CPC C12N 15/907 (2013.01) [A01H 6/4684 (2018.05); A01K 67/027 (2013.01); A61K 38/465 (2013.01); C12N 9/22 (2013.01); C12N 15/102 (2013.01); C12N 15/111 (2013.01); C12N 15/113 (2013.01); C12N 15/63 (2013.01); C12N 15/70 (2013.01); C12N 15/746 (2013.01); C12N 15/90 (2013.01); C12N 15/902 (2013.01); C12Q 1/686 (2013.01); H01L 21/02312 (2013.01); H01L 21/205 (2013.01); H01L 33/0075 (2013.01); A61K 48/00 (2013.01); C12N 2310/11 (2013.01); C12N 2310/13 (2013.01); C12N 2310/14 (2013.01); C12N 2310/20 (2017.05); C12N 2310/31 (2013.01); C12N 2310/32 (2013.01); C12N 2310/33 (2013.01); C12N 2310/3519 (2013.01); C12N 2310/531 (2013.01); C12N 2800/80 (2013.01); C12Y 301/04 (2013.01)] | 165 Claims |
1. A method of producing a modified prokaryotic cell, the method comprising: introducing into a target prokaryotic cell comprising a target DNA:
(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 DNA-targeting RNA that comprises:
(i) a targeter-RNA that is capable of hybridizing with a target sequence of the 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,
wherein the DNA-targeting RNA targets the chimeric Cas9 protein to the target sequence of the target DNA, and wherein said introducing produces a modified prokaryotic cell.
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