Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription

Abstract

The present disclosure provides a DNA-targeting RNA that comprises a targeting sequence and, together with a modifying polypeptide, provides for site-specific modification of a target DNA and/or a polypeptide associated with the target DNA. The present disclosure further provides site-specific modifying polypeptides. The present disclosure further provides methods of site-specific modification of a target DNA and/or a polypeptide associated with the target DNA The present disclosure provides methods of modulating transcription of a target nucleic acid in a target cell, generally involving contacting the target nucleic acid with an enzymatically inactive Cas9 polypeptide and a DNA-targeting RNA. Kits and compositions for carrying out the methods are also provided. The present disclosure provides genetically modified cells that produce Cas9; and Cas9 transgenic non-human multicellular organisms.

1 . A prokaryotic cell comprising a chimeric Cas9 protein comprising a Cas9 polypeptide fused to a transcriptional activator polypeptide or a transcription repressor, wherein the Cas9 polypeptide comprises one or more mutations in a RuvC domain and/or an HNH domain. 
     
     
         2 . A single-molecule DNA-targeting RNA having the structure: 
       
         
           
           
               
               
           
         
       
       wherein the linker is nnnn such that the single-molecule DNA-targeting RNA has the nucleotide sequence nnnnnnnnnnnnnnnnnnnnGUUUUAGAGCUAnnnnUAGCAAGUUAAAAUAAGGCUAGUCCG (SEQ ID NO: 680). 
     
     
         3 . The single-molecule DNA-targeting RNA of  claim 2 , wherein the linker is GAAA such that the single-molecule DNA-targeting RNA has the structure: 
       
         
           
           
               
               
           
         
         wherein “20-nt targeting seq” is nnnnnnnnnnnnnnnnnnnn. 
       
     
     
         4 . A method of cleaving a target DNA, the method comprising:
 contacting a target DNA with:   (a) the single molecule DNA-targeting RNA of  claim 29 , wherein nnnnnnnnnnnnnnnnnnnn is a DNA-targeting segment that hybridizes with a target sequence of the target DNA; and   (b) a Cas9 protein comprising the  S. pyogenes  Cas9 amino acid sequence set forth as SEQ ID NO.: 2,   wherein said contacting does not take place inside of a cell, and   wherein the target DNA is cleaved.   
     
     
         5 . A method of assembling a complex, the method comprising:
 (A) generating an engineered targeter-RNA that comprises: (a) a DNA-targeting segment comprising a nucleotide sequence that is not found in naturally occurring crRNA and is complementary to a desired target sequence of a target DNA such that the engineered targeter-RNA is capable of hybridizing to the desired target sequence, and (b) a duplex-forming segment that is capable of hybridizing with an activator-RNA; and   (B) contacting a Cas9 protein, in vitro outside of a cell, with the engineered targeter-RNA and the activator-RNA, which hybridize to form a DNA-targeting RNA,   wherein the DNA-targeting RNA forms a complex with the Cas9 protein and is capable of guiding the complex in a site-specific manner to said desired target sequence for modification of the target DNA.   
     
     
         6 . The method of  claim 5 , wherein the Cas9 protein is produced from a recombinant expression vector or by using an automated synthesizer. 
     
     
         7 . The method of  claim 5 , wherein the Cas9 protein is produced from a recombinant expression vector in a genetically modified prokaryotic host cell 
     
     
         8 . The method of  claim 5 , wherein the nucleotide sequence of the engineered targeter-RNA that is complementary to the desired target sequence of the target DNA is about 20 nucleotides long. 
     
     
         9 . The method of  claim 5 , wherein the engineered targeter-RNA is generated by chemical synthesis. 
     
     
         10 . The method of  claim 5 , wherein the Cas9 protein comprises a mutation in a RuvC and/or an HNH domain. 
     
     
         11 . A method of assembling a complex, the method comprising:
 contacting a Cas9 protein, in vitro outside of a cell, with an engineered targeter-RNA and an activator-RNA,   wherein the engineered targeter-RNA comprises: (a) a DNA-targeting segment comprising a nucleotide sequence that is not found in naturally occurring crRNA and is complementary to a desired target sequence of a target DNA such that the engineered targeter-RNA is capable of hybridizing to the desired target sequence, and (b) a duplex-forming segment that hybridizes with the activator-RNA to form a DNA-targeting RNA,   wherein the DNA-targeting RNA forms a complex with the Cas9 protein and is capable of guiding the complex to said desired target sequence for modification of the target DNA.   
     
