins (i.e., Cs from various species) are used. as9 protein) takes full advantage of the various enzymatic characteristics of different Cas9 proteins Therefore, it may be advantageous to use it in various provided methods (e.g., different P due to AM sequence preference; due to increased or decreased enzyme activity; due to increased or decreased cytotoxicity levels due to the decrease in NHEJ, homology-directed repair, single-strand breaks, double-strand breaks, etc. Cas9 proteins from various species (SEQ ID NOS: 1-256 and 7) 95-1346) may require different PAM sequences in the target DNA. For a given optimal Cas9 protein, the PAM sequence requirement is the 5'-XGG-3 ' may differ from the array.

[0202] Although many Cas9 orthologs from a wide variety of species have been identified herein, The proteins share only a few identical amino acids. The as9 orthologues contain a central HNH endonuclease domain and a split Ruv It has the same domain structure as the C / RNaseH domain (Figures 3A and 3B, Figure 5, (See Table 1 and Table 1). The Cas9 protein shares a conserved structure and four key motifs. Motifs 1, 2, and 4 are RuvC-like motifs, while motif 3 is an HNH motif. In some instances, a suitable site-directed modifying polypeptide is It contains an amino acid sequence having four motifs, each of which is a motif 1 to 4 shown in FIG. 3A. Motifs 1 to 4 of the Cas9 / Csn1 amino acid sequence (SEQ ID NOs: 260 to 269, respectively) are 263, as shown in Table 1), or SEQ ID NOs: 1 to 256 and 795 to 134 6. The corresponding portion of any of the amino acid sequences set forth in 6 (divergent Cas9 sequences) For the alignment of motifs 1-4 from the nucleotide sequence (see Figure 5), 5%, at least about 80%, at least about 85%, at least about 90%, at least about 9 5%, at least about 99% or 100% amino acid sequence identity. In one example, a suitable site-directed modifying polypeptide is the Cas9 / Csn polypeptide shown in FIG. For amino acids 7 to 166 or 731 to 1003 of one amino acid sequence, or SEQ ID NO: In either of the amino acid sequences described as 1 to 256 and 795 to 1346 At least about 75%, at least about 80%, at least about 85% of the corresponding portion in , at least about 90%, at least about 95%, at least about 99%, or 100% of the mesh Any of the above Cas9 proteins may contain amino acid sequences that share amino acid sequence identity with the corresponding Cas9 protein. As a site-specifically modified polypeptide or as a chimeric site-specifically modified polypeptide of the subject method It can be used as part of

[0203] The nuclease activity cleaves the target DNA to generate double-strand breaks. These breaks then by cells in one of two ways: non-homologous end joining and homology-directed repair. In non-homologous end joining (NHEJ), the double-strand break is repaired by The new nucleic acid material is repaired by direct ligation to the new site. No insertion occurs, although some nucleic acid material may be lost, resulting in a deletion. In DNA repair, a donor polynucleotide with homology to the cleaved target DNA sequence is inserted. The donor polynucleotide is used as a template to repair the cut target DNA sequence. This results in the transfer of genetic information from the nucleic acid to the target DNA. In some instances, the target DNA may be inserted / replicated into the subject donor polynucleotide. In some instances, the subject donor polynucleotide is contacted with a subject Modification of the target DNA by NHEJ and / or homology-directed repair is For example, gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, It causes gene disruption, gene mutations, etc.

[0204] Therefore, by using DNA cleavage with site-specific modifying polypeptides, target DNA The sequence is excised and expressed in cells in the absence of exogenously supplied donor polynucleotide. By repairing the sequence, nucleic acid material is removed from the target DNA sequence (e.g., by infecting cells). Genes that predispose T cells to infection (e.g., CCR5 or CXCR4, which predispose T cells to infection by HIV) 4 gene) to remove the triplet repeat sequence that causes the disease in neurons. As mentioned above, gene knockout and gene mutation have been used as disease models in research. Thus, using the subject method, genes can be knocked out. quenching (resulting in a complete absence of transcription or altered transcription), or within the target DNA Genetic material can be knocked in at the optimal locus.

[0205] Alternatively, the DNA-targeting RNA and the site-specific modifying polypeptide may be used to target the target DNA sequence. a donor polynucleotide sequence comprising at least one segment having homology to When co-administered to a cell, the subject method allows the nucleic acid molecules to target DNA sequences. Addition (i.e., insertion or substitution) of a target gene (e.g., a protein, siRNA, miRNA, etc.) "Knock-in" the encoding nucleic acid), tag (e.g., 6xHis), fluorescent protein (e.g., For example, green fluorescent protein, yellow fluorescent protein, etc.), hemagglutinin (HA), FLAG, etc. ) and add regulatory sequences to the gene (e.g., promoters, polyadenylation signals, Internal ribosome entry sequence (IRES), 2A peptide, start codon, stop codon, supramolecular structure It is possible to add a nucleic acid sequence (e.g., a nucleic acid sequence signal, a localization signal, etc.), modify the nucleic acid sequence (e.g., introduce a mutation), etc. Therefore, a complex comprising a DNA-targeting RNA and a site-specific modifying polypeptide are used in, for example, gene therapy (e.g., to treat a disease), or antiviral therapy, anti-disease as a therapeutic or anti-cancer treatment), the creation of genetically modified organisms in agriculture, therapy, and diagnosis or large-scale production of proteins from cells for research purposes, induction of iPS cells, and biological site-specific gene deletion or replacement, used in biological studies, and targeted deletion or replacement of pathogen genes or "targeted" methods (e.g., gene knockout, gene knockin, genetic Any in vitro or in vivo method in which it is desired to modify DNA (e.g., gene editing, gene tagging, etc.) is also useful in in vivo applications.

[0206] In some embodiments, the site-directed modifying polypeptide is a Cas9 / Csn1 polypeptide. In some instances, modifications of the Cas9 / Csn1 protein Decorated forms reduce the spontaneous nuclease activity of the Cas9 / Csn1 protein This includes amino acid changes (e.g., deletions, insertions, or substitutions). Therefore, modified forms of Cas9 / Csn1 proteins are more efficient than the corresponding wild-type Cas9 / Csn1 proteins. Less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% of the peptide or less than 1% of the nuclease activity. Modified forms of the n1 polypeptide do not have substantial nuclease activity. The modified polypeptide is a Cas9 / Csn1 polypeptide that does not have substantial nuclease activity. When the peptide is a modified form of the peptide, it may be referred to as "dCas9."

[0207] In some embodiments, the modified form of the Cas9 / Csn1 polypeptide is a target D It is capable of cleaving the complementary strand of DNA but has a reduced ability to cleave the non-complementary strand of the target DNA. (thus resulting in a single-strand break (SSB) rather than a DSB; see Figure 11), 10A (aspartic acid to alanine at amino acid position 10 of SEQ ID NO: 8) mutation (or or any of the proteins set forth as SEQ ID NOs: 1 to 256 and 795 to 1346 In some embodiments, the Cas9 / Csn1 polypeptide is Modified forms of the peptide are capable of cleaving non-complementary strands of target DNA, but not of target DNA. Reduced ability to break the complementary strand (thus generating single-strand breaks (SSBs) rather than DSBs) 11), H840A (His at amino acid position 840 of SEQ ID NO:8), alanine to cysteine) mutations (also listed as SEQ ID NOS: 1-256 and 795-1346) (D10A of Cas9 or the corresponding mutation of one of the proteins carried by the is the H840A mutant (also listed as SEQ ID NOs: 1-256 and 795-1346) In contrast to SSB, When a DSB is present, non-homologous end joining (NHEJ) is even more likely to occur Therefore, the likelihood of DSBs can be altered. It is desirable to reduce (and therefore reduce the likelihood of NHEJ) some In some examples, the D10A or H840A mutants of Cas9 can be used. residues can be mutated to achieve the same effect (i.e., one or the other nuclease moiety). Non-limiting examples include residues D10, G12, G17, E76, 2, H840, N854, N863, H982, H983, A984, D986, and / or A987 (or SEQ ID NOs: 1-256 and 795-1346) The corresponding mutation in either of the proteins can be altered (i.e., substituted) (For more information on the conservation of Cas9 amino acid residues, see Figures 3, 5, and 1. 1A and Table 1). Mutations other than alanine substitutions are also appropriate. When a polypeptide (e.g., a site-directed modified polypeptide) has reduced catalytic activity (For example, the Cas9 protein is D10, G12, G17, E762, H840, N85 4, N863, H982, H983, A984, D986, and / or A987 mutations , for example, D10A, G12A, G17A, E762A, H840A, N854A, N8 If you have 63A, H982A, H983A, A984A, and / or D986A In some embodiments of the method described above, the polypeptide is a DNA-targeting R As long as it retains the ability to interact with NA, it will still bind to the target DNA in a site-specific manner. (The polypeptide can still be directed to the target DNA sequence by the DNA-targeting RNA.) (Because it can be).

[0208] In some embodiments, the modified form of the Cas9 / Csn1 polypeptide comprises the polypeptide The peptide appears to have a reduced ability to cleave both complementary and non-complementary strands of target DNA. (i.e., the mutant may not have substantial nuclease activity), D10 Both the A mutation and the H840A mutation (or SEQ ID NOs: 1-256 and 795-1346) (corresponding mutations in any of the proteins described as and achieve the same effect (i.e., inactivating one or the other nuclease moiety). Non-limiting examples include residues D10, G12, G17, E762, H84 0, N854, N863, H982, H983, A984, D986, and / or A 987 (or proteins set forth as SEQ ID NOS: 1-256 and 795-1346) (C For further information on the conservation of as9 amino acid residues, see Figures 3, 5, 11A, and (See Table 1.) Mutations other than alanine substitutions are also appropriate.

[0209] In some embodiments, the site-directed modifying polypeptide comprises a heterologous sequence (e.g., In some embodiments, the heterologous sequence is a sequence of the site-directed modifying polypeptide. It can confer intracellular localization (e.g., a nuclear localization signal (NLS) for targeting to the nucleus; a mitochondrial localization signal for targeting to mitochondrial cells; chloroplast localization signal; ER retention signal; etc.). The seed sequence may provide a tag (e.g., a fluorescent protein) to facilitate tracking or purification. , e.g., green fluorescent protein (GFP), YFP, RFP, CFP, mCherry, tdTomato, etc.; His tag, e.g., 6XHis tag; hemagglutinin (HA) tag ; FLAG tag; Myc tag; etc.). In some embodiments, the heterologous sequence is a stabilizing may give an increase or decrease in

[0210] In some embodiments, the subject site-directed modified polypeptides are codon-optimized. This type of optimization is known in the art and allows for the creation of sequences encoding the same protein. Mutations in the foreign DNA to mimic the codon preferences of the desired host organism or cell Thus, the codon is changed, but the encoded protein remains unchanged. For example, if the target cells of interest are human cells, human codon-optimized Cas9 ( or mutants, e.g., enzymatically inactive mutants) are suitable site-directed modified polypeptides. (See, for example, SEQ ID NO: 256.) Any suitable site-specifically modified polypeptide may be used. peptides (e.g., any of the sequences set forth in SEQ ID NOS: 1-256 and 795-1346) Any Cas9 (e.g., any of the above) can be codon-optimized. If the intended host cell is a mouse cell, use a mouse codon-optimized Cas9 (or a variant, For example, enzymatically inactive mutants) would be suitable site-directed modified polypeptides. Codon optimization is not required, but may be acceptable in certain cases. Yes, and in some cases it may be preferable.

[0211] In some embodiments, the subject DNA-targeting RNAs and the subject site-specific modifications The polypeptides can be used as inducible systems to silence gene expression in bacterial cells. In some instances, a suitable DNA-targeting RNA and / or a suitable site can be used. The nucleic acid encoding the specific polypeptide is integrated into the chromosome of the target cell and expressed as an inducible promoter. DNA-targeting RNA and / or site-specific polypeptides are induced When introduced, the target DNA is transformed with the DNA-targeting RNA and the site-specific modifying polypeptide. When both are present and form a complex, the target site (e.g., target on a separate plasmid) Thus, in some instances, bacterial expression The strain comprises a nucleic acid sequence encoding the appropriate site-directed modifying polypeptide within the bacterial genome, and and / or a suitable DNA target on a plasmid (e.g., under the control of an inducible promoter). The strain was engineered to contain a targeting RNA (expressed from a separate plasmid introduced into the strain). Any targeted gene expression can be achieved by DNA-targeting RNA and site-specific polypeptides. This allows for experiments in which the expression of the peptide can be controlled by inducing it.

[0212] In some instances, the site-directed modifying polypeptide performs other functions than introducing a double-strand break. The target DNA has an enzymatic activity that modifies the target DNA in a manner that A chimeric site-specific modification polypeptide is formed by fusing a heterologous polypeptide containing the site-specific modification polypeptide to the site-specific modification polypeptide. The enzyme activity of interest that can be used to modify (by producing a differentially modified polypeptide) The properties include, but are not limited to, methyltransferase activity, demethylase activity, DNA A repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, decomposition activity Purine activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposon activity ase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, DNA damage and repair activity (including photolyase activity or glycosylase activity). are essential for cell survival and proper genome maintenance in response to environmental stress, Methylation and demethylation are recognized in the art as important methods of epigenetic gene regulation. It is recognized as such.

[0213] Thus, the methods herein are useful in epigenetic modification of target DNA, A. Transfecting a desired complementary nucleic acid sequence into the DNA-targeting segment of the targeting RNA to control epigenetic modifications of target DNA at any position within the target DNA. The methods herein can also be used to cleave any desired position within a target DNA. These methods are useful in the deliberate and controlled damage of DNA in various applications. The method also allows for sequence-specific and nucleotide sequence modification of DNA at any desired location within the target DNA. It is useful for controlled repair by targeting DNA modifying enzyme activity to specific locations within the target DNA. The targeting method is useful in both research and clinical applications.

