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 . 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 GAUUUCUUCUUGCGCUUUUUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAG UCCG (SEQ ID NO: 321).
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 taatgaattccccaatacccaaaaagcgcaagaagaaatcaaccagcgca (SEQ ID NO: 302) hybridized to a nucleotide sequence tgcgctggttgatttcttcttgcgctttttgggtattggggaattcatta (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.