METHODS AND COMPOSITIONS FOR RNA-DIRECTED TARGET DNA MODIFICATION AND FOR RNA-DIRECTED MODULATION OF TRANSCRIPTION
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 .- 1144 . (canceled)
1145 . A system comprising:
(i) a Cas9 protein, or a nucleic acid comprising a nucleotide sequence encoding said Cas9 protein; and
(ii) a single molecule DNA-targeting RNA, or a nucleic acid comprising a nucleotide sequence encoding said single molecule DNA-targeting RNA, wherein the single molecule DNA-targeting RNA comprises
(a) a targeter-RNA that hybridizes with a target sequence in a target DNA molecule, and
(b) 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 the single molecule DNA-targeting RNA is capable of forming a complex with the Cas9 protein, thereby targeting the Cas9 protein to the target DNA molecule,
whereby said complex is capable of cleaving or editing the target DNA molecule or modulating transcription of at least one gene encoded by the target DNA molecule.
1146 . The system according to claim 1145 , wherein said double-stranded RNA duplex has a length of from 8 base pairs (bp) to 30 bp.