[0214] In some instances, the site-directed modifying polypeptide is a chimeric site-directed In the case of modified polypeptides, etc., they have the activity of regulating the transcription of target DNA. Heterologous polypeptides (e.g., transcriptional activators) that exhibit the ability to increase or decrease transcription in The chimeric site-directed modifying polypeptide containing the transcription factor or transcription repressor polypeptide is Specific targeting within the target DNA, guided by the DNA-targeting segment of the NA-targeting RNA It is used to increase or decrease transcription of target DNA at a specific site. Examples of source polypeptides that confer transcriptional regulatory activity on the heterologously modified polypeptide include those containing the following: Although not widely used, light-inducible transcription factors, small molecule / drug-responsive transcription factors, transcription factors, transcription factors In some instances, the subject methods involve targeting coding regions. RNA (protein-coding genes) and / or targeted non-coding RNA (e.g. , tRNA, rRNA, snoRNA, siRNA, miRNA, long ncRNA, etc.) Used to control expression.

[0215] In some instances, the site-directed modifying polypeptide is a polypeptide bound to DNA. In some embodiments, the enzyme has an enzymatic activity that modifies a protein (e.g., a histone). Enzyme activity includes methyltransferase activity, demethylase activity, and acetyltransferase activity. acetylase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin Ligase activity (i.e., ubiquitination activity), deubiquitination activity, adenylation activity, Deadenylation activity, sumoylation activity, desumoylation activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity, glycosylation activity (e.g., OG lcNAc transferase) or deglycosylation activity. The enzymatic activity catalyzes covalent modifications to proteins. Altering the stability or activity of target proteins is known in the art. (e.g., phosphorylation by kinase activity stimulates protein activity depending on the target protein. Histones are particularly important as protein targets. Proteins bind to DNA to form complexes known as nucleosomes. Histone modifications (e.g., methylation, acetylation, ubiquitination) are known in the art. By cleaving the DNA, the transcription factor (chitinylation, phosphorylation) induces structural changes in the surrounding DNA, which in turn accelerates transcription. accessibility of potentially large portions of DNA to interacting factors such as proteins, polymerases, etc. A single histone can be regulated in many different ways and in many different combinations. In combination, modifications can be made (e.g., at lysine 27 of histone 3 (H3K27)). Trimethylation is associated with transcriptionally repressed DNA regions, whereas lysine trimethylation of histone 3 Trimethylation of H3K4 (H3K4) is associated with transcriptionally active DNA regions. Site-specific modifying polypeptides with histone-modifying activity regulate DNA structure in a site-specific manner It is useful in altering histone modification patterns in selected regions of target DNA. Such methods are useful in both research and clinical applications. It is useful.

[0216] In some embodiments, multiple DNA-targeting RNAs target the same target DNA or They are used simultaneously to modify different locations on different target DNAs. In some embodiments, two or more DNA-targeting RNAs target the same gene or transcript or In some embodiments, two or more DNA-targeting RNAs target a locus. In some embodiments, two or more D The NA-targeting RNAs target different but related loci.

[0217] In some instances, the site-directed modified polypeptide is provided directly as a protein. As one non-limiting example, fungi (e.g., yeast) can be transformed by spheroplast transformation. can be used to transform with foreign proteins and / or nucleic acids (Kawai et al. , Bioeng Bugs. 2010 Nov-Dec;1(6):395-403 : “Transformation of Saccharomyces cer evisiae and other fungi: methods and possible underlying mechanisms”; and Tank a et al., Nature. 2004 Mar 18;428(6980):323-8: “Conformational variations in an See "Infectious Protein Determine Prion Strain Differences" (these Both are incorporated herein by reference in their entireties. A decorative polypeptide (e.g., Cas9) can be incorporated into the spheroplasts (D The presence or absence of a nucleic acid encoding the NA-targeting RNA and the presence or absence of a donor polynucleotide Regardless of the type of yeast cell, spheroplasts can be used to introduce the contents into yeast cells. The site-directed modified polypeptide can be introduced into cells by any convenient method. can be administered to cells; such methods are known to those skilled in the art. By way of example, the site-specific modifying polypeptide can be administered to cells (e.g., cells of zebrafish embryos, It can be injected directly into the pronucleus of a fertilized mouse oocyte (e.g., DNA targeting). with or without nucleic acid encoding the targeting RNA, and with or without donor polynucleotide ).

[0218] Target cells of interest In some of the above applications, the subject methods can be used to perform in vivo and / or experimental In vivo and / or in vitro, in mitotic or postmitotic cells can induce DNA cleavage, DNA modification, and / or transcriptional regulation (e.g., individual (Genetically modified cells can be produced that can be reintroduced into the body.) DNA-targeted R NAs confer specificity by hybridizing to target DNA, and therefore are useful in the methods of the present disclosure. The mitotic and / or postmitotic cells of interest in this study may be cells of any organism. can include (e.g., bacterial cells, archaeal cells, unicellular eukaryotic cells, plant cells, algae cells , e.g., Botryococcus braunii ), Chlamydomonas reinhardtii, Nanno Nannochloropsis gaditana, Chlorella pyrenoidosa, Yatsumatamoku (Sargassum patens), C. Agardh, etc., fungi cells (e.g., yeast cells), animal cells, invertebrate cells (e.g., Drosophila, Cnidaria) cells derived from vertebrates (e.g., fish, amphibians, reptiles, birds, mammals, etc.); Mammalian cells, mammalian cells, rodent cells, human cells, etc. ).

[0219] Any type of cell can be of interest (e.g., stem cells, e.g., embryonic stem (ES) cells, etc. , induced pluripotent stem (iPS) cells, germ cells; somatic cells, e.g., fibroblasts, hematopoietic cells, Neurons, muscle cells, bone cells, liver cells, pancreatic cells; in vitro or in vivo Embryonic cells of all stages of embryos, e.g., zebrafish embryos at the 1-cell, 2-cell, 4-cell, 8-cell, etc. stages Cells may be derived from established cell lines or may be primary cells. As used herein, the terms "primary cell," "primary cell line," and "primary culture" are synonymous. are used commercially and are obtained from a subject and cultured for a limited number of passages (i.e., divisions) refers to cells and cell cultures that have been propagated in vitro. For example, primary cultures , may have been passaged 0, 1, 2, 4, 5, 10, or 15 times, Cultures that have not been passaged enough times to reach a crisis stage Typically, the primary cell lines of the invention are maintained in vitro for fewer than 10 passages. Target cells, in many embodiments, are single-celled organisms or grown in culture.

[0220] If the cells are primary cells, they may be obtained from an individual by any convenient method. For example, white blood cells can be obtained by apheresis, leukapheresis, density gradient separation, etc. Therefore, it may be convenient to collect the tissue from the skin, muscle, bone marrow, spleen, liver, pancreas, lung, etc. Cells from tissues such as the intestine, stomach, etc. are most conveniently obtained by biopsy. The solution may be used to disperse or suspend the harvested cells. Typically, fetal bovine blood is diluted with an acceptable buffer at a low concentration, typically 5-25 mM. balanced salt solutions, which may conveniently be supplemented with serum or other naturally occurring factors, e.g. For example, physiological saline, phosphate buffered saline (PBS), Hank's balanced salt solution, etc. Suitable buffers include HEPES, phosphate buffer, lactate buffer, etc. Cells should be used immediately. Alternatively, it can be stored for a long period of time, frozen, and then thawed and reused. In such cases, cells are typically cultured in 10% DMSO, 50% serum, 40% buffered medium, or , and methods commonly used in the art for preserving cells at such freezing temperatures. and several other solutions known in the art for thawing frozen cultured cells. The data is then decompressed in a known manner.

[0221] A subject DNA-targeting RNA and / or a subject site-specific modifying polypeptide are encoded by the subject DNA-targeting RNA and / or the subject site-specific modifying polypeptide. Nucleic acid In some embodiments, the subject methods include converting the target DNA into a DNA-targeting RNA and and / or site-specifically modified polypeptides and / or donor polynucleotides. contacting a cell with one or more nucleic acids containing a nucleotide sequence that encodes the or cell population) by administering DNA-targeting RNA and / or site-specific modifying polypeptides and and / or one or more polynucleotides containing a nucleotide sequence encoding the donor polynucleotide. The introduction of multiple nucleic acids includes DNA-targeting RNA and / or site-specific Suitable nucleic acids containing a nucleotide sequence encoding a modified polypeptide include expression vectors. Rarely, where the DNA-targeting RNA and / or the site-specific modifying polypeptide are encoded An expression vector containing a nucleotide sequence is a "recombinant expression vector."

[0222] In some embodiments, the recombinant expression vector is a viral construct, e.g., Recombinant adeno-associated virus constructs (see, e.g., U.S. Patent No. 7,078,387) , recombinant adenoviral constructs, recombinant lentiviral constructs, etc.

[0223] Suitable expression vectors include, but are not limited to, viral vectors (e.g., vaccinia vectors). Viral vectors based on rhesus monkey viruses; poliovirus; adenovirus (e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6 :515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gen e Ther 5:1088 1097, 1999; WO 94 / 12649, WO 93 / 03769; No. 93 / 19191; No. 94 / 28938; No. 95 / 11984 and No. 95 / 00655); adeno-associated viruses (see, e.g., Ali et al., Hum Gene Ther r 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Inves t Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997 , Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:59 1 594, 1996; Srivastava, Samulski et al., J. Vir. in WO 93 / 09239. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus Viruses that cause epidemic deficiency (e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al. , J Virol 73:7812 7816, 1999); retroviral vectors (e.g., mouse leukemia vectors) Disease virus, spleen necrosis virus, and Rous sarcoma virus, Harvey sarcoma virus, avian Leukemia viruses, lentiviruses, human immunodeficiency viruses, myeloproliferative sarcoma viruses (m erythroproliferative sarcoma virus, and breast cancer virus vectors derived from retroviruses such as rabies);

[0224] Large numbers of suitable expression vectors are known to those of skill in the art, and many are commercially available. The following vectors are described: eukaryotic host cells: pXT1, pSG5 (Stratagene) ), pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia). However, any other vector may be used so long as it is compatible with the host cell.

[0225] In some embodiments, the DNA-targeting RNA and / or the site-specifically modified polynucleotide The nucleotide sequence encoding the peptide may be linked to a transcriptional regulatory region, such as a promoter. The transcriptional regulatory region is operably linked to the transcriptional regulatory region in eukaryotic cells (e.g., mammalian cells), Alternatively, it may be functional in a prokaryotic cell (e.g., a bacterial cell or an archaeal cell). In some embodiments, the DNA-targeting RNA and / or the site-specifically modified polypeptide The nucleotide sequence encoding the nucleotide sequence can be used to identify the DNA target in both prokaryotic and eukaryotic cells. and expression of nucleotide sequences encoding modified RNA and / or site-specifically modified polypeptides. The gene is operably linked to multiple regulatory regions that enable expression of the gene.

[0226] Depending on the host / vector system used, several appropriate transcriptional and translational regulatory regions may be used. Any of the regions, e.g., constitutive and inducible promoters, transcription enhancers, Ser elements, transcription terminators, etc. may be used in the expression vector (for example, U6 promoter). promoter, H1 promoter, etc.; see above) (e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544).

[0227] In some embodiments, the DNA-targeting RNA and / or the site-specifically modified polynucleotide The peptide may be provided as an RNA. In such an example, DNA-targeting RNA and RNA encoding the site-specifically modified polypeptide can be synthesized by direct chemical synthesis. or in vitro from DNA encoding the DNA-targeting RNA. Methods for synthesizing RNA from a template DNA are well known in the art. In some instances, the DNA-targeting RNA and / or the site-specifically modified polynucleotide The RNA encoding the peptide is synthesized by an RNA polymerase enzyme (e.g., T7 polymerase, It is synthesized in vitro using a polymerase such as T3 polymerase or SP6 polymerase. Afterwards, the RNA may be directly contacted with the target DNA or the nucleic acid may be introduced into the cell. well-known techniques for transfecting cells (e.g., microinjection, electroporation, transfection, etc.) The vector may be introduced into the cell by any of the following methods: transfection, immunoprecipitation, etc.

[0228] DNA-targeting RNA (delivered as DNA or RNA) and / or site-specific modifications Polypeptides (delivered as DNA or RNA) and / or donor polynucleotides Nucleotides encoding the vectors can be transfected using well-developed transfection techniques (e.g., An See Gel and Yanik (2010) PLoS ONE 5(7): e11756), and Qiagen n) TransMessenger® Reagent, sold by Stemgent, Inc. Stemfect™ RNA transfection kit available from Stemgent Application kits and commercially available from Mirus Bio LLC Transfection of cells using the TransIT®-mRNA transfection kit Beumer et al. (2008) Efficient gene targeting in Drosophila a by direct embryo injection with zinc-finger nucleases. PNAS 105(50):19821-1982 See also 6. Alternatively, DNA-targeting RNA and / or site-specifically modified polypeptides peptide and / or chimeric site-directed modified polypeptide and / or donor polypeptide The nucleic acid encoding the peptide may be provided on a DNA vector. Many vectors are available for introducing the Vectors containing the nucleic acid(s) can be, for example, plasmids, viruses, etc. mide, minicircle DNA, viruses, e.g., cytomegalovirus, adenovirus or may be maintained episomally as a nucleotide sequence or may be expressed by homologous recombination or random insertion. Integrating retroviral vectors such as MMLV, HIV-1, and ALV The vector may be integrated into the target cell genome by the target vector.

[0229] The vector may be directly administered to the subject's cells. In other words, the cells may be DNA-targeting RNA and / or site-specifically modified polypeptides are prepared for uptake by cells. The chimeric site-directed modified polypeptide and / or donor polynucleotide The cells are contacted with a vector containing a nucleic acid encoding a nucleotide. a method for contacting the target with the target, such as electroporation, calcium chloride transfer, transfection (calcium chloride transfection), Microinjection and lipofection are well known in the art. In viral vector delivery, cells are transfected with DNA-targeting RNA and / or site-specific Chimeric site-specifically modified polypeptides and / or chimeric site-specifically modified polypeptides and / or The virus is contacted with a viral particle containing nucleic acid encoding a retrovirus polynucleotide. Genes (e.g., lentiviruses) are particularly suitable for the methods of the present invention. Retroviral vectors are "defective," i.e., contain the viral components required for productive infection. Rather, replication of the vector occurs in the packaging cell. In order to produce viral particles containing the nucleic acid of interest, the nucleic acid must be propagated in a system. The retroviral nucleic acid containing the nucleotide sequence is packaged into a viral capsid by a packaging cell line. Different packaging cell lines produce different endonucleases that are incorporated into the capsid. Envelope proteins (e.g., ecotropic, amphotropic, or xenotropic) are provided, and this envelope The loop protein determines the specificity of the virus particle to the cell (mouse and rat allotropic for most mammalian cell types, including human, canine, and murine species-tropic; and xenotropic for most mammalian cell types except mouse cells). By using a suitable packaging cell line, the cells become the target for the packaged viral particles. Reprogramming factors can be used to ensure that the target gene is targeted. A method for introducing a rovirus vector into a packaging cell line and a method for introducing the rovirus vector into a packaging cell line Methods for harvesting the viral particles produced thereby are well known in the art. Acids can also be introduced by direct microinjection (e.g., RNA in zebrafish). (injection into embryos).

