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 - 2 . (canceled)
3 . A method of targeting and binding a nucleic acid comprising:
contacting a target DNA molecule having a target sequence with an engineered and/or non naturally occurring Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)—CRISPR associated (Cas) (CRISPR-Cas) system comprising
a) a Cas9 protein; and
b) a DNA-targeting RNA comprising
i) a targeter-RNA that hybridizes with the target sequence, and ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment,
wherein (I) the DNA-targeting RNA comprises a nucleotide sequence that is modified relative to naturally occurring DNA-targeting RNAs, and/or (II) the Cas9 protein comprises an amino acid sequence that is modified relative to naturally occurring Cas9 proteins
wherein the DNA-targeting RNA forms a complex with the Cas9 protein,
whereby the DNA-targeting RNA targets the target sequence, and the Cas9 protein binds the target DNA molecule; and
wherein said contacting occurs in a eukaryotic cell.
4 . The method of claim 3 , wherein, prior to the contacting step, the method comprises:
introducing into a eukaryotic cell containing the target DNA molecule:
1) the DNA-targeting RNA, or one or more DNA molecules comprising one or more nucleotide sequences that (i) encode the DNA-targeting RNA and (ii) are operably linked to regulatory elements operable in said eukaryotic cell; and
2) the Cas9 protein, an RNA molecule comprising a nucleotide sequence encoding the Cas9 protein, or a DNA molecule comprising a nucleotide sequence that (i) encodes the Cas9 protein and (ii) is operably linked to a regulatory element operable in said eukaryotic cell.
5 . The method of claim 4 , wherein the genome of said eukaryotic cell comprises the target DNA molecule.
6 . The method of claim 4 , wherein the DNA-targeting RNA is a double-molecule DNA-targeting RNA such that said targeter-RNA and said activator-RNA are present on different RNA molecules.
7 . The method of claim 3 , wherein the method comprises: introducing the Cas9 protein or an RNA that encodes the Cas9 protein into the eukaryotic cell.
8 . The method of claim 3 , wherein the method comprises: introducing the DNA-targeting RNA into the eukaryotic cell as RNA.
9 . The method of claim 3 , wherein the method comprises introducing into the eukaryotic cell:
the DNA-targeting RNA as RNA; and
the Cas9 protein or an RNA that encodes the Cas9 protein.
10 . The method of claim 9 , wherein the eukaryotic cell is in vivo and said introducing comprises microinjection.
11 . The method of claim 9 , wherein the eukaryotic cell is a mammalian cell and said introducing comprises lipofection.
12 . The method of claim 3 , wherein the method comprises introducing a plasmid that encodes the DNA-targeting RNA into the eukaryotic cell.
13 . The method of claim 3 , wherein the method comprises introducing a plasmid that encodes the Cas9 protein into the eukaryotic cell.
14 . The method of claim 3 , wherein the method comprises introducing into the eukaryotic cell:
a plasmid that encodes the DNA-targeting RNA, and
a plasmid that encodes the Cas9 protein.
15 . The method of claim 14 , wherein the eukaryotic cell is a mammalian cell and said introducing comprises lipofection.
16 . The method of claim 14 , wherein the eukaryotic cell is a single-cell organism.
17 . The method of claim 15 , wherein the Cas9 protein comprises the amino acid sequence set forth in SEQ ID NO: 2.
18 . The method of claim 3 , wherein the method comprises introducing a viral vector that encodes the DNA-targeting RNA into the eukaryotic cell.
19 . The method of claim 3 , wherein the method comprises introducing a viral vector that encodes the Cas9 protein into the eukaryotic cell.
20 . The method of claim 3 , wherein the method comprises introducing one or more viral vectors into the eukaryotic cell, wherein said one or more viral vectors comprises:
a first nucleotide sequence that encodes the DNA-targeting RNA, and
a second nucleotide sequence that encodes the Cas9 protein.
21 . The method of claim 20 , wherein the eukaryotic cell is a mammalian cell and said introducing comprises lipofection.
22 . The method of claim 20 , wherein the eukaryotic cell is a single-cell organism.
