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.

What is claimed is: 
     
         1 . A single-molecule DNA-targeting RNA having the structure: 
       
         
           
           
               
               
           
         
       
       wherein the linker is GAAA and the nucleotide sequence of the single-molecule DNA-targeting RNA is 
       
         
           
                 
               
                   (SEQ ID NO: 321) 
                 
                   GAUUUCUUCUUGCGCUUUUUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAU 
                 
                     
                 
                   AAGGCUAGUCCG. 
                 
             
                
                
                
                
               
            
           
         
       
     
     
         2 . A method of cleaving a target DNA, the method comprising:
 contacting a target DNA with a complex comprising:   (a) the single molecule DNA-targeting RNA of  claim 1 ; and   (b) a Cas9 protein comprising the  S. pyogenes  Cas9 amino acid sequence set forth as SEQ ID NO.: 2,   wherein prior to said contacting the target DNA is double stranded and comprises a nucleotide sequence taatgaattccccaatacccaaaagcgcaagaagaaatcaaccagcgca (SEQ ID NO: 302) hybridized to a nucleotide sequence tgcgctggttgatttcttgcgctttttgggtattggggaattcatta (SEQ ID NO: 301),   wherein said contacting is in vitro and does not take place inside of a cell, and   wherein the method results in cleavage of the target DNA.