[0230] DNA-targeting RNA and / or site-specific modifying polypeptides and / or chimeras Nucleic acids encoding the site-directed modifying polypeptide and / or donor polynucleotide are The vector used to deliver to the subject cells typically directs the expression (i.e., In other words, the nucleic acid of interest contains a promoter suitable for driving the transcriptional activity of the nucleic acid. is operably linked to a promoter. ously acting) promoters (e.g., CMV-β-actin promoter) ), or inducible promoters (e.g., active in specific cell populations or These promoters may include promoters that respond to the presence of drugs such as tracycline. transcription is increased by at least about 10-fold, at least about 100-fold, and more usually by at least about 1 It is intended that the level of the antibody be increased by 000-fold above the basal level in the target cells. NA-targeting RNA and / or site-specific modifying polypeptide and / or chimeric moiety To provide specific modified polypeptides and / or donor polynucleotides to the subject cells, The vectors used for this purpose include DNA-targeting RNA and / or site-specific modified polypeptides. The chimeric site-directed modified polypeptide and / or donor polynucleotide A nucleic acid encoding a selectable marker in target cells to identify cells that have taken up the oxidase. It may also contain an array.

[0231] a subject DNA-targeting RNA and / or site-specific modifying polypeptide and / or Alternatively, a chimeric site-directed modifying polypeptide is used to contact the DNA; or It may be introduced into cells as RNA. Methods for introducing RNA into cells are well known in the art. are known for their use in, for example, direct injection, transfection, or introduction of DNA. Any other method that can be used may be included.

[0232] The subject site-directed modified polypeptides may alternatively be provided to cells as polypeptides. Such polypeptides may optionally be used in combination with polypeptides that increase the solubility of the product. The domain may be fused to a regulatory domain that is cleaved by the TEV protease. linked to the polypeptide via a specific protease cleavage site (e.g., a TEV sequence). The linker may comprise one or more flexible sequences, for example, 1 to 10 glycine residues. In some embodiments, cleavage of the fusion protein may include cleaving the product. In a buffer that maintains solubility, for example, in the presence of 0.5-2M urea, poly(A)-1,2-diol, and poly(A)-1,2-diol, which increases solubility, The domain of interest may be a polypeptide or a polypeptide fragment, such as a polypeptide fragment or a polypeptide fragment, or a polypeptide fragment or a polypeptide fragment. , endosomolytic domains, e.g., influenza the HA domain; and other polypeptides that aid in production, e.g., IF2 domain, GS The polypeptide contains a T domain, a GRPE domain, etc. For example, the peptides may be PEGylated or may be formulated to The olefin group provides extended life in the bloodstream.

[0233] Additionally, or alternatively, the subject site-directed modified polypeptides may be modified to enhance uptake by cells. To facilitate penetration, some polypeptides may be fused to a permeabilizing domain. Penetrating domains are known in the art and are suitable for use in the non-integrating (non-int) egating polypeptides, including peptides, peptidomimetics, Drugs and non-peptide carriers are included. For example, a penetrating peptide may have the amino acid sequence RQI KIWFQNRRMKWKK (SEQ ID NO: / / ), called penetratin, Drosophila melanogaster transcription factor ante It may originate from the third alpha helix of Antennapaedia. The penetrating peptide may be, for example, amino acids 49-57 of the naturally occurring tat protein. Other penetrating domains include the HIV-1 tat basic region amino acid sequence, which may include: , poly-arginine motifs, e.g., amino acids 34 to 38 of the HIV-1 rev protein 56 regions, including nona-arginine, octa-arginine, etc. (e.g., Futaki e t al. (2003) Curr Protein Pept Sci. 2003 Apr; 4(2): 87-9 and 446; and Wender et al. al. (2000) Proc. Natl. Acad. Sci. USA 2000 Nov. 21; 97(24):13003-8; U.S. Patent Publication No. 20030220334; Publication No. 20030083256; Publication No. 20030 Nos. 032593 and 20030022831 (which describe transit peptides and See, for example, the US Pat. No. 6,229,999, which is expressly incorporated herein by reference for its teaching of transitional peptoids. The nona-arginine (R9) sequence is one of the more efficient PTDs characterized. The site where the fusion occurs is the site of the fusion of this polypeptide (Wender et al. 2000; Uemura et al. 2002). The polypeptides may be selected to optimize the biological activity, secretion, or binding characteristics of the polypeptide. The site is determined by routine experimentation.

[0234] The subject site-specifically modified polypeptides can be expressed in vitro or by eukaryotic cells, and can be produced by prokaryotic cells and can be unfolded, e.g., by heat denaturation, DTT reduction, etc. and may be further processed by refolding using methods known in the art. You may receive further dings.

[0235] Desired modifications that do not alter primary sequence include chemical derivatization of polypeptides, e.g., acylation. Glycosylation modifications, such as carboxylation, acetylation, carboxylation, and amidation, are also included. For example, during polypeptide synthesis and processing or during further processing steps, For example, a polypeptide can be converted to a glycosylating enzyme, such as a mammalian glycosylating enzyme or a deglycosylating enzyme. The glycosylation pattern of a polypeptide can be altered by exposing it to enzymes that affect glycosylation. The term also includes modifications such as phosphorylated amino acid residues, e.g., phosphatidylcholinesterase (PHO), ... Also embraced are sequences which contain phosphotyrosine, phosphoserine, or phosphothreonine.

[0236] DNA-targeting RNAs and RNAs modified using conventional molecular biology techniques and synthetic chemistry Site-specifically modified polypeptides may also be used to improve their resistance to proteolysis. , to alter target sequence specificity, to optimize solubility properties, to alter protein activity (e.g., for example, to alter transcriptional regulatory activity, enzymatic activity, etc., or to make them more suitable as therapeutic agents. Analogs of such polypeptides include, but are not limited to, those derived from the subject invention. Residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring synthetic amino acids Some or all of the amino acid residues are D-amino acids. It may be substituted.

[0237] Site-directed modified polypeptides can be synthesized in vitro using conventional methods known in the art. They can be prepared synthetically. Various commercially available devices are available, e.g. Manufactured by Applied Biosystems, Inc. The synthesis equipment is an automatic synthesis equipment manufactured by Beckman, etc. Thus, naturally occurring amino acids can be substituted with non-natural amino acids. The type and method of preparation are determined by convenience, economy, required purity, etc.

[0238] If desired, various groups may be added during synthesis or expression to allow for linkage to other molecules or surfaces. Thus, cysteine ​​may be introduced into the peptide in order to link the metal ion complex. carboxyl groups to form thioethers, histidine, amides or esters of It may also be used to create amino groups for forming amides, etc.

[0239] The site-directed modified polypeptide may be isolated and purified according to standard techniques for recombinant synthesis. Lysates of expression hosts are prepared and analyzed by HPLC, exclusion chromatography, gel electrophoresis, The lysate is purified using affinity chromatography or other purification methods. Typically, the compositions used are free of contaminants associated with the preparation of the product and its purification. In relation to the above, at least 20% by weight of the desired product, more generally at least about 75% by weight % by weight, preferably at least about 95% by weight of the desired product, and for therapeutic purposes, contains at least about 99.5% by weight of the desired product. Generally, this percentage depends on the total protein amount.

[0240] to induce DNA cleavage and recombination, or any desired modification to the target DNA or for any desired modification to the polypeptide bound to the target DNA, NA-targeting RNA and / or site-specific modifying polypeptide and / or donor polypeptide The nucleotides, whether introduced as nucleic acids or polypeptides, Regardless of the duration, the time may be from about 30 minutes to about 24 hours, for example, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, The cells are given 18 hours, 20 hours, or any other period between about 30 minutes and about 24 hours. , about every day to about every 4 days, for example, every 1.5 days, every 2 days, every 3 days, or is repeated at any other frequency from about every day to about every four days. The subject cells are administered one or more times, e.g., one, two, three, or more than three times. The cells may be allowed to stand for some time (e.g., 16-24 hours) after each contact event. The medium is incubated with the agent(s) for 1 hour, after which time the medium is replaced with fresh medium. The cells are then replaced with the soil and further cultured.

[0241] Two or more different targeting complexes (e.g., different targeting complexes within the same or different target DNA) When two different DNA-targeting RNAs (each complementary to a different sequence) are given to a cell, These complexes may be provided simultaneously (e.g., as two polypeptides and / or nucleic acids). Alternatively, two or more different targets may be delivered simultaneously. The targeting complexes may be administered sequentially (e.g., the targeting complex is administered first, and and then a second targeting complex is administered, or vice versa).

[0242] Typically, an effective amount of a DNA-targeting RNA and / or a site-specific modifying polypeptide and and / or donor polynucleotides are inserted into target DNA or cells to induce cleavage. DNA-targeting RNA and / or site-specific modifying polypeptides and / or The effective amount of the donor polynucleotide depends on the amount of target modification observed between the two homologous sequences. In addition, negative controls (e.g., cells contacted with an empty vector or an irrelevant polypeptide) That is, the amount of DNA-targeting RNA and and / or the effectiveness of the site-specific modified polypeptide and / or donor polynucleotide. The amount or effective dose is a two-fold increase in the amount of target modification observed in the target DNA region. Inducing an increase, a three-fold increase, a four-fold increase or more, and in some instances , a five-fold increase, a six-fold increase or more in the amount of recombination observed, sometimes A 7-fold or 8-fold increase or greater, e.g., 10-fold, 50-fold, or 100-fold Inducing an increase of more than 200-fold in the amount of recombination observed, in some cases , 500x, 700x, or 1000x or more, e.g., 5000x or 10,00 The amount of target modification can be measured by any conventional method. For example, reconstituting a nucleic acid that encodes an active reporter when recombined, The targeting segment of the DNA-targeting RNA is flanked by repeat sequences (targeting sequences). A silent reporter construct containing a sequence complementary to the nucleotide sequence (column 1) was co-transfected into the cells. The amount of reporter protein may be adjusted to the amount of DNA-targeting RNA and / or site-specific After contact with the heterologously modified polypeptide and / or donor polynucleotide, e.g., DNA-targeting RNA and / or site-specific modifying polypeptide and / or donor polypeptide 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 36 hours after contact with ribonucleotides After 48, 72 or more hours, the test is evaluated. For example, the extent of recombination in a genomic DNA region of interest containing a target DNA sequence can be determined. The degree of DNA targeting RNA and / or site-specific modifying polypeptide and / or DNA After contact with the target polynucleotide, e.g., DNA targeting RNA and / or site 2 hours after contact with the specific modified polypeptide and / or donor polynucleotide, 4 Hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours or more After the interval, the region is evaluated by PCR or Southern hybridization. can be done.

[0243] Cell- and DNA-targeting RNA and / or site-specific modifying polypeptides and / or Contact of the donor polynucleotide promotes cell survival in any medium, and The cells can be cultured under any culture conditions. For example, the cells can be cultured in fetal bovine serum or heat. Inactivated goat serum (approximately 5-10%), L-glutamine, thiols, especially 2-mercapto Add ethanol, and antibiotics, such as penicillin and streptomycin. Any convenient suitable nutrient medium, such as Iscove's modified DMEM or RPMI 1640, may be used. The medium may also contain growth factors to which the cells are responsive. Growth factors as defined herein act to mediate the growth of cells through specific effects on transmembrane receptors. It can promote cell survival, proliferation and / or differentiation in the soil or in intact tissue. Growth factors include polypeptide factors and non-polypeptide factors. Conditions that promote cell survival typically permit non-homologous end joining and homology-directed repair. Accept.

[0244] In applications where it is desirable to insert a polynucleotide sequence into a target DNA sequence, the insertion A polynucleotide containing a donor sequence to be transduced is also provided to the cell. The "donor polynucleotide" refers to the cleavage site induced by the site-directed modifying polypeptide. A donor polynucleotide refers to a nucleic acid sequence to be inserted at a position to assist in homologous recombination repair between the gene and the genomic sequence to which it has homology. have sufficient homology to the genomic sequence at the site, e.g., adjacent to the cleavage site, e.g. For example, within about 50 bases of the cleavage site, e.g., within about 30 bases, within about 15 bases, or within about 10 bases. 70% of the nucleotide sequence within 1 base, within about 5 bases, or immediately adjacent to the cleavage site , 80%, 85%, 90%, 95%, or 100% homology. Approximately 25, 50, 100, or 200 genes with sequence homology between the genome and the nucleotides, or more than 200 nucleotides (or any integer between 10 and 200) nucleotides) assists in homologous recombination repair. The nucleotide sequence may be of any length, for example, 10 nucleotides or more, up to 50 nucleotides. nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 500 nucleotides or more It may be 1000 nucleotides or more, 5000 nucleotides or more, etc. in length.

[0245] The donor sequence is typically not identical to the genomic sequence being replaced. The ner sequence is a genomic DNA fragment, provided there is sufficient homology to support homologous recombination repair. The sequence contains at least one or more single base changes, insertions, deletions, inversions or rearrangements. In some embodiments, the donor sequence may comprise a target DNA region and and homology-directed repair between two flanking sequences results in the insertion of a non-homologous sequence in the target region. The donor sequence contains a non-homologous sequence flanked by two homologous regions, such as the DNA region of interest. vectors containing sequences that are not homologous to and are not intended for insertion into the DNA region of interest Generally, the homologous region(s) of the donor sequence are the desired sequence for recombination. In certain embodiments, the sequence has at least 50% sequence identity to a genomic sequence contained within the genome. is a distribution of 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9%. There is sequence identity between 1% and 100%, depending on the length of the donor polynucleotide. Any degree of sequence identity may be present.

