Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription

A method of producing a modified prokaryotic cell, the method comprising:

• contacting a population of target prokaryotic cells with:
• (a) a chimeric Cas9 protein, or a nucleic acid encoding the chimeric Cas9 protein, wherein the chimeric Cas9 protein comprises a Cas9 polypeptide fused to a heterologous polypeptide, wherein the Cas9 polypeptide comprises one or more mutations in a RuvC domain and/or an HNH domain;
• and
• (b) a single-molecule DNA-targeting RNA, or a nucleic acid encoding the single-molecule DNA-targeting RNA, wherein the single-molecule DNA-targeting RNA comprises, in 5′ to 3′ order:
• (i) a targeter-RNA that is capable of hybridizing with a target sequence of a target DNA inside of one or more cells of said population of target prokaryotic cells, and
• (ii) an activator-RNA that is capable of hybridizing with the targeter-RNA to form a double-stranded duplex,
• wherein (i) and (ii) are covalently linked by intervening nucleotides,
• wherein the single-molecule DNA-targeting RNA targets the chimeric Cas9 protein to the target sequence of the target DNA, and wherein said contacting produces one or more modified prokaryotic cells.

A method of modulating transcription in a prokaryotic cell, the method comprising:

• contacting a target DNA inside of a prokaryotic cell with:
• (a) a chimeric Cas9 protein comprising a Cas9 polypeptide fused to a heterologous polypeptide, wherein the Cas9 polypeptide comprises one or more mutations in a RuvC domain and/or an HNH domain;
• and
• (b) a single-molecule DNA-targeting RNA comprising, 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 RNA duplex,
• wherein (i) and (ii) are covalently linked by intervening nucleotides,
• wherein the single-molecule DNA-targeting RNA targets the chimeric Cas9 protein to the target sequence of the target DNA, and wherein said contacting results in modulating transcription from the target DNA in the prokaryotic cell.

A prokaryotic cell comprising:

• (a) a chimeric Cas9 protein, or a nucleic acid encoding the chimeric Cas9 protein, wherein the chimeric Cas9 protein comprises a Cas9 polypeptide fused to a heterologous polypeptide, wherein the Cas9 polypeptide comprises one or more mutations in a RuvC domain and/or an HNH domain;
• and
• (b) a single-molecule DNA-targeting RNA, or a nucleic acid encoding the single-molecule DNA-targeting RNA, wherein the single-molecule DNA-targeting RNA comprises, in 5′ to 3′ order:
• (i) a targeter-RNA that is 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 (i) and (ii) are covalently linked by intervening nucleotides,
• wherein the single-molecule DNA-targeting RNA is capable of forming a complex with a chimeric Cas9 protein and hybridization of the targeter-RNA to the target sequence is capable of targeting the complex to the target DNA.