     
         12 . The method of  claim 11 , wherein the engineered targeter-RNA is obtained by identifying the desired target sequence and generating said nucleotide sequence that is complementary to said desired target sequence. 
     
     
         13 . The method of  claim 11 , wherein the engineered targeter-RNA is generated by chemical synthesis. 
     
     
         14 . The method of  claim 11 , wherein the Cas9 protein comprises a mutation in a RuvC and/or an HNH domain. 
     
     
         15 . The method of  claim 11 , wherein the nucleotide sequence of the engineered targeter-RNA that is complementary to the desired target sequence of the target DNA is about 20 nucleotides long. 
     
     
         16 . A method of assembling a complex, the method comprising:
 (A) identifying a desired target sequence of a target DNA;   (B) generating an engineered targeter-RNA that comprises: (a) a DNA-targeting segment comprising a nucleotide sequence that is not found in naturally occurring crRNA and is complementary to the desired target sequence such that the engineered targeter-RNA is capable of hybridizing to the desired target sequence, and (b) a duplex-forming segment that is capable of hybridizing with an activator-RNA; and   (C) contacting a Cas9 protein, in vitro outside of a cell, with the engineered targeter-RNA and the activator-RNA, which hybridize to form a DNA-targeting RNA,   wherein the DNA-targeting RNA forms a complex with the Cas9 protein and is capable of guiding the complex in a site-specific manner to said desired target sequence for modification of the target DNA.   
     
     
         17 . The method of  claim 16 , wherein the Cas9 protein comprises a mutation in a RuvC and/or an HNH domain. 
     
     
         18 . The method of  claim 16 , wherein the Cas9 protein is produced from a recombinant expression vector in a genetically modified prokaryotic host cell or by using an automated synthesizer. 
     
     
         19 . A method of assembling a complex, the method comprising:
 contacting a recombinant Cas9 protein, in vitro outside of a cell, with an engineered targeter-RNA and an activator-RNA,   wherein the engineered targeter-RNA comprises: (a) a DNA-targeting segment comprising a nucleotide sequence that is not found in naturally occurring crRNA and is complementary to a desired target sequence of a target DNA such that the engineered targeter-RNA is capable of hybridizing to the desired target sequence, and (b) a duplex-forming segment that hybridizes with the activator-RNA to form a DNA-targeting RNA,   wherein the DNA-targeting RNA forms a complex with the recombinant Cas9 protein and is capable of guiding the complex in a site-specific manner to said desired target sequence for modification of the target DNA.   
     
     
         20 . The method of  claim 19 , wherein the Cas9 protein comprises a mutation in a RuvC and/or an HNH domain. 
     
     
         21 . A method of assembling a complex, the method comprising:
 (A) generating an engineered targeter-RNA that comprises: (a) a DNA-targeting segment comprising a nucleotide sequence that is complementary to a desired target sequence of a target DNA such that the engineered targeter-RNA is capable of hybridizing to the desired target sequence, and (b) a duplex-forming segment that is capable of hybridizing with an activator-RNA, wherein the DNA-targeting segment is heterologous to the duplex-forming segment such that the engineered targeter-RNA has a nucleotide sequence that is not found in naturally occurring crRNA; and   (B) contacting a Cas9 protein, in vitro outside of a cell, with the engineered targeter-RNA and the activator-RNA, which hybridize to form a DNA-targeting RNA,   wherein the DNA-targeting RNA forms a complex with the Cas9 protein and is capable of guiding the complex in a site-specific manner to said desired target sequence for modification of the target DNA.   
     
     
         22 . The method of  claim 21 , wherein the Cas9 protein is produced from a recombinant expression vector or by using an automated synthesizer. 
     
     
         23 . The method of  claim 21 , wherein the Cas9 protein is produced from a recombinant expression vector in a genetically modified prokaryotic host cell 
     
     
         24 . The method of  claim 21 , wherein the nucleotide sequence of the engineered targeter-RNA that is complementary to the desired target sequence of the target DNA is about 20 nucleotides long. 
     
     
         25 . The method of  claim 21 , wherein the engineered targeter-RNA is generated by chemical synthesis. 
     
     
         26 . The method of  claim 21 , wherein the Cas9 protein comprises a mutation in a RuvC and/or an HNH domain. 
     