[0246] The donor sequence may contain certain sequence differences compared to the genomic sequence, e.g. , can be used to assess the success of insertion of the donor sequence at the cleavage site, or In some instances, the target gene may be used for other purposes (e.g., to detect expression at a target genomic locus). restriction enzyme recognition sites, nucleotide polymorphisms, selectable markers (e.g., For example, a drug resistance gene, a fluorescent protein, an enzyme, etc. In the present invention, when located within the coding region, such nucleotide sequence differences do not alter or make silent amino acid changes (i.e., (changes that do not affect structure or function). Alternatively, these sequence differences may be containing flanking recombination sequences such as FLP and loxP sequences that can be subsequently activated for removal of the It's fine.

[0247] The donor sequence may be single-stranded DNA, single-stranded RNA, double-stranded DNA, or double-stranded RNA. The donor sequence may be introduced into the cell in a linear or circular form. When introduced in a linear form, the ends of the donor sequence may be ligated by methods known to those skilled in the art. For example, one or more added to the 3' end of the linear molecule, and / or Self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang et al. al. (1987) Proc. Natl. Acad Sci USA 84:4959-4963; Nehls et al. (1996) Science 2 72:886-889. Additional methods for protecting exogenous polynucleotides from degradation Methods include, but are not limited to, the addition of terminal amino group(s), as well as the addition of modified nucleic acids. Internucleotide linkages (e.g., phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues). As an alternative to protecting the ends of the The donor sequence may include sequences outside the homologous region. Vector molecules containing additional sequences such as motors and genes encoding antibiotic resistance The donor sequence can be introduced into a cell as part of a DNA-targeting RNA. and / or site-specifically modifying polypeptides and / or donor polynucleotides. As described above for loading nucleic acids, as naked nucleic acids, liposomes or poloxamers or can be delivered as a nucleic acid complexed with an agent such as a virus (e.g., For example, delivery can be by adenovirus (AAV).

[0248] According to the above method, the DNA region of interest can be excised and modified ex vivo, i.e., "genetic In some cases, a selectable marker may be inserted into the DNA region of interest. In one embodiment, the cell population is isolated by separating the genetically modified cells from the remainder of the population. The cells can be enriched for cells containing the genetic modification. The cells represent about 1% or more (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, % or more, 7% or more, 8% or more, 9% or more, 10% or more, 15% or more, or 20% or more) The isolation of "genetically modified" cells depends on whether the selection marker used is suitable. This can also be achieved by any convenient separation technique. For example, When activated, cells can be separated by fluorescence-activated cell sorting, while cell surface markers When the marker is inserted, the cells can be separated by affinity separation techniques, e.g., magnetic separation, affinity chromatography, etc. affinity chromatography, "panning" with affinity reagents attached to a solid matrix, can be separated from the heterogeneous population by other convenient techniques. The technologies include fluorescence activated cell sorters, which use multiple color channels to Various sophistications including channels, low-angle and obtuse-angle light scattering detection channels, and impedance channels The cells may utilize a dye (e.g., propidium iodide) bound to dead cells. This may be used to select for dead cells. Any technique can be used that is not highly enriched for cells containing modified DNA. A highly enriched cell composition is thus achieved. The product contains 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more of genetically modified cells For example, about 95% or more, or 98% or more of the cellular composition. In other words, the composition can be a substantially pure composition of genetically modified cells.

[0249] The genetically modified cells produced by the methods described herein are ready for immediate use. Alternatively, the cells may be frozen at liquid nitrogen temperature for long-term storage, and then thawed and reused. In such cases, cells are typically cultured in 10% dimethyl sulfoxide (DMSO ), 50% serum, 40% buffered medium, or storing cells at such freezing temperatures and frozen in several other solutions commonly used in the art for freezing. The cells are thawed using methods well known in the art for thawing cultured cells.

[0250] The genetically modified cells can be cultured in vitro under a variety of culture conditions. The modified cells are grown in culture, i.e., grown under conditions that promote their growth. The medium may be a liquid or semi-solid medium containing, for example, agar, methylcellulose, etc. The cell population may be supplemented with fetal bovine serum (about 5-10%), L-glutamine, thiol, antimicrobials, especially 2-mercaptoethanol, and antibiotics, such as penicillin and strontium. A suitable medium such as Iscove's modified DMEM or RPMI 1640, usually supplemented with leptomycin, is used. The cells can be suspended in an appropriate nutrient medium, which may contain growth factors to which the regulatory T cells are responsive. Growth factors as defined herein may contain specific for transmembrane receptors. through various effects on cell survival, proliferation and / or differentiation in culture or in intact tissues. Growth factors are molecules that can promote growth. Growth factors include polypeptide factors and non-polypeptide factors. Contains tid factors.

[0251] Such genetically modified cells can be used, for example, to treat disease or as an antiviral. For purposes such as gene therapy, as an anti-cancer, anti-pathogen or anti-inflammatory drug for the production of genetically modified organisms in agriculture or for biological research, The subject may be a neonate, juvenile, or adult. Mammalian subjects are particularly Importantly, mammalian species that may be treated using the methods of the present invention include dogs and cats; horses; Animal models, especially small mammals, include cattle, sheep, etc., as well as primates, especially humans. (e.g., mice, rats, guinea pigs, hamsters, lagomorphs (e.g., rabbits), etc.) may be used in experimental investigations.

[0252] The cells may be used alone or, for example, to enhance their proliferation and / or proliferation in the tissue into which they are implanted. or provide the subject with an appropriate substrate or matrix to support organization. Typically, at least 1×10 3 cells, e.g., 5 x 10 3 cells, 1×1 0 4 cells, 5 x 10 4 cells, 1 x 10 5 cells, 1 x 10 6 A cell or cells At least one such cell is administered. The cells may be introduced into the subject via one of the following routes: The cells can be administered parenterally, subcutaneously, intravenously, intracranially, intraspinally, intraocularly, or intracerebrospinal fluid. , catheter, etc. Local delivery, i.e., delivery to the site of injury. Examples of methods for this include, for example, Ommaya reservoirs (e.g., See, for example, U.S. Patent Nos. 5,222,982 and 5,385,582 (incorporated herein by reference). bolus administration, e.g., by syringe, e.g., into a joint; e.g., Continuous infusion by cannulation, e.g., with convection (see, e.g., U.S. Pat. App. No. 6,239,623). No. 0070254842 (incorporated herein by reference); or Implantation of locally attached devices (see, e.g., U.S. Patent Application Publication No. 20080081064) and 20090196903 (incorporated herein by reference) The cells can be used to generate transgenic animals (e.g., transgenic mice). For this purpose, they may also be introduced into an embryo (eg, a blastocyst).

[0253] The number of therapeutic administrations to a subject can vary. however, in certain circumstances, such treatment may be for a limited period of time. In other situations, it may result in improvement in the condition and require a series of repeated treatments. Multiple injections of genetically modified cells may be required before results are observed. The dosage will vary depending on the disease or condition, the stage of the disease, and the parameters of the individual subject being treated. different.

[0254] In another aspect of the invention, the DNA-targeting RNA and / or the site-specifically modified polypeptide The gene and / or donor polynucleotides may again be used in, for example, gene therapy ( For example, to treat a disease or as an antiviral, antipathogenic, or anticancer treatment. for the production of genetically modified organisms in agriculture, or for biological research. In these in vivo embodiments, D NA-targeting RNA and / or site-specific modifying polypeptide and / or donor polypeptide Nucleotides are administered directly to an individual. DNA-targeting RNA and / or site-specific The modified polypeptide and / or donor polynucleotide may be administered to a subject by administering to the subject a peptide, small molecule, or and by any of several well-known methods in the art for administration of nucleic acids. The DNA-targeting RNA and / or site-specific modifying polypeptide may be administered. And / or the donor polynucleotide can be incorporated into a variety of formulations. Specifically, the DNA-targeting RNA and / or site-specific modifying polypeptide of the present invention and The donor polynucleotide and / or the donor polynucleotide may be combined with a suitable pharmaceutically acceptable carrier or diluent. The combination can be formulated into a pharmaceutical composition.

[0255] The pharmaceutical agent comprises one or more DNA-targeting R&D molecules present in a pharmaceutically acceptable vehicle. NA and / or site-specifically modified polypeptide and / or donor polynucleotide A "pharmaceutically acceptable vehicle" is a composition comprising a pharmaceutical agent or a pharmaceutical composition as defined by federal or state regulations. Approved by a regulatory authority or in accordance with the United States Pharmacopoeia or other regulations for use in mammals, including humans. The term "vehicle" may be any vehicle described in any generally recognized pharmacopoeia of the United States. Diluents, adjuvants with which the compounds of the present invention are formulated for administration to mammals Such pharmaceutical vehicles include lipids, e.g., liposomes. liquids such as water and oils, e.g., petroleum, animal, vegetable or are of synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil, etc., saline solution; Gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents may be used. Compositions include tablets, capsules, powders, granules, ointments, liquids, suppositories, injections, inhalants, and gels. Preparations in solid, semi-solid, liquid or gaseous form such as capsules, microspheres, and aerosols Thus, the DNA-targeting RNA and / or the site-specifically modified polynucleotide may be formulated in a Administration of peptides and / or donor polynucleotides can be oral, buccal, rectal, or parenteral. This can be achieved by a variety of methods, including intraperitoneal, intradermal, transdermal, intratracheal, intraocular, etc. The active agent may be systemic after administration or may be administered regionally. al administration), use of intramural administration, or at the implant site It may be localized by the use of an implant that acts to hold the active dose. The drugs may be formulated for immediate activity or may be formulated for sustained release. Good too.

[0256] In some conditions, particularly those of the central nervous system, It may be necessary to formulate the agent as follows: One strategy for achieving this is by osmotic means such as mannitol or leukotrienes. Disruption of the BBB by biochemical or vasoactive substances such as bradykinin The possibility of exploiting BBB opening to target specific drugs to brain tumors is also being explored. The BBB disruption agent is one option when the therapeutic composition of the present invention is administered by intravascular injection. Other strategies for crossing the BBB include administering the composition simultaneously with the BBB. , caveolin-1-mediated transcytosis, glucose carriers, amino acid carriers, etc. carrier-mediated transporters, receptor-mediated transactivators for insulin or transferrin Use of endogenous transport systems, including cytosis and active diffusion transporters such as p-glycoprotein Active transport moieties are used in the present invention to facilitate transport across the endothelial wall of a blood vessel. Alternatively, the compound may be conjugated to a therapeutic compound for use across the BBB. Drug delivery of the therapeutic agent can be by local delivery, for example, by an Ommaya reservoir. , by intrathecal delivery (see, e.g., U.S. Pat. Nos. 5,222,982 and 5,222,982). See, e.g., U.S. Pat. No. 3,855,822 (incorporated herein by reference); e.g., intravitreal or or intracranially, e.g., by syringe, by bolus administration; e.g., by convection, e.g. For example, by cannulation, by continuous infusion (see, e.g., U.S. Patent Application Publication No. 2007 see US Pat. No. 0254842 (hereby incorporated by reference); or the agent is reversible by implantation of an electrically attached device (see, e.g., U.S. Patent Application Publication No. 20080080808). See US Pat. Nos. 1064 and 20090196903, which are incorporated herein by reference. It can be)).

[0257] Typically, an effective amount of a DNA-targeting RNA and / or a site-specific modifying polypeptide and and / or donor polynucleotides are provided. As described above, in vivo DNA-targeting RNA and / or site-specifically modified polypeptides An effective amount or effective dose of the nucleotide and / or donor polynucleotide is a polynucleotide that binds to the nucleotide sequence of the two homologous sequences. The amount of recombination observed in the negative control (e.g., empty vector or unrelated polypeptide) The amount is the amount required to induce a two-fold or greater increase in the number of cells in contact with the recombinant peptide. The amount of can be determined by any convenient method, for example, by the methods described above and those known in the art. The amount of DNA-targeting RNA administered and the amount of DNA-targeting RNA administered can be measured by known methods. and / or an effective amount of the site-specific modifying polypeptide and / or donor polynucleotide Calculation of the effective dose is within the skill of those skilled in the art and is routine for those skilled in the art. The ultimate amount administered will depend on the route of administration and the nature of the disorder or condition being treated. Varies depending on the

[0258] The effective amount given to a particular patient will vary depending on a variety of factors, some of which may vary from patient to patient. A competent clinician may administer the drug to the patient to stop or improve the progression of the disease as needed. The effective amount of a therapeutic agent to reverse the progression can be determined using LD50 animal data, and Using other information about the drug that is available to the clinician, the clinician can determine the For example, the maximum safe dose for an individual can be determined by the The volume is administered intrathecally in view of the larger fluid into which the therapeutic composition is administered. Similarly, compositions that are rapidly cleared from the body may be used in combination with other compositions that are rapidly cleared from the body. may be administered in higher doses or in repeated doses to maintain therapeutic concentrations. Using ordinary skill, a competent clinician may administer a particular therapeutic agent during a routine clinical trial. The amount can be optimized.

[0259] DNA-targeting RNA and / or site-specifically modified polypeptides for inclusion in pharmaceuticals. The gene and / or donor polynucleotides may be obtained from suitable commercial sources. As a general rule, parenterally administered DNA-targeting RNA and / or site per dose The total amount of the specific modified polypeptide and / or donor polynucleotide in a pharmaceutically effective amount is , within a range that can be measured by a dose-response curve.

[0260] DNA-targeting RNA and / or site-specific modifying polypeptide and / or donor Polynucleotide-based therapeutic agents, i.e., DNA-targeted RNAs for therapeutic administration A and / or the site-specific modifying polypeptide and / or donor polynucleotide The preparation must be sterile. Sterility is confirmed by filtration through a sterile membrane (e.g., 0.2 μm membrane). This is readily accomplished by filtration through a sterile access port. a container having a stopper that can be pierced by a hypodermic needle, for example, an intravenous injection The injection solution is placed in a bag or vial. DNA-targeting RNA and / or site Therapeutic agents based on specific modified polypeptides and / or donor polynucleotides may be administered in a single dose. or as an aqueous solution in a multi-dose container (e.g., a sealed ampule or vial). The formulation may be stored in a lyophilized form or as a lyophilized formulation for reconstitution. 0 mL vials were filled with 5 mL of sterile-filtered 1% (w / v) aqueous compound solution. The infusion solution is prepared by reconstituting the lyophilized compound with bacteriostatic water for injection. It is prepared by synthesizing

[0261] The pharmaceutical composition may be formulated for administration to animals or humans, depending on the desired formulation. pharmaceutically acceptable, non-toxic vehicles, which are defined as vehicles commonly used to Carriers of diluents may be included. The diluent is selected so as not to affect the biological activity of the combination. Examples include distilled water, buffered water, saline, PBS, Ringer's solution, dextrose solution, and and Hank's solution. In addition, the pharmaceutical composition or formulation may contain other carriers, adjuvants, or The composition may also include nontoxic, nontherapeutic, nonimmunogenic stabilizers, excipients, and the like. Contains additives that mimic physiological conditions, such as pH adjusters and buffers, toxicity adjusters, wetting agents and surfactants. It may also contain additional materials for making the product.

