Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
First Claim
1. A method of producing a genetically modified cell comprising:
- (1) introducing into a target cell comprising a target DNA;
(a) a Cas9 protein or a nucleic acid encoding the Cas9 protein; and
(b) a DNA-targeting RNA that comprises;
(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 the targeter-RNA and the activator-RNA are covalently linked,wherein said introducing results in modification of the target DNA thereby producing the genetically modified cell; and
then(2) culturing said genetically modified cell to produce a population of cells.
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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.
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Citations
34 Claims
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1. A method of producing a genetically modified cell comprising:
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(1) introducing into a target cell comprising a target DNA; (a) a Cas9 protein or a nucleic acid encoding the Cas9 protein; and (b) a DNA-targeting RNA that comprises; (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 the targeter-RNA and the activator-RNA are covalently linked, wherein said introducing results in modification of the target DNA thereby producing the genetically modified cell; and
then(2) culturing said genetically modified cell to produce a population of cells. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. A method of making a genetically modified cell, the method comprising:
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contacting a population of target cells with; (a) a Cas9 protein; and (b) a DNA-targeting RNA that comprises; (i) a targeter-RNA comprising a nucleotide sequence that is complementary to, and hybridizes with, a target sequence of a target DNA inside of one or more target cells of said population of target cells, and (ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA duplex, wherein the targeter-RNA and the activator-RNA are covalently linked, wherein said contacting results in modification of the target DNA inside of said one or more target cells of said population of target cells, thereby producing one or more genetically modified cells. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)
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Specification