     
         27 . A method of assembling a complex, the method comprising:
 contacting a Cas9 protein, in vitro outside of a cell, with an engineered targeter-RNA and an activator-RNA,   wherein the engineered targeter-RNA comprises: (a) a DNA-targeting segment comprising a nucleotide sequence that is complementary to a desired target sequence of a target DNA such that the engineered targeter-RNA is capable of hybridizing to the desired target sequence, and (b) a duplex-forming segment that hybridizes with the activator-RNA to form a DNA-targeting RNA, wherein the DNA-targeting segment is heterologous to the duplex-forming segment such that the engineered targeter-RNA has a nucleotide sequence that is not found in naturally occurring crRNA,   wherein the DNA-targeting RNA forms a complex with the Cas9 protein and is capable of guiding the complex to said desired target sequence for modification of the target DNA.   
     
     
         28 . The method of  claim 27 , wherein the engineered targeter-RNA is obtained by identifying the desired target sequence and generating said nucleotide sequence that is complementary to said desired target sequence. 
     
     
         29 . The method of  claim 27 , wherein the engineered targeter-RNA is generated by chemical synthesis. 
     
     
         30 . The method of  claim 27 , wherein the Cas9 protein comprises a mutation in a RuvC and/or an HNH domain. 
     
     
         31 . The method of  claim 27 , wherein the nucleotide sequence of the engineered targeter-RNA that is complementary to the desired target sequence of the target DNA is about 20 nucleotides long. 
     
     
         32 . A method of assembling a complex, the method comprising:
 (A) identifying a desired target sequence of a target DNA;   (B) generating an engineered targeter-RNA that comprises: (a) a DNA-targeting segment comprising a nucleotide sequence that is complementary to the desired target sequence such that the engineered targeter-RNA is capable of hybridizing to the desired target sequence, and (b) a duplex-forming segment that is capable of hybridizing with an activator-RNA, wherein the DNA-targeting segment is heterologous to the duplex-forming segment such that the engineered targeter-RNA has a nucleotide sequence that is not found in naturally occurring crRNA; and   (C) contacting a Cas9 protein, in vitro outside of a cell, with the engineered targeter-RNA and the activator-RNA, which hybridize to form a DNA-targeting RNA,   wherein the DNA-targeting RNA forms a complex with the Cas9 protein and is capable of guiding the complex in a site-specific manner to said desired target sequence for modification of the target DNA.   
     
     
         33 . The method of  claim 32 , wherein the Cas9 protein comprises a mutation in a RuvC and/or an HNH domain. 
     
     
         34 . The method of  claim 32 , wherein the Cas9 protein is produced from a recombinant expression vector in a genetically modified prokaryotic host cell or by using an automated synthesizer. 
     
     
         35 . A method of assembling a complex, the method comprising:
 contacting a recombinant Cas9 protein, in vitro outside of a cell, with an engineered targeter-RNA and an activator-RNA,   wherein the engineered targeter-RNA comprises: (a) a DNA-targeting segment comprising a nucleotide sequence that is complementary to a desired target sequence of a target DNA such that the engineered targeter-RNA is capable of hybridizing to the desired target sequence, and (b) a duplex-forming segment that hybridizes with the activator-RNA to form a DNA-targeting RNA, wherein the DNA-targeting segment is heterologous to the duplex-forming segment such that the engineered targeter-RNA has a nucleotide sequence that is not found in naturally occurring crRNA,   wherein the DNA-targeting RNA forms a complex with the recombinant Cas9 protein and is capable of guiding the complex in a site-specific manner to said desired target sequence for modification of the target DNA.   
     
     
         36 . The method of  claim 35 , wherein the Cas9 protein comprises a mutation in a RuvC and/or an HNH domain. 
     
     
         37 . A method of assembling a complex, the method comprising:
 (A) generating an engineered targeter-RNA that comprises: (a) a DNA-targeting segment comprising a nucleotide sequence that is not found in naturally occurring crRNA and is complementary to a desired target sequence of a target DNA such that the engineered targeter-RNA is capable of hybridizing to the desired target sequence, and (b) a duplex-forming segment that is capable of hybridizing with an activator-RNA; and   (B) contacting a Cas9 protein in vitro with the engineered targeter-RNA and the activator-RNA, which hybridize to form a DNA-targeting RNA,   wherein the DNA-targeting RNA forms a complex with the Cas9 protein and is capable of guiding the complex in a site-specific manner to said desired target sequence for modification of the target DNA.   
     
     
         38 . The method of  claim 37 , wherein the Cas9 protein is produced from a recombinant expression vector or by using an automated synthesizer. 
     