[0262] The composition can include any of a variety of stabilizing agents, such as, for example, antioxidants. When the pharmaceutical composition comprises a polypeptide, the polypeptide may be administered in a manner that enhances the in vivo stability of the polypeptide. enhance or otherwise enhance its pharmacological properties (e.g., by enhancing the half-length of a polypeptide) Various well-known Examples of such modifying or complexing agents include sulfur compounds. The nucleic acid or polynucleotide of the composition may be a salt thereof. Peptides can also be conjugated to molecules that enhance their properties in vivo. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions, (e.g., sodium, potassium, calcium, magnesium, manganese), and fat Quality is one of the key factors.

[0263] Further guidance on formulations suitable for various types of administration can be found at Remington's Pharmaceuticals See utical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985). For a brief overview of drug delivery methods, see Langer, Science 249:1527-153 3 (1990).

[0264] The pharmaceutical compositions can be administered for prophylactic and / or therapeutic treatments. The toxicity and therapeutic effect of the active ingredient can be measured, for example, by the LD50 (the dose lethal to 50% of the population) and determining the ED50 (the dose that is therapeutically effective in 50% of the population). In cell cultures and / or experimental animals, the efficacy and safety of the product can be determined according to standard pharmaceutical procedures. The dose ratio between toxic and therapeutic effects is the therapeutic index, which is expressed as the LD50 / ED50 ratio. Therapies that exhibit large therapeutic indices are preferred.

[0265] Data obtained from cell culture and / or animal studies may support the use of a range of doses in humans. The dosage of the active ingredient is typically low in toxicity. The dose will be within a range of blood concentrations that includes the ED50. It may vary within this range depending on the route of administration.

[0266] The ingredients used to formulate the pharmaceutical compositions are preferably of high purity and have potential Substantially free from harmful contaminants (e.g., at least as defined by the National Food and Drug Administration) Food (NF) grade, generally at least analytical grade, more typically (For compositions intended for in vivo use, at least pharmaceutical grade is used.) Typically, they are sterile. To the extent that a given compound must be synthesized before use, The product being analyzed typically does not include any potentially useful components that may be present during the synthesis or purification process. Substantially free of toxic agents, and in particular of any endotoxins. ) Compositions for administration are also sterile, substantially isotonic and made under GMP conditions.

[0267] The effective amount of the therapeutic composition given to a particular patient will vary depending on a variety of factors, including Some vary between patients. A competent clinician will administer the drug to the patient and monitor the progression of the disease as needed. The effective amount of a therapeutic agent to stop or reverse the LD50 animal Using the data and other information about the drug that is available to the clinician, the clinician can: The maximum safe dose for an individual can be determined depending on the route of administration. For example, intravenous The dose administered to the patient is larger in volume than the fluid into which the therapeutic composition is administered. The dose administered intrathecally may also be higher than the dose administered intrathecally. The compositions administered may be administered at higher doses or in repeated doses to maintain therapeutic concentrations. Using ordinary skill, a competent clinician may identify specific The dosage of the therapeutic agent can be optimized.

[0268] Genetically modified host cells The present disclosure provides genetically modified host cells, including isolated genetically modified host cells, and the subject The genetically modified host cells include: 1) an exogenous DNA-targeting RNA; 2) a host cell that encodes the DNA-targeting RNA; 3) an exogenous site-specific modifying polypeptide ( For example, naturally occurring Cas9; modified, i.e., mutant or variant, Cas9; chimeric Cas9; etc.); 4) a nucleotide sequence encoding a site-specific modifying polypeptide or 5) any combination of the above, including (and genetically modified with) The subject genetically modified cells can be prepared by modifying a host cell, for example, by: 1) introducing an exogenous DNA-targeting R NA; 2) exogenous nucleic acid comprising a nucleotide sequence encoding a DNA-targeting RNA; 3) exogenous 3) a nucleic acid encoding a site-specifically modified polypeptide; 4) genetically modifying the target gene with an exogenous nucleic acid containing a nucleotide sequence; or 5) any combination of the above. It is produced by

[0269] All cells suitable to be target cells are also suitable to be genetically modified host cells. For example, the genetically modified host cell of interest can be a cell of any cell origin (e.g., a cell Bacterial cells, archaeal cells, unicellular eukaryotic cells, plant cells, algal cells, e.g., Botryococcus Cass braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrethrum noidosa, Yatsumatamoku, C. agarde, etc., fungal cells (e.g., yeast cells), animal cells, Cells derived from invertebrates (e.g., Drosophila, Cnidaria, Echinoderms, Nematodes, etc.), vertebrates Cells derived from animals (e.g., fish, amphibians, reptiles, birds, mammals), mammals (e.g., , pigs, cows, goats, sheep, rodents, rats, mice, non-human primates, (e.g., cells derived from mammals, etc.)

[0270] In some embodiments, the genetically modified host cell expresses the site-specifically modified polypeptide ( For example, naturally occurring Cas9; modified, i.e., mutant or variant, Cas9; ; chimeric Cas9; etc.) The DNA of the genetically modified host cell is then transferred to the cell via the DNA-targeting RNA (or modified DNA (DNA encoding the targeting RNA) and the desired gene location / sequence to be By introducing donor nucleic acid via cleavage, modification can be targeted. In embodiments, the nucleotide sequence encoding the site-directed modifying polypeptide is an inducible Promoters (e.g., heat shock promoters, tetracycline-regulated promoters, Steroid-regulated promoters, metal-regulated promoters, estrogen receptor-regulated promoters In some embodiments, the site-specifically modified polymer is operably linked to a target molecule (e.g., a target molecule). The nucleotide sequence encoding the peptide may be a spatially and / or temporally restricted promoter. Operable with a promoter (e.g., tissue-specific promoter, cell type-specific promoter, etc.) In some embodiments, the site-specific modifying polypeptide is encoded by The nucleotide sequence is operably linked to a constitutive promoter.

[0271] In some embodiments, a subject genetically modified host cell is in vitro. In some embodiments, the subject genetically modified host cells are present in vivo. In some embodiments, the subject genetically modified host cells are prokaryotic cells or In some embodiments, the subject genetically modified host cells are bacterial cells. In some embodiments, the subject genetically modified host is a bacterial cell. The cell is an archaeal cell or is derived from an archaeal cell. Thus, the subject genetically modified host cells are eukaryotic cells or are derived from eukaryotic cells. In some embodiments, the subject genetically modified host cells are plant cells or plant cells. In some embodiments, the subject genetically modified host cells are derived from animal cells. or derived from an animal cell. In some embodiments, the subject genetic modifications The host cell is an invertebrate cell or is derived from an invertebrate cell. In embodiments, the subject genetically modified host cells are vertebrate cells or are vertebrate cells. In some embodiments, the subject genetically modified host cells are derived from mammalian cells. In some embodiments, the subject genes are derived from mammalian cells. The modified host cells are rodent cells or are derived from rodent cells. In embodiments, the subject genetically modified host cells are human cells or derived from human cells. is.

[0272] The disclosure further provides progeny of the subject genetically modified cells, which progeny are the same as the subject genetically modified cells from which they were derived. The present disclosure further provides a method for producing a genetically modified cell comprising the steps of: The present invention provides a composition comprising a genetically modified host cell of the present invention.

[0273] Genetically modified stem and progenitor cells In some embodiments, the subject genetically modified host cells are genetically modified stem cells or progenitor cells. Suitable host cells include, for example, stem cells (adult stem cells, embryonic stem cells, iPS cells, etc.) These include stem cells (e.g., myocardial progenitor cells, neural progenitor cells, etc.) and progenitor cells (e.g., cardiac progenitor cells, neural progenitor cells, etc.). Host cells include, for example, rodent stem cells, rodent progenitor cells, human stem cells, human progenitor cells, Suitable host cells include mammalian stem and progenitor cells, including in vitro host cells, e.g., isolated host cells.

[0274] In some embodiments, the subject genetically modified host cells contain exogenous DNA-targeting RNA. In some embodiments, the subject genetically modified host comprises a DNA-targeting RNA nucleic acid. The cell contains an exogenous gene containing a nucleotide sequence encoding a DNA-targeting RNA. In some embodiments, a subject genetically modified host cell contains an exogenous nucleic acid. Site-specific modifying polypeptides (e.g., naturally occurring Cas9; modified, i.e., mutant In some embodiments, the Cas9 gene is a heterologous or heterologous Cas9; chimeric Cas9; etc. Thus, the subject genetically modified host cells contain nucleotides encoding site-specifically modified polypeptides. In some embodiments, the subject genetically modified host cells include an exogenous nucleic acid comprising a gene sequence. The cells contain 1) a DNA-targeting RNA and 2) a nucleic acid encoding a site-specific modifying polypeptide. The exogenous nucleic acid comprises a nucleic acid sequence.

[0275] In some instances, the site-directed modifying polypeptide is a Cas9 / C polypeptide, as shown in FIG. to amino acids 7 to 166 or 731 to 1003 of the sn1 amino acid sequence, or Any of the amino acid sequences set forth as numbers 1 to 256 and 795 to 1346 At least about 75%, at least about 80%, at least about 8% of the corresponding portion in 5%, at least about 90%, at least about 95%, at least about 99%, or 100% and amino acid sequences having an amino acid sequence identity of

[0276] composition The present invention includes the subject DNA-targeting RNA and / or site-specific modifying polypeptides. In some instances, the site-directed modifying polypeptide is a subject The subject compositions are useful in the methods of the disclosure, e.g., for site-specific target DNA sequencing. method for site-specific modification of polypeptides bound to target DNA; It is useful to

[0277] Compositions Comprising DNA-Targeting RNA The present invention provides compositions comprising the subject DNA-targeting RNA. In addition to the targeted RNA, salts, e.g., NaCl, MgCl, KCl, MgSO, etc.; buffers agents, such as Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethylhexyl)piperazine 2-(N-morpholino)ethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES) , MES sodium salt, 3-(N-morpholino)propanesulfonic acid (MOPS), N- Tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; Solubilizers; surfactants, such as nonionic surfactants, e.g., Tween-20; For example, in some instances, the compound may include one or more of the following: a lyase inhibitor; The subject compositions include a subject DNA-targeting RNA and a buffer for stabilizing nucleic acids. include.

[0278] In some embodiments, the DNA-targeting RNA present in the subject compositions is pure For example, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or is greater than 99% pure, where "% pure" means that the DNA-targeting RNA is free of other macromolecules, or the stated percentage of contaminants that may be present during the manufacture of DNA-targeting RNA. It means not to include.

[0279] Compositions Comprising the Subject Chimeric Polypeptides The present invention provides the subject chimeric polypeptide compositions. The compositions comprise a DNA-targeting R In addition to NA, salts such as NaCl, MgCl2, KCl, MgSO4, etc.; buffering agents, For example, Tris buffer, HEPES, MES, MES sodium salt, MOPS, TAPS etc.; solubilizers; surfactants, for example, nonionic surfactants, for example, Tween-20 etc.; protease inhibitors; reducing agents (e.g., dithiothreitol); etc. may include multiple.

[0280] In some embodiments, a subject chimeric polypeptide present in a subject composition is , pure, e.g., at least about 75%, at least about 80%, at least about 85% , at least about 90%, at least about 95%, at least about 98%, at least about 99% or greater than 99% pure, where "% pure" means that the site-directed modified polypeptide is , other proteins, other macromolecules, or contaminants that may be present during the production of the chimeric polypeptide. This means that the product does not contain the stated percentage of the ingredient.

[0281] Compositions Comprising DNA-Targeting RNA and Site-Specific Modifying Polypeptides The present invention provides (i) a DNA-targeting RNA or a DNA polynucleotide encoding the same; and ii) a site-directed modified polypeptide, or a polynucleotide encoding the same. In some instances, the site-directed modifying polypeptide is a subject In another example, the site-directed modified polypeptide is a chimeric site-directed modified polypeptide. A tide is a naturally occurring site-specifically modified polypeptide. The specific modifying polypeptide exhibits an enzymatic activity that modifies the target DNA. The site-specific modifying polypeptide has an enzymatic activity that modifies the polypeptide bound to the target DNA. In yet another example, the site-specific modifying polypeptide regulates transcription of the target DNA. do.

[0282] The present invention provides a method for detecting a target DNA fragment comprising: (i) (a) a nucleotide sequence complementary to a sequence in the target DNA; and (b) a second segment that interacts with the site-directed modifying polypeptide. the DNA-targeting RNA or a DNA polynucleotide encoding the same; and (ii) (a) an RNA-binding portion that interacts with the DNA-targeting RNA; and (b) a portion An active site that exhibits site-specific enzymatic activity, wherein the site of enzymatic activity is determined by DNA-targeting RNA. A site-directed modified polypeptide containing an active site determined by the method of the present invention, or a polypeptide encoding the same. The present invention provides a composition comprising:

[0283] In some instances, the subject compositions comprise: (i) (a) a sequence complementary to a sequence within the target DNA; (b) a first segment comprising a site-specifically modified polypeptide; and a subject DNA-targeting RNA comprising a second segment that interacts with (ii)(a) (b) an RNA-binding site that interacts with the DNA-targeting RNA; and (b) a site-specific enzymatic activity. Active sites showing the enzyme activity are determined by the DNA-targeting RNA. The present invention provides a composition comprising a site-directed modifying polypeptide comprising a site.

