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 - 2 . (canceled) 
     
     
         3 . A method of introducing a site-specific, double-stranded break in a target DNA in a mammalian cell, the method comprising:
 introducing into a mammalian cell that comprises a target DNA:   (a) a nucleic acid comprising a nucleotide sequence encoding a Cas9 protein that is covalently linked to a protein transduction domain (PTD), wherein the PTD is a polypeptide that aids in traversal of the Cas9 protein from the mammalian cell's cytosol to within an organelle, wherein the nucleotide sequence encoding the Cas9 protein is modified to replace one or more codons of a wild-type nucleotide sequence with one or more different codons encoding the same amino acid; and   (b) a single-molecule DNA-targeting RNA, or a nucleic acid encoding the single-molecule DNA-targeting RNA, where in the single-molecule DNA-targeting RNA comprises, in 5′ to 3′ order:
 (i) a targeter-RNA comprising a nucleotide sequence that is complementary to, and hybridizes with, a target sequence of the target DNA, and 
 (ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded duplex; 
 wherein the targeter-RNA and the activator-RNA are covalently linked by intervening nucleotides, 
   wherein the single-molecule DNA-targeting RNA forms a complex with the Cas9 protein, the complex is guided to the target sequence of the target DNA via the single-molecule DNA-targeting RNA, and the Cas9 protein cleaves the target DNA, thereby producing a site-specific double-stranded break in the target DNA.   
     
     
         4 . The method of  claim 3 , wherein the PTD comprises the amino acid sequence YGRKKRRQRRR (SEQ ID NO: 264), RRQRRTSKLMKR (SEQ ID NO: 265), RQIKIWFQNRRMKWKK (SEQ ID NO: 268), or RKKRRQRRR (SEQ ID NO: 269). 
     
     
         5 . The method of  claim 3 , wherein the PTD comprises the amino acid sequence RKKRRQRRR (SEQ ID NO: 269). 
     
     
         6 . The method of  claim 3 , wherein the PTD is covalently linked to the carboxyl terminus (C-terminus) of the Cas9 protein. 
     
     
         7 . The method of  claim 3 , wherein the mammalian cell is a human cell. 
     
     
         8 . The method of  claim 3 , wherein the target DNA is chromosomal DNA of the mammalian cell and said double-stranded break results in gene mutation. 
     
     
         9 . The method of  claim 3 , wherein the method comprises introducing the single-molecule DNA-targeting RNA into the mammalian cell as RNA, and wherein the single-molecule DNA-targeting is synthesized in vitro using an RNA polymerase enzyme before introduction into the mammalian cell. 
     
     
         10 . The method of  claim 3 , wherein the Cas9 protein is a  Streptococcus pyogenes  Cas9. 
     
     
         11 . The method of  claim 3 , wherein nucleotide sequence that is complementary to, and hybridizes with, the target sequence of the target DNA is 20 nucleotides long. 
     
     
         12 . The method of  claim 4 , wherein the PTD is covalently linked to the carboxyl terminus (C-terminus) of the Cas9 protein. 
     
     
         13 . The method of  claim 4 , wherein the mammalian cell is a human cell. 
     
     
         14 . The method of  claim 4 , wherein the target DNA is chromosomal DNA of the mammalian cell and said double-stranded break results in gene mutation. 
     
     
         15 . The method of  claim 4 , wherein the method comprises introducing the single-molecule DNA-targeting RNA into the mammalian cell as RNA, and wherein the single-molecule DNA-targeting is synthesized in vitro using an RNA polymerase enzyme before introduction into the mammalian cell. 
     
     
         16 . The method of  claim 4 , wherein the Cas9 protein is a  Streptococcus pyogenes  Cas9. 
     
     
         17 . The method of  claim 4 , wherein nucleotide sequence that is complementary to, and hybridizes with, the target sequence of the target DNA is 20 nucleotides long. 
     
     
         18 . The method of  claim 4 , wherein (a) is introduced into the mammalian cell before (b). 
     
     
         19 . An isolated genetically modified mammalian cell comprising:
 (a) a nucleic acid comprising a nucleotide sequence encoding a Cas9 protein that is covalently linked to a protein transduction domain (PTD), wherein the PTD is a polypeptide that aids in traversal of the Cas9 protein from the mammalian cell's cytosol to within an organelle, wherein the nucleotide sequence encoding the Cas9 protein is modified to replace one or more codons of a wild-type nucleotide sequence with one or more different codons encoding the same amino acid; and   (b) a single-molecule DNA-targeting RNA, or a nucleic acid encoding the single-molecule DNA-targeting RNA, where in the single-molecule DNA-targeting RNA comprises, in 5′ to 3′ order:
 (i) a targeter-RNA comprising a nucleotide sequence that is complementary to, and capable of hybridizing with, a target sequence of a target DNA, and 
 (ii) an activator-RNA that is capable of hybridizing with the targeter-RNA to form a double-stranded duplex, 
 wherein the targeter-RNA and the activator-RNA are covalently linked by intervening nucleotides, 
   wherein the single-molecule DNA-targeting RNA is capable of forming a complex with the Cas9 protein and guiding said complex to the target sequence of the target DNA, and wherein the Cas9 protein is capable of cleaving the target DNA, thereby producing a site-specific double-stranded break in the target DNA.   
     
     
         20 . The mammalian cell of  claim 19 , wherein the PTD comprises the amino acid sequence YGRKKRRQRRR (SEQ ID NO: 264), RRQRRTSKLMKR (SEQ ID NO: 265), RQIKIWFQNRRMKWKK (SEQ ID NO: 268), or RKKRRQRRR (SEQ ID NO: 269). 
     
     
         21 . The method of  claim 19 , wherein the PTD comprises the amino acid sequence RKKRRQRRR (SEQ ID NO: 269). 
     
     
         22 . The mammalian cell of  claim 19 , wherein the PTD is covalently linked to the carboxyl terminus (C-terminus) of the Cas9 protein. 
     
     
         23 . The mammalian cell of  claim 19 , wherein the single-molecule DNA-targeting RNA is an in vitro transcribed RNA. 
     
     
         24 . The mammalian cell of  claim 19 , wherein the target DNA is chromosomal DNA of the mammalian cell and the target sequence is a genomic sequence. 
     
     
         25 . The mammalian cell of  claim 19 , wherein the Cas9 protein is a  Streptococcus pyogenes  Cas9. 
     
     
         26 . The mammalian cell of  claim 19 , wherein nucleotide sequence that is complementary to, and capable of hybridizing with, the target sequence of the target DNA is 20 nucleotides long. 
     
     
         27 . The mammalian cell of  claim 20 , wherein the PTD is covalently linked to the carboxyl terminus (C-terminus) of the Cas9 protein. 
     
     
         28 . The mammalian cell of  claim 20 , wherein the mammalian cell is a human cell. 
     
     
         29 . The mammalian cell of  claim 20 , wherein the single-molecule DNA-targeting RNA is an in vitro transcribed RNA 
     
     
         30 . The mammalian cell of  claim 20 , wherein the target DNA is chromosomal DNA of the mammalian cell and the target sequence is a genomic sequence. 
     
     
         31 . The mammalian cell of  claim 20 , wherein the Cas9 protein is a  Streptococcus pyogenes  Cas9. 
     
     
         32 . The method of  claim 20 , wherein nucleotide sequence that is complementary to, and capable of hybridizing with, the target sequence of the target DNA is 20 nucleotides long.