     
         39 . The method of  claim 37 , wherein the Cas9 protein is produced from a recombinant expression vector in a genetically modified prokaryotic host cell 
     
     
         40 . The method of  claim 37 , wherein the nucleotide sequence of the engineered targeter-RNA that is complementary to the desired target sequence of the target DNA is about 20 nucleotides long. 
     
     
         41 . The method of  claim 37 , wherein the engineered targeter-RNA is generated by chemical synthesis. 
     
     
         42 . The method of  claim 37 , wherein the Cas9 protein comprises a mutation in a RuvC and/or an HNH domain. 
     
     
         43 . A method of assembling a complex, the method comprising:
 contacting a Cas9 protein in vitro with an engineered targeter-RNA and an activator-RNA,   wherein the engineered targeter-RNA comprises: (a) a DNA-targeting segment comprising a nucleotide sequence that is not found in naturally occurring crRNA and is complementary to a desired target sequence of a target DNA such that the engineered targeter-RNA is capable of hybridizing to the desired target sequence, and (b) a duplex-forming segment that hybridizes with the activator-RNA to form a DNA-targeting RNA,   wherein the DNA-targeting RNA forms a complex with the Cas9 protein and is capable of guiding the complex to said desired target sequence for modification of the target DNA.   
     
     
         44 . The method of  claim 43 , wherein the engineered targeter-RNA is obtained by identifying the desired target sequence and generating said nucleotide sequence that is complementary to said desired target sequence. 
     
     
         45 . The method of  claim 43 , wherein the engineered targeter-RNA is generated by chemical synthesis. 
     
     
         46 . The method of  claim 43 , wherein the Cas9 protein comprises a mutation in a RuvC and/or an HNH domain. 
     
     
         47 . The method of  claim 43 , wherein the nucleotide sequence of the engineered targeter-RNA that is complementary to the desired target sequence of the target DNA is about 20 nucleotides long. 
     
     
         48 . A method of assembling a complex, the method comprising:
 (A) identifying a desired target sequence of a target DNA;   (B) generating an engineered targeter-RNA that comprises: (a) a DNA-targeting segment comprising a nucleotide sequence that is not found in naturally occurring crRNA and is complementary to the desired target sequence such that the engineered targeter-RNA is capable of hybridizing to the desired target sequence, and (b) a duplex-forming segment that is capable of hybridizing with an activator-RNA; and   (C) contacting a Cas9 protein in vitro with the engineered targeter-RNA and the activator-RNA, which hybridize to form a DNA-targeting RNA,   wherein the DNA-targeting RNA forms a complex with the Cas9 protein and is capable of guiding the complex in a site-specific manner to said desired target sequence for modification of the target DNA.   
     
     
         49 . The method of  claim 48 , wherein the Cas9 protein comprises a mutation in a RuvC and/or an HNH domain. 
     
     
         50 . The method of  claim 48 , wherein the Cas9 protein is produced from a recombinant expression vector in a genetically modified prokaryotic host cell or by using an automated synthesizer. 
     
     
         51 . A method of assembling a complex, the method comprising:
 contacting a recombinant Cas9 protein in vitro with an engineered targeter-RNA and an activator-RNA,   wherein the engineered targeter-RNA comprises: (a) a DNA-targeting segment comprising a nucleotide sequence that is not found in naturally occurring crRNA and is complementary to a desired target sequence of a target DNA such that the engineered targeter-RNA is capable of hybridizing to the desired target sequence, and (b) a duplex-forming segment that hybridizes with the activator-RNA to form a DNA-targeting RNA,   wherein the DNA-targeting RNA forms a complex with the recombinant Cas9 protein and is capable of guiding the complex in a site-specific manner to said desired target sequence for modification of the target DNA.   
     
     
         52 . The method of  claim 51 , wherein the Cas9 protein comprises a mutation in a RuvC and/or an HNH domain. 
     
     
         53 . A method of assembling a complex, the method comprising:
 (A) generating an engineered targeter-RNA that comprises: (a) a DNA-targeting segment comprising a nucleotide sequence that is complementary to a desired target sequence of a target DNA such that the engineered targeter-RNA is capable of hybridizing to the desired target sequence, and (b) a duplex-forming segment that is capable of hybridizing with an activator-RNA, wherein the DNA-targeting segment is heterologous to the duplex-forming segment such that the engineered targeter-RNA has a nucleotide sequence that is not found in naturally occurring crRNA; and   (B) contacting a Cas9 protein in vitro with the engineered targeter-RNA and the activator-RNA, which hybridize to form a DNA-targeting RNA,   wherein the DNA-targeting RNA forms a complex with the Cas9 protein and is capable of guiding the complex in a site-specific manner to said desired target sequence for modification of the target DNA.   
     