23 . The method of claim 4 , wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s)), wherein the one or more PTD(s) comprise an amino acid sequence selected from the group consisting of SEQ ID NOs:268 and 269.
24 . The method of claim 4 , wherein the Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain.
25 . The method of claim 4 , wherein the activator-RNA comprises the 88 nucleotide tracrRNA sequence set forth in SEQ ID NO:433.
26 . A method of cleaving or editing a target DNA molecule or modulating transcription of at least one gene encoded thereon, the method comprising
contacting a target DNA molecule having a target sequence with an engineered and/or non-naturally-occurring Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)—CRISPR associated (Cas) (CRISPR-Cas) system comprising:
(a) a Cas9 protein; and
(b) a DNA-targeting RNA comprising:
(i) a targeter-RNA that hybridizes with the target sequence, and
(ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment,
wherein (I) the DNA-targeting RNA comprises a nucleotide sequence that is modified relative to naturally occurring DNA-targeting RNAs, and/or (II) the Cas9 protein comprises an amino acid sequence that is modified relative to naturally occurring Cas9,
wherein the DNA-targeting RNA forms a complex with the Cas9 protein, whereby said target DNA molecule is cleaved or edited or transcription of at least one gene encoded by the target DNA molecule is modulated,
wherein said contacting occurs in a eukaryotic cell,
wherein, prior to the contacting step, the method comprises:
introducing into the eukaryotic cell containing the target DNA molecule:
1) the DNA-targeting RNA, or one or more DNA molecules comprising one or more nucleotide sequences that (i) encode the DNA-targeting RNA and (ii) are operably linked to regulatory elements operable in said eukaryotic cell; and
2) the Cas9 protein, or a DNA molecule comprising a nucleotide sequence that (i) encodes the Cas9 protein and (ii) is operably linked to a regulatory element operable in said eukaryotic cell.
27 . The method of claim 26 , wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s)) that aid in traversal of an organelle membrane, wherein the one or more PTD(s) comprise an amino acid sequence selected from the group consisting of SEQ ID NOs:268 and 269.
28 . The method of claim 26 , wherein the activator-RNA comprises the 88 nucleotide tracrRNA sequence set forth in SEQ ID NO:433.
29 . The method of claim 26 , wherein said Cas9 protein cleaves only one strand of DNA and comprises one or more mutations in a RuvC domain and/or a HNH domain.
30 . The method of claim 26 , wherein the method comprises creation of a double strand break in the target DNA molecule which is repaired by a homology-directed repair mechanism which incorporates a sequence of a donor polynucleotide into the target DNA molecule, thereby editing the target DNA molecule.
31 . The method of claim 26 , wherein the eukaryotic cell is an in vivo plant cell or in vivo animal cell, and wherein the method comprises introducing into the in vivo plant cell or in vivo animal:
(1) the DNA-targeting RNA, or one or more DNA molecules comprising nucleotide sequences that (i) encode the DNA-targeting RNA and (ii) are operably linked to a regulatory element operable in said eukaryotic cell; and
(2) the Cas9 protein, or a DNA molecule comprising a nucleotide sequence that (i) encodes the Cas9 protein and (ii) is operably linked to a regulatory element operable in said eukaryotic cell,
wherein the method results in editing of the target DNA, wherein the method is performed to treat a disease, to produce a gene knock-out, to produce a gene knock-in, or to produce a genetically modified organism for agriculture.
32 . A method of cleaving or editing a target DNA molecule, the method comprising
contacting a target DNA molecule having a target sequence with an engineered and/or non-naturally-occurring Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)—CRISPR associated (Cas) (CRISPR-Cas) system comprising:
(a) a Cas9 protein; and
(b) a DNA-targeting RNA comprising:
(i) a targeter-RNA that hybridizes with the target sequence, and
(ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment,
wherein (I) the DNA-targeting RNA comprises a nucleotide sequence that is modified relative to naturally occurring DNA-targeting RNAs, and/or (II) the Cas9 protein comprises an amino acid sequence that is modified relative to naturally occurring Cas9,
wherein the DNA-targeting RNA forms a complex with the Cas9 protein, whereby said target DNA molecule is cleaved or edited,
wherein said contacting occurs in a eukaryotic cell.