[0284] In other embodiments, the subject compositions comprise: (i) (a) a sequence complementary to a sequence within the target DNA; (b) a first segment comprising a nucleotide sequence; and (c) a segment corresponding to the site-directed modifying polypeptide. A polynucleotide encoding the subject DNA-targeting RNA containing an interacting second segment. and (ii) (a) an RNA-binding site that interacts with the DNA-targeting RNA; and b) an active site that exhibits site-specific enzymatic activity, wherein the site of enzymatic activity is a DNA-targeting RNA A polynucleotide encoding a site-specifically modified polypeptide containing an active site, as determined by including leotide.

[0285] In some embodiments, the subject compositions comprise a double-molecule DNA-targeting RNA. Thus, in some embodiments, the subject compositions comprise both targeted RNA molecules. A double-stranded segment complementary to the double-stranded segment of A is included. (See Figure 1A.) The duplex-forming segments of the activator-RNA and targeter-RNA are hybridization to form a dsRNA duplex of the protein-binding segment of the DNA-targeting RNA. The targeting RNA further comprises a DNA-targeting segment (single-stranded ) to target the DNA-targeting RNA to a specific sequence within the target DNA. In one non-limiting example, the duplex-forming segment of the activator RNA has the sequence 5'-UAGC AAGUUAAAAU-3' (SEQ ID NO: 562) and at least about 70%, at least about 8 0%, at least about 90%, at least about 95%, at least about 98%, or 100% As another non-limiting example, the double stranded targeting RNA The forming segment has the sequence 5'-GUUUUAGAGCUA-3' (SEQ ID NO: 679) and a small At least about 70%, at least about 80%, at least about 90%, at least about 95%, It includes nucleotide sequences with at least about 98%, or 100% identity.

[0286] The present disclosure provides a method for detecting a target DNA fragment comprising: (i) (a) a nucleotide sequence complementary to a sequence in the target DNA; and (b) a second segment that interacts with the site-directed modifying polypeptide. a DNA-targeting RNA comprising the following, or a DNA polynucleotide encoding the same; and ii) (a) an RNA-binding site that interacts with the DNA-targeting RNA; and (b) a target DNA The active site that regulates transcription in A is the site where transcription is regulated in the target DNA. a site-directed modifying polypeptide containing an active site determined by a targeting RNA; or and a polynucleotide encoding the same.

[0287] For example, in some instances, the subject compositions (i) (a) target a sequence within the target DNA; and (b) a first segment comprising a nucleotide sequence complementary to the site-specifically modified polynucleotide. (ii) a DNA-targeting RNA comprising a second segment that interacts with the peptide; and ) an RNA-binding site that interacts with the DNA-targeting RNA; and (b) a transcription factor within the target DNA. The site in the target DNA where transcription is regulated is a DNA-targeted RNA. A site-directed modified polypeptide comprising an active site determined by A.

[0288] As another example, in some instances, the subject compositions comprise: (i) (a) a target DNA fragment; (b) a first segment comprising a nucleotide sequence complementary to the site-specific modification sequence; D encodes a DNA-targeting RNA containing a second segment that interacts with the decorative polypeptide. (ii) a DNA-targeting RNA that interacts with the DNA-targeting RNA; and (ii) a DNA-targeting RNA that interacts with the DNA-targeting RNA. (b) an active site that regulates transcription within the target DNA; The site where transcription is regulated is determined by the DNA-targeting RNA, including the active site. It includes polynucleotides that encode specific modified polypeptides.

[0289] The subject compositions include: i) a subject DNA-targeting RNA, or a DNA polypeptide encoding the same; nucleotides; and ii) site-specifically modified polypeptides, or polypeptides encoding same. In addition to nucleotides, salts, e.g., NaCl, MgCl, KCl, MgSO, etc.; buffer Buffers, such as Tris buffer, HEPES, MES, MES sodium salt, MOPS, T APS, etc.; solubilizers; surfactants, for example, nonionic surfactants, for example, Tween -20, etc.; protease inhibitors; reducing agents (e.g., dithiothreitol); It may include one or more.

[0290] In some instances, the components of the composition are each pure, e.g., each component is: At least about 75%, at least about 80%, at least about 90%, at least about 95%, At least about 98%, at least about 99%, or at least 99% pure. In some instances, each component of the subject composition is pure prior to being added to the composition. be.

[0291] For example, in some embodiments, the site-specifically modified polynucleotides present in the subject compositions The peptides are pure, e.g., at least about 75%, at least about 80%, at least about 100% pure. at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least The site-specifically modified polynucleotides are also about 99% or greater than 99% pure, where "% pure" refers to the site-specifically modified polynucleotides. The peptide may be a polypeptide of another protein (e.g., a protein other than the site-directed modified polypeptide). , other macromolecules, or contaminants that may be present during the production of the site-directed modified polypeptide. This means that the listed percentage is excluded.

[0292] kit The present disclosure provides kits for carrying out the subject methods. The subject kits include site-specific modified polypeptides; nucleic acids comprising nucleotides encoding site-specifi...

Claims

1. (i) a first segment comprising a nucleotide sequence complementary to a sequence in the target DNA; and Beauty (ii) a second segment that interacts with the site-directed modifying polypeptide; A DNA-targeting RNA comprising:

2. The first segment is composed of eight nucleotides that are 100% complementary to a sequence in the target DNA. The DNA-targeting RNA of claim 1 , comprising a nucleotide.

3. the second segment is any one of the nucleotide sequences set forth in SEQ ID NOs: 563-682 For each of the sequences, at least 6 sequences are present over a stretch of at least 8 consecutive nucleotides.

10. The nucleotide sequence of claim 1, wherein the nucleotide sequence has 0.0% identity to the nucleotide sequence of claim 1, or a complement thereof. NA targeting RNA.

4. the second segment is any one of the nucleotide sequences set forth in SEQ ID NOs: 431 to 562 For each of the sequences, at least 6 sequences are present over a stretch of at least 8 consecutive nucleotides.

10. The nucleotide sequence of claim 1, wherein the nucleotide sequence has 0.0% identity to the nucleotide sequence of claim 1, or a complement thereof. NA targeting RNA.

5. The site-directed modifying polypeptide has the Cas9 / Csn1 amino acid sequence shown in FIG. or to amino acids 7 to 166 or 731 to 1003 of SEQ ID NOs: 1 to 256 and and the corresponding portion of any of the amino acid sequences set forth as 795-1346.

3. The method of claim 1, further comprising:

2. The DNA-targeting RNA according to claim 1.

6. A DNA polymerase comprising a nucleotide sequence encoding the DNA-targeting RNA of claim 1. Ligands.

7. A recombinant expression vector comprising the DNA polynucleotide of claim 6.

8. the nucleotide sequence encoding the DNA-targeting RNA is operable with a promoter The recombinant expression vector of claim 7, wherein the

9. The recombinant expression vector of claim 8, wherein the promoter is an inducible promoter. -.

10. The nucleotide sequence encoding the DNA-targeting RNA of claim 1 is The recombinant expression vector of claim 7, further comprising a coding site.

11. An in vitro genetically modified host cell comprising the DNA polynucleotide of claim 6.

12. (i) (a) a first segment comprising a nucleotide sequence complementary to a sequence in the target DNA; and Call (b) a second segment that interacts with the site-directed modifying polypeptide; a nucleotide sequence encoding a DNA-targeting RNA comprising: (ii) (a) an RNA-binding site that interacts with the DNA-targeting RNA; and (b) an active site that exhibits site-specific enzymatic activity, the site of enzymatic activity being related to the DNA target; The active site is determined by the RNA a nucleotide sequence encoding the site-directed modifying polypeptide comprising A recombinant expression vector comprising:

13. (i) (a) a first segment comprising a nucleotide sequence complementary to a sequence in the target DNA; and Call (b) a second segment that interacts with the site-directed modifying polypeptide; a nucleotide sequence encoding a DNA-targeting RNA comprising: (ii) (a) an RNA-binding site that interacts with the DNA-targeting RNA; and (b) an active site that regulates transcription within the target DNA; The site at which the DNA-targeting RNA is regulated is determined by the active site. a nucleotide sequence encoding the site-directed modifying polypeptide, comprising: A recombinant expression vector comprising:

14. (i) a DNA-targeting RNA comprising a nucleotide sequence complementary to a sequence within the target DNA; interacting RNA binding sites; and (ii) an active site that exhibits reduced site-specific enzymatic activity, wherein the site of enzymatic activity is Active site determined by DNA-targeting RNA A mutant site-directed modified polypeptide comprising:

15. The H840A mutation in the S. pyogenes sequence (SEQ ID NO: 8) or any of the amino acid sequences set forth as SEQ ID NOs: 1-256 and 795-1346 15. The mutant site-directed modifying polypeptide of claim 14, comprising a corresponding mutation in 。

16. The D10A mutation of the S. pyogenes sequence (SEQ ID NO: 8), or SEQ ID NOs: 1-256 and 795-1346 The mutant site-directed modified polypeptide of claim 14.

17. (i) the D10A mutation of the S. pyogenes sequence (SEQ ID NO: 8), or SEQ ID NOs: 1-256 and the corresponding mutation in any of the amino acid sequences set forth as 795-1346 and (ii) the H840A mutation of the S. pyogenes sequence (SEQ ID NO: 8), or SEQ ID NO: 1-256 and 795-1346 of the amino acid sequence 15. The mutant site-directed modified polypeptide of claim 14, comprising both of the corresponding mutations.

18. (i) a DNA-targeting RNA comprising a nucleotide sequence complementary to a sequence within the target DNA; interacting RNA binding sites; and (ii) an active site that exhibits site-specific enzymatic activity, the site of enzymatic activity being the DNA target The active site is determined by the RNA A chimeric site-directed modified polypeptide comprising:

19. Amino acids 7 to 166 or 731 of the Cas9 / Csn1 amino acid sequence shown in FIG. to 1003, or as set forth as SEQ ID NOs: 1 to 256 and 795 to 1346 and at least about 75% amino acid identity to the corresponding portion of any of the amino acid sequences 19. The chimeric site-directed modification of claim 18, comprising an amino acid sequence having amino acid sequence identity with Polypeptides

20. the DNA-targeting RNA is selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOs: 563-682 at least one of the following over a stretch of at least 8 consecutive nucleotides:

19. The chimeric moiety of claim 18, further comprising a nucleotide sequence having at least 60% identity to said Site-specifically modified polypeptides.

21. the DNA-targeting RNA is selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOs: 431 to 562 at least one of the following over a stretch of at least 8 consecutive nucleotides:

19. The chimeric moiety of claim 18, further comprising a nucleotide sequence having at least 60% identity to said Site-specifically modified polypeptides.

22. 19. The chimeric site-specific modification of claim 18, wherein the enzymatic activity modifies the target DNA. Polypeptide.

23. The enzyme activity is selected from the group consisting of nuclease activity, methyltransferase activity, demethylase activity, and the like. activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkyl Depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, Transposase activity, recombinase activity, polymerase activity, ligase activity, helical activity 23. The chimera of claim 22, wherein the chimera has a glycosylase activity, a photolyase activity, or a glycosylase activity. Site-directed modified polypeptides.

24. 24. The chimeric site-specific modification polymer of claim 23, wherein the enzymatic activity is a nuclease activity. Lipeptide.

25. 25. The method of claim 24, wherein the nuclease activity causes a double-strand break in the target DNA. The chimeric site-directed modified polypeptide described above.

26. 19. The method of claim 18, wherein the enzymatic activity modifies a target polypeptide bound to the target DNA.

2. A chimeric site-directed modified polypeptide as described above.

27. The enzyme activity is a methyltransferase activity, a demethylase activity, an acetyltransfer ... acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin activity Chitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, SUM O-desumoylation activity, desumoylation activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylating activity. Chid.

28. the target polypeptide is a histone and the enzymatic activity is a methyltransferase activity activity, demethylase activity, acetyltransferase activity, deacetylase activity, enzyme activity, phosphatase activity, ubiquitin ligase activity, or deubiquitinating activity 27. The chimeric site-directed modifying polypeptide of claim 26.

29. A nucleotide sequence encoding the chimeric site-directed modifying polypeptide of claim 18. An RNA polynucleotide comprising:

30. A nucleotide sequence encoding the chimeric site-directed modifying polypeptide of claim 18. A DNA polynucleotide comprising:

31. A recombinant expression vector comprising the polynucleotide of claim 30.

32. 32. The method of claim 31 , wherein the polynucleotide is operably linked to a promoter. Recombinant expression vectors.

33. 33. The recombinant expression vector of claim 32, wherein the promoter is an inducible promoter. Tar.

34. 31. An in vitro genetically modified host cell comprising the polynucleotide of claim 30.

35. (i) a DNA-targeting RNA comprising a nucleotide sequence complementary to a sequence within the target DNA; interacting RNA binding sites; and (ii) an active site that regulates transcription within the target DNA, The site at which the DNA-targeting RNA is regulated is determined by the active site. A chimeric site-directed modified polypeptide comprising:

36. 36. The chimeric site of claim 35, wherein the active site increases transcription in the target DNA. Specific modified polypeptides.

37. 36. The chimeric site of claim 35, wherein the active site reduces transcription in the target DNA. Specific modified polypeptides.

38. an RNA-binding site that interacts with the DNA-targeting RNA; and exhibits site-specific enzymatic activity. an active site, the site of enzymatic activity being determined by said DNA-targeting RNA; A genetically modified cell comprising a recombinant site-specifically modified polypeptide comprising the site.

39. The site-directed modifying polypeptide has the Cas9 / Csn1 amino acid sequence shown in FIG. or to amino acids 7 to 166 or 731 to 1003 of SEQ ID NOs: 1 to 256 and and the corresponding portion of any of the amino acid sequences set forth as 795-1346 2. A method for producing a medicament comprising the steps of: Item 39. The genetically modified cell of item 38.

40. The cell is an archaeal cell, a bacterial cell, a eukaryotic cell, a eukaryotic unicellular organism, a somatic cell, a germ cell, Stem cells, plant cells, algae cells, animal cells, invertebrate cells, vertebrate cells, fish cells, mosquitoes Human cells, bird cells, mammalian cells, porcine cells, bovine cells, goat cells, sheep cells, rodent cells a mammalian cell, a rat cell, a mouse cell, a non-human primate cell, and a human cell; The genetically modified cell of claim 38, wherein the cell is selected from the group consisting of:

41. The genome comprises: (i) an RNA-binding site that interacts with a DNA-targeting RNA; and (ii) an active site that exhibits site-specific enzymatic activity, the site of enzymatic activity being located within the DNA-targeting RNA; A nucleic acid encoding a recombinant site-specifically modified polypeptide containing an active site, as determined by Transgenic non-human organisms containing a transgene comprising a nucleotide sequence.