     
         54 . The method of  claim 53 , wherein the Cas9 protein is produced from a recombinant expression vector or by using an automated synthesizer. 
     
     
         55 . The method of  claim 53 , wherein the Cas9 protein is produced from a recombinant expression vector in a genetically modified prokaryotic host cell 
     
     
         56 . The method of  claim 53 , wherein the nucleotide sequence of the engineered targeter-RNA that is complementary to the desired target sequence of the target DNA is about 20 nucleotides long. 
     
     
         57 . The method of  claim 53 , wherein the engineered targeter-RNA is generated by chemical synthesis. 
     
     
         58 . The method of  claim 53 , wherein the Cas9 protein comprises a mutation in a RuvC and/or an HNH domain. 
     
     
         59 . A method of assembling a complex, the method comprising:
 contacting a Cas9 protein in vitro with an engineered targeter-RNA and an activator-RNA,   wherein the engineered targeter-RNA comprises: (a) a DNA-targeting segment comprising a nucleotide sequence that is complementary to a desired target sequence of a target DNA such that the engineered targeter-RNA is capable of hybridizing to the desired target sequence, and (b) a duplex-forming segment that hybridizes with the activator-RNA to form a DNA-targeting RNA, wherein the DNA-targeting segment is heterologous to the duplex-forming segment such that the engineered targeter-RNA has a nucleotide sequence that is not found in naturally occurring crRNA,   wherein the DNA-targeting RNA forms a complex with the Cas9 protein and is capable of guiding the complex to said desired target sequence for modification of the target DNA.   
     
     
         60 . The method of  claim 59 , wherein the engineered targeter-RNA is obtained by identifying the desired target sequence and generating said nucleotide sequence that is complementary to said desired target sequence. 
     
     
         61 . The method of  claim 59 , wherein the engineered targeter-RNA is generated by chemical synthesis. 
     
     
         62 . The method of  claim 59 , wherein the Cas9 protein comprises a mutation in a RuvC and/or an HNH domain. 
     
     
         63 . The method of  claim 59 , wherein the nucleotide sequence of the engineered targeter-RNA that is complementary to the desired target sequence of the target DNA is about 20 nucleotides long. 
     
     
         64 . A method of assembling a complex, the method comprising:
 (A) identifying a desired target sequence of a target DNA;   (B) generating an engineered targeter-RNA that comprises: (a) a DNA-targeting segment comprising a nucleotide sequence that is complementary to the desired target sequence such that the engineered targeter-RNA is capable of hybridizing to the desired target sequence, and (b) a duplex-forming segment that is capable of hybridizing with an activator-RNA, wherein the DNA-targeting segment is heterologous to the duplex-forming segment such that the engineered targeter-RNA has a nucleotide sequence that is not found in naturally occurring crRNA; and   (C) contacting a Cas9 protein in vitro with the engineered targeter-RNA and the activator-RNA, which hybridize to form a DNA-targeting RNA,   wherein the DNA-targeting RNA forms a complex with the Cas9 protein and is capable of guiding the complex in a site-specific manner to said desired target sequence for modification of the target DNA.   
     
     
         65 . The method of  claim 64 , wherein the Cas9 protein comprises a mutation in a RuvC and/or an HNH domain. 
     
     
         66 . The method of  claim 64 , wherein the Cas9 protein is produced from a recombinant expression vector in a genetically modified prokaryotic host cell or by using an automated synthesizer. 
     
     
         67 . A method of assembling a complex, the method comprising:
 contacting a recombinant Cas9 protein in vitro with an engineered targeter-RNA and an activator-RNA,   wherein the engineered targeter-RNA comprises: (a) a DNA-targeting segment comprising a nucleotide sequence that is complementary to a desired target sequence of a target DNA such that the engineered targeter-RNA is capable of hybridizing to the desired target sequence, and (b) a duplex-forming segment that hybridizes with the activator-RNA to form a DNA-targeting RNA, wherein the DNA-targeting segment is heterologous to the duplex-forming segment such that the engineered targeter-RNA has a nucleotide sequence that is not found in naturally occurring crRNA,   wherein the DNA-targeting RNA forms a complex with the recombinant Cas9 protein and is capable of guiding the complex in a site-specific manner to said desired target sequence for modification of the target DNA.   
     
     
         68 . The method of  claim 67 , wherein the Cas9 protein comprises a mutation in a RuvC and/or an HNH domain.