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 nucleic acid comprising a nucleotide sequence encoding a single molecule DNA-targeting RNA, wherein the single molecule DNA-targeting RNA comprises, in 5′ to 3′ order:
(i) a targeter-RNA comprising a first nucleotide sequence