42. The site-directed modifying polypeptide has the Cas9 / Csn1 amino acid sequence shown in FIG. or to amino acids 7 to 166 or 731 to 1003 of SEQ ID NOs: 1 to 256 and and the corresponding portion of any of the amino acid sequences set forth as 795-1346 2. A method for producing a medicament comprising the steps of: Item 42. A transgenic organism according to Item 41.

43. The organism may be an archaea, a bacterium, a eukaryotic single-cell organism, an alga, a plant, an animal, an invertebrate, a fly, or an insect. , Cnidarians, Vertebrates, Fish, Frogs, Birds, Mammals, Ungulates, Rodents, Rats, 42. The gene of claim 41, selected from the group consisting of mouse, and non-human primate. Introduced organisms.

44. (i) (a) a first segment comprising a nucleotide sequence complementary to a sequence in the target DNA; and Call (b) a second segment that interacts with the site-directed modifying polypeptide; or a DNA polynucleotide encoding the same; and (ii) (a) an RNA-binding site that interacts with a DNA-targeting RNA; and (b) an active site that exhibits site-specific enzymatic activity, the site of enzymatic activity being related to the DNA target; The active site is determined by the RNA or a polynucleotide encoding the site-directed modified polypeptide, A composition comprising:

45. The first segment of the DNA-targeting RNA is at least partially related to a sequence within the target DNA.

45. The composition of claim 44, comprising at least 8 nucleotides with 100% complementarity. thing.

46. the second segment of the DNA-targeting RNA is a nucleic acid sequence set forth in SEQ ID NOs: 563-682 For any one of the nucleotide sequences, a series of at least 8 consecutive nucleotides 45. The method of claim 44, comprising a nucleotide sequence having at least 60% identity across the entire region. The composition described.

47. The second segment of the DNA-targeting RNA is a nucleic acid sequence set forth in SEQ ID NOs: 431-562. For any one of the nucleotide sequences, a series of at least 8 consecutive nucleotides 45. The method of claim 44, comprising a nucleotide sequence having at least 60% identity across the entire region. The composition described.

48. The site-directed modifying polypeptide has the Cas9 / Csn1 amino acid sequence shown in FIG. or to amino acids 7 to 166 or 731 to 1003 of SEQ ID NOs: 1 to 256 and and the corresponding portion of any of the amino acid sequences set forth as 795-1346 2. A method for producing a medicament comprising the steps of: Item 45. The composition according to item 44.

49. 45. The composition of claim 44, wherein the enzymatic activity modifies the target DNA.

50. The enzyme activity is selected from the group consisting of nuclease activity, methyltransferase activity, demethylase activity, and the like. activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkyl Depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, Transposase activity, recombinase activity, polymerase activity, ligase activity, helical activity 50. The composition of claim 49, wherein the activity is a glycosylase, a photolyase, or a glycosylase. 。

51. 51. The composition of claim 50, wherein the enzymatic activity is a nuclease activity.

52. 52. The method of claim 51, wherein the nuclease activity causes a double-strand break in the target DNA. The composition described above.

53. 45. The method of claim 44, wherein the enzymatic activity modifies a target polypeptide bound to the target DNA. The composition described.

54. The enzyme activity is a methyltransferase activity, a demethylase activity, an acetyltransfer ... acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin activity Chitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, SUM O-desumoylation activity, desumoylation activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylating activity.

55. the target polypeptide is a histone and the enzymatic activity is a methyltransferase activity activity, demethylase activity, acetyltransferase activity, deacetylase activity, enzyme activity, phosphatase activity, ubiquitin ligase activity, or deubiquitinating activity 54. The composition of claim 53.

56. The DNA-targeting RNA is a double-molecule DNA-targeting RNA, and the composition is a targeting RNA. The double-stranded segments of the RNA are complementary to each other.

45. The method of claim 44, wherein the DNA-targeting RNA is hybridized to form the second segment of the DNA-targeting RNA. The composition described.

57. The duplex-forming segment of the activator-RNA is a nucleic acid sequence set forth in SEQ ID NOs: 431-682. For any one of the nucleotide sequences, a series of at least 8 consecutive nucleotides 57. The method of claim 56, comprising a nucleotide sequence having at least 60% identity across the entire region. The composition described.

58. (i) a DNA-targeting RNA according to claim 44, or a DNA polynucleotide encoding the same; leotide; and (ii) Buffer for stabilizing nucleic acids A composition comprising:

59. (i) A site-directed modified polypeptide according to claim 44, or a polypeptide encoding the same. nucleotides; and (ii) Buffers for stabilizing nucleic acids and / or proteins A composition comprising:

60. (i) (a) a first segment comprising a nucleotide sequence complementary to a sequence in the target DNA; and Call (b) a second segment that interacts with the site-directed modifying polypeptide; or a DNA polynucleotide encoding the same; and (ii) (a) an RNA-binding site that interacts with the DNA-targeting RNA; and (b) an active site that regulates transcription within the target DNA; The site at which the DNA-targeting RNA is regulated is determined by the active site. or a polynucleotide encoding the site-directed modified polypeptide, A composition comprising:

61. 61. The composition of claim 60, wherein the active site increases transcription in the target DNA.

62. 61. The composition of claim 60, wherein the active site reduces transcription in the target DNA.

63. (i) A site-directed modified polypeptide according to claim 60, or a polypeptide encoding the same. nucleotides; and (ii) Buffers for stabilizing nucleic acids and / or proteins A composition comprising:

64. 1. A method for site-specific modification of target DNA, comprising: The target DNA (i) (a) a first segment comprising a nucleotide sequence complementary to a sequence within said target DNA; and (b) a second segment that interacts with the site-directed modifying polypeptide; or a DNA polynucleotide encoding the same; and (ii) (a) an RNA-binding site that interacts with the DNA-targeting RNA; and (b) Active site exhibiting site-specific enzymatic activity or a polynucleotide encoding the same. The method as described above, comprising contacting the

65. 65. The method of claim 64, wherein the target DNA is present extrachromosomally.

66. The target DNA contains a PAM sequence on the complementary strand that is 5'-CCY-3', where Y is any DNA nucleotide, and Y is immediately 5' to the target sequence on the complementary strand of said target DNA.

65. The method of claim 64, wherein:

67. 65. The method of claim 64, wherein the target DNA is part of a chromosome in vitro.

68. 65. The method of claim 64, wherein the target DNA is part of a chromosome in vivo.

69. 65. The method of claim 64, wherein the target DNA is part of a chromosome in a cell.

70. The cell is an archaeal cell, a bacterial cell, a eukaryotic cell, a eukaryotic unicellular organism, a somatic cell, a germ cell, Stem cells, plant cells, algae cells, animal cells, invertebrate cells, vertebrate cells, fish cells, mosquitoes Human cells, bird cells, mammalian cells, porcine cells, bovine cells, goat cells, sheep cells, rodent cells a mammalian cell, a rat cell, a mouse cell, a non-human primate cell, and a human cell; 70. The method of claim 69, wherein

71. the DNA-targeting RNA is selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOs: 563-682 at least one of the following over a stretch of at least 8 consecutive nucleotides:

65. The method of claim 64, wherein the sequence comprises a nucleotide sequence having at least 60% identity to the sequence.

72. the DNA-targeting RNA is selected from the group consisting of the nucleotide sequences set forth in SEQ ID NOs: 431 to 562 at least one of the following over a stretch of at least 8 consecutive nucleotides:

65. The method of claim 64, wherein the sequence comprises a nucleotide sequence having at least 60% identity to the sequence.

73. The DNA-modified polypeptide has the amino acid sequence of Cas9 / Csn1 shown in FIG. to amino acids 7-166 or 731-1003, or to SEQ ID NOs: 1-256 and 795-1346 of the corresponding portion of any of the amino acid sequences set forth in and an amino acid sequence having at least about 75% amino acid sequence identity with the target gene.

4. The method according to claim 4.

74. 65. The method of claim 64, wherein the enzymatic activity modifies the target DNA.

75. The enzyme activity is a nuclease activity, a methyltransferase activity, a demethylase activity, or activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, Transposase activity, recombinase activity, polymerase activity, ligase activity, helical activity 75. The method of claim 74, wherein the activity is a photolyase activity, a photolyase activity, or a glycosylase activity.

76. 76. The method of claim 75, wherein the DNA modifying enzymatic activity is a nuclease activity.

77. 77. The method of claim 76, wherein the nuclease activity causes a double-strand break in the target DNA. How to post.

78. 8. The method of claim 7, wherein the contacting occurs under conditions permissive for non-homologous end joining or homology directed repair.

7. The method according to claim 7.

79. contacting the target DNA with a donor polynucleotide, Polynucleotide, a portion of the donor polynucleotide, A copy, or a portion of a copy of the donor polynucleotide, is integrated into the target DNA.

79. The method of claim 78,

80. The method does not involve contacting a cell with a donor polynucleotide, and does not involve the detection of nucleotides within the target DNA.

79. The method of claim 78, wherein the target DNA is modified so that a nucleotide is deleted.

81. 65. The method of claim 64, wherein the enzymatic activity modifies a target polypeptide bound to the target DNA. The method described.

82. The enzyme activity is a methyltransferase activity, a demethylase activity, an acetyltransfer ... acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin activity Chitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, SUM O-desumoylation activity, desumoylation activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity.

83. the target polypeptide is a histone and the enzymatic activity is a methyltransferase activity activity, demethylase activity, acetyltransferase activity, deacetylase activity, enzyme activity, phosphatase activity, ubiquitin ligase activity, or deubiquitinating activity The method of claim 81.

84. 65. The method of claim 64, wherein the complex further comprises an activator-RNA.

85. The activator-RNA is any one of the nucleotide sequences set forth in SEQ ID NOs: 431 to 682. For each of them, at least 60 over a stretch of at least 8 consecutive nucleotides 85. The method of claim 84, comprising a nucleotide sequence having % identity to the sequence of claim 84.

86. 1. A method for modulating site-specific transcription in a target DNA, comprising: (i) (a) a first segment comprising a nucleotide sequence complementary to a sequence within said target DNA; and (b) a second segment that interacts with the site-directed modifying polypeptide; or a DNA polynucleotide encoding the same; and (ii) (a) an RNA-binding site that interacts with the DNA-targeting RNA; and (b) Active sites that regulate transcription or a polynucleotide encoding the same. wherein said contacting results in modulation of transcription within said target DNA. , the above method.

87. 87. The method of claim 86, wherein transcription within the target DNA is increased.

88. 87. The method of claim 86, wherein transcription within the target DNA is reduced.

89. 1. A method for site-specific modification in target DNA, comprising: The target DNA (i) (a) a first segment comprising a nucleotide sequence complementary to a sequence within said target DNA; and (b) a second segment that interacts with the site-directed modifying polypeptide; or a DNA polynucleotide encoding the same; and (ii) (a) an RNA-binding site that interacts with the DNA-targeting RNA; and (b) an active site within the target DNA that regulates transcription; or a polynucleotide encoding the same. The method as described above, comprising contacting the

90. 86. Claim 86, wherein said site-directed modifying polypeptide increases transcription in said target DNA. The method described below.

91. 86. Claim 86, wherein said site-directed modifying polypeptide reduces transcription in said target DNA. The method described below.

92. 1. A method for promoting site-specific cleavage and modification of target DNA in a cell, comprising: The cells (i) (a) a first segment comprising a nucleotide sequence complementary to a sequence within said target DNA; and (b) a second segment that interacts with the site-directed modifying polypeptide; or a DNA polynucleotide encoding the same; and (ii) (a) an RNA-binding site that interacts with the DNA-targeting RNA; and (b) an active site that exhibits nuclease activity that generates a double-strand break in the target DNA; or a polynucleotide encoding the same; including introducing The site of the double-strand break is determined by the DNA-targeting RNA, and the contact is between the non-homologous ends. occurs under conditions permissive for end-joining or homology-directed repair, and the target DNA is cleaved and reassembled. ligated to produce a modified DNA sequence; The above method.

93. contacting the target DNA with a donor polynucleotide, Polynucleotide, a portion of the donor polynucleotide, A copy, or a portion of a copy of the donor polynucleotide, is integrated into the target DNA.

93. The method of claim 92.

94. a nucleic acid sequence within the target DNA, the nucleic acid sequence not including contacting the cell with a donor polynucleotide; 93. The method of claim 92, wherein the target DNA is modified so that a nucleotide is deleted.

95. The cell is an archaeal cell, a bacterial cell, a eukaryotic cell, a eukaryotic unicellular organism, a somatic cell, a germ cell, Stem cells, plant cells, algae cells, animal cells, invertebrate cells, vertebrate cells, fish cells, mosquitoes Human cells, bird cells, mammalian cells, porcine cells, bovine cells, goat cells, sheep cells, rodent cells a mammalian cell, a rat cell, a mouse cell, a non-human primate cell, and a human cell; 93. The method of claim 92, wherein

96. 93. The method of claim 92, wherein the cell is in vitro.

97. 93. The method of claim 92, wherein the cell is in vivo.

98. 1. A method of producing genetically modified cells in a subject, comprising: (I) a cell, (i) (a) a first segment comprising a nucleotide sequence complementary to a sequence in the target DNA; and Call (b) a second segment that interacts with the site-directed modifying polypeptide; or a DNA polynucleotide encoding the same; and (ii) (a) an RNA-binding site that interacts with the DNA-targeting RNA; and (b) an active site that exhibits nuclease activity that generates a double-strand break in the target DNA; A site-specific modified polypeptide comprising the compound or a polynucleotide encoding the same is introduced. ； wherein the site of the double-strand break is determined by the DNA-targeting RNA and the contact is occurs under conditions permissive for non-homologous end joining or homology-directed repair, resulting in cleavage of the target DNA. and recombined to produce a modified DNA sequence; thereby producing said genetically modified cell; and (II) transplanting the genetically modified cells into the subject. The above method, comprising:

99. contacting the cells with a donor polynucleotide, Nucleotides, portions of the donor polynucleotides, and copies of the donor polynucleotides or a portion of a copy of the donor polynucleotide is integrated into the target DNA; 99. The method of claim 98.

