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 .- 155 . (canceled) 
     
     
         156 . A method of cleaving a nucleic acid comprising contacting a target DNA molecule having a target sequence with
 a) a Cas9 protein; and   b) a single molecule DNA-targeting RNA comprising
 i) a targeter-RNA that hybridizes with the target sequence, and 
 ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment, wherein the activator-RNA comprises the nucleotide sequence set forth as SEQ ID NO: 1347, 
 wherein the activator-RNA and the targeter-RNA are covalently linked to one another with intervening nucleotides, 
   
       wherein said contacting is in vitro outside of a cell, 
       wherein the single molecule DNA-targeting RNA forms a complex with the Cas9 protein, 
       whereby the single molecule DNA-targeting RNA targets the target sequence, and the Cas9 protein cleaves the target DNA molecule. 
     
     
         157 . The method of  claim 156 , wherein said double-stranded RNA duplex has a length of from 8 base pairs (bp) to 30 bp.