100. a nucleic acid sequence within the target DNA, the nucleic acid sequence not including contacting the cell with a donor polynucleotide; 99. The method of claim 98, wherein the target DNA is modified so that a nucleotide is deleted.

101. The cell is an archaeal cell, a bacterial cell, a eukaryotic cell, a eukaryotic unicellular organism, a somatic cell, a germ cell, Stem cells, plant cells, algae cells, animal cells, invertebrate cells, vertebrate cells, fish cells, amphibian mammalian cells, ungulate cells, rodent cells, non-human primate cells, and and human cells.

102. Genetically modified cells containing a nucleotide sequence encoding an exogenous site-specifically modified polypeptide.

1. A method for modifying target DNA in a cell, comprising: The genetically modified cells are then transfected with a DNA-targeting RNA or a DNA polynucleotide encoding the same. including introducing an octide, (i) the DNA-targeting RNA is (a) a first segment comprising a nucleotide sequence complementary to a sequence within said target DNA; and (b) a second segment that interacts with the site-directed modifying polypeptide; (ii) the site-directed modifying polypeptide is (a) an RNA-binding site that interacts with the DNA-targeting RNA; and (b) comprises an active site that exhibits nuclease activity; The above method.

103. The site-directed modifying polypeptide has the Cas9 / Csn1 amino acid sequence shown in FIG. or to amino acids 7 to 166 or 731 to 1003 of SEQ ID NOs: 1 to 256 and and the corresponding portion of any of the amino acid sequences set forth as 795-1346 2. A method for producing a medicament comprising the steps of: Item 103. The method according to item 102.

104. The cell is an archaeal cell, a bacterial cell, a eukaryotic cell, a eukaryotic unicellular organism, a somatic cell, a germ cell, Stem cells, plant cells, algae cells, animal cells, invertebrate cells, vertebrate cells, fish cells, amphibian mammalian cells, ungulate cells, rodent cells, non-human primate cells, and and human cells.

105. 103. The method of claim 102, wherein the cell is in vivo.

106. 103. The method of claim 102, wherein the cell is in vitro.

107. wherein the expression of the site-directed modifying polypeptide is under the control of an inducible promoter.

102. The method according to claim 102.

108. wherein expression of the site-specific modifying polypeptide is under the control of a cell type-specific promoter. The method of claim 102.

109. (i) The DNA-targeting RNA of claim 1 or a DNA polynucleotide encoding the same. Ochid; and (ii) Reconstitution and / or dilution reagents Includes a kit.

110. Buffers for introducing the DNA-targeting RNA into cells, washing buffers, control reagents, control Expression vector or RNA polynucleotide, transcribing DNA-targeting RNA from DNA and combinations thereof. The kit of claim 109.

111. (i) A site-directed modified polypeptide according to claim 44, or a polypeptide encoding the same. nucleotides; and (ii) Reconstitution and / or dilution reagents Includes a kit.

112. A buffer for introducing the site-specific modifying polypeptide into cells, a washing buffer, a control buffer, the site-specific modification of the drug, control expression vector or RNA polynucleotide, DNA Reagents for in vitro production of polypeptides, and combinations thereof 112. The kit of claim 111, further comprising a selected reagent.

113. (i) A site-directed modified polypeptide according to claim 60, or a polypeptide encoding the same. nucleotides; and (ii) Reconstitution and / or dilution reagents Includes a kit.

114. (i) (a) a first segment comprising a nucleotide sequence complementary to a sequence in the target DNA; and Call (b) a second segment that interacts with the site-directed modifying polypeptide; or a DNA polynucleotide encoding the same; and (ii) (a) an RNA-binding site that interacts with the DNA-targeting RNA; and (b) an active site that exhibits site-specific enzymatic activity, the site of enzymatic activity being related to the DNA target; The active site is determined by the RNA or a polynucleotide encoding the site-directed modified polypeptide, Includes a kit.

115. (i) (a) a first segment comprising a nucleotide sequence complementary to a sequence in the target DNA; and Call (b) a second segment that interacts with the site-directed modifying polypeptide; or a DNA polynucleotide encoding the same; and (ii) (a) an RNA-binding site that interacts with the DNA-targeting RNA; and (b) an active site that regulates transcription within the target DNA; The site at which the DNA-targeting RNA is regulated is determined by the active site. or a polynucleotide encoding the site-directed modified polypeptide, Includes a kit.

116. (i) the recombinant expression vector of claim 12; and (ii) Reconstitution and / or dilution reagents Includes a kit.

117. (i) the recombinant expression vector of claim 13; and (ii) Reconstitution and / or dilution reagents Includes a kit.

118. (i) the recombinant expression vector of claim 7; and (ii) Reconstitution and / or dilution reagents Includes a kit.

119. (i) the recombinant expression vector of claim 7; and (ii) (a) an RNA-binding site that interacts with a DNA-targeting RNA; and (b) an active site that exhibits site-specific enzymatic activity, the site of enzymatic activity being related to the DNA target; The active site is determined by the RNA A recombinant expression vector comprising a nucleotide sequence encoding a site-specifically modified polypeptide comprising Kutar Includes a kit.

120. (i) the recombinant expression vector of claim 7; and (ii) (a) an RNA-binding site that interacts with a DNA-targeting RNA; and (b) an active site that regulates transcription in the target DNA, The site of the targeting is determined by the DNA-targeting RNA. A recombinant expression vector comprising a nucleotide sequence encoding a site-specifically modified polypeptide comprising Kutar Includes a kit.

121. Two or more DNA-targeting RNAs according to claim 1, or DNA fragments encoding them. A kit for targeting target DNA containing nucleotides, comprising: At least one first segment of the DNA-targeting RNA is linked to the two or more DNA targets. and at least one nucleoside. The above kit, only the chid is different.

122. A method for selectively regulating transcription of a target DNA in a host cell, comprising: 、 a) i) a first segment comprising a nucleotide sequence complementary to a target sequence within said target DNA; to; ii) a second segment that interacts with the site-directed polypeptide; and iii) Stability control sequence or a nucleotide sequence encoding said DNA-targeting RNA. a nucleic acid comprising a sequence; and b) i) an RNA-binding site that interacts with the DNA-targeting RNA; and ii) an active site that exhibits reduced endodeoxyribonuclease activity or a mutant Cas9 site-specific polypeptide comprising the mutant Cas9 site-specific polypeptide a nucleic acid comprising a nucleotide sequence encoding a polypeptide including introducing The DNA-targeting RNA and the mutant Cas9 polypeptide form a complex within the cell. and said complex selectively regulates transcription of target DNA in said cell.

123. The mutant Cas9 site-directed polypeptide cleaves the non-complementary strand of the target DNA.

10. The method of claim 1, wherein the target DNA is capable of cleaving the complementary strand of the target DNA but has a reduced ability to cleave the complementary strand of the target DNA.

23. The method according to claim 22.

124. The mutant site-directed polypeptide has the amino acid sequence H840A shown in FIG. Mutations or mutations of the amino acid sequences set forth in SEQ ID NOS: 1-256 and 795-1346 124. The method of claim 123, comprising a corresponding mutation in any of the following:

125. The mutant site-directed polypeptide cannot cleave the complementary strand of the target DNA.

123. The method of claim 122, wherein the target DNA fragment has a reduced ability to cleave non-complementary strands of the target DNA. How to post.

126. The mutant site-directed polypeptide is a D10A mutation of the amino acid sequence shown in FIG. or the amino acid sequences set forth in SEQ ID NOs: 1 to 256 and 795 to 1346 126. The method of claim 125, comprising a corresponding mutation in any of:

127. The mutant site-directed polypeptide has reduced ability to cleave the complementary strand of the target DNA.

13. The method of claim 12, wherein the target DNA has a reduced ability to cleave non-complementary strands of the target DNA.

2. The method according to claim 2.

128. The mutant site-directed polypeptide comprises: (i) D10 of the amino acid sequence shown in FIG. A mutation, or the amino acid sequences set forth in SEQ ID NOs: 1-256 and 795-1346 and (ii) the H840A mutation in SEQ ID NO: 8, or or any of the amino acid sequences set forth in SEQ ID NOs: 1 to 256 and 795 to 1346 and a corresponding mutation in either

129. 123. The method of claim 122, wherein the host cell is a prokaryotic cell.

130. 123. The method of claim 122, wherein the host cell is a eukaryotic cell.

131. 131. The method of claim 130, wherein the eukaryotic cell is a mammalian cell.

132. the transcription of the target DNA is greater than or equal to the transcription of the target DNA in the absence of the complex; 123. The method of claim 122, wherein the expression is inhibited by at least about 10%.

133. 122. The mutant Cas9 site-directed polypeptide comprises a heterologous polypeptide. The method described below.

134. The heterologous polypeptide may comprise a subcellular localization sequence, a detectable label, a stability control peptide, a transcription factor, a 134. The method of claim 133, comprising a transcription regulatory sequence, a protein binding sequence, or a combination thereof. How to post.

135. 135. The method of claim 134, wherein the heterologous polypeptide comprises a stability control peptide.

136. 136. The method of claim 135, wherein the stability control peptide comprises a degron sequence.

137. The heterologous polypeptide is a transcription activator, a transcription repressor, a histone lysine methyltransferase, lysine demethylase, histone lysine acetyltransferase enzymes, histone lysine deacetylases, DNA methylases, DNA demethylases, Boundary elements, peripheral recruitment elements, protein docking elements, and combinations thereof.

138. The transcription of the target DNA is increased compared to the transcription of the target DNA in the absence of the complex.

134. The method of claim 133, wherein the IL-10 level is increased by at least about 1.2 times.

139. The heterologous polypeptide is selected from the group consisting of a transcription activator, a histone lysine methyltransferase, , histone lysine demethylase, histone lysine acetyltransferase, DNA Demethylases, protein docking elements, and their combinations 139. The method of claim 138, selected from the group:

140. 123. The method of claim 122, wherein the DNA-targeting RNA is a single-molecule DNA-targeting RNA. method.

141. The method of claim 122, wherein the DNA-targeting RNA is a bimolecular DNA-targeting RNA. Law.

142. 123. The method of claim 122, wherein the host cell is transfected with a second DNA-targeting RNA or or a nucleic acid comprising a nucleotide sequence encoding said second DNA-targeting RNA. wherein the second DNA-targeting RNA comprises: a first segment comprising a nucleotide sequence complementary to a second target sequence within said target DNA; to; a second segment that interacts with the site-directed polypeptide; and Stability control sequence The above method, comprising:

143. the second segment has a length of about 30 nucleotides to about 60 nucleotides; 1) 5'-GUUUUAGAGCUA-(linker)-UAGCAAGUUAAAA-3 ’； 2) 5'-GUUUAGAGCUG-(linker)-CAGCAAGUUAAA-3'; 3) 5'-GUUUUAGAGCUG-(linker)-CAGCGAGUUAAA-3' ； 4) 5'-GUUUUUGUACUCU-(linker)-AGAAGCUACAAAGA U-3′; 5) 5'-GUUGUAGCUCC-(linker)-GUUGCUACAAU-3'; Call 6) 5'-GUUUUAGAGCUAGAAAAUAGCAAGUUAAAAAAAGGC UAGUCCG-3' at least about 95% nucleotide sequence identity to a nucleotide sequence selected from 123. The method of claim 122, comprising a nucleotide sequence having the following structure:

144. a) a first segment comprising a nucleotide sequence complementary to a target sequence within the target DNA; b) a second segment that interacts with the site-directed polypeptide; and c) Stability control sequence An isolated nucleic acid comprising a nucleotide sequence encoding a DNA-targeting RNA comprising:

145. the nucleotide sequence encoding the DNA-targeting RNA is operable with a promoter 145. The isolated nucleic acid of claim 144, wherein the nucleic acid is linked to

146. The nucleic acid has reduced endonuclease activity compared to wild-type Cas9. Further, a nucleotide sequence encoding a mutant Cas9 site-specific polypeptide exhibiting a specificity is provided.

145. The isolated nucleic acid of claim 144, comprising:

147. The nucleotide sequence encoding the mutant Cas9 site-directed polypeptide is 147. The isolated nucleic acid of claim 146, operably linked to a motor.

148. 25. A recombinant expression vector comprising the nucleic acid of claim 24.

149. 25. The isolated nucleic acid of claim 24, or the recombinant expression vector of claim 28. , genetically modified host cells.

150. A library of nucleic acids comprising a plurality of members, each nucleic acid member comprising: a) a first segment comprising a nucleotide sequence complementary to a target sequence within the target DNA; b) a second segment that interacts with the site-directed polypeptide; and c) Stability control sequence a nucleotide sequence encoding a DNA-targeting RNA comprising: Each member is identical to the other members of the library in the nucleotide sequence of the first segment. Members of the above library are different.

151. the nucleotide sequence encoding the DNA-targeting RNA is operable with a promoter 151. The library of claim 150, wherein the

152. a) i) a first segment comprising a nucleotide sequence complementary to a target sequence within the target DNA; ii) a second segment that interacts with the site-directed polypeptide; and iii) Stability control sequence or a nucleotide sequence encoding said DNA-targeting RNA. a nucleic acid comprising a sequence; and b) Buffer Includes a kit.

153. Mutations that exhibit reduced endodeoxyribonuclease activity compared to wild-type Cas9 153. The kit of claim 152, further comprising a Cas9 site-directed polypeptide.

154. The nucleic acid comprising a nucleotide sequence encoding the DNA-targeting RNA is a wild-type Ca Mutant Cas9 moieties exhibiting reduced endodeoxyribonuclease activity compared to Cas9 153. The method of claim 152, further comprising a nucleotide sequence encoding a site-specific polypeptide. Kit.

155. Mutations that exhibit reduced endodeoxyribonuclease activity compared to wild-type Cas9 The method further includes the step of preparing a Cas9 site-specific polypeptide comprising a nucleic acid comprising a nucleotide sequence encoding the Cas9 site-specific polypeptide. The kit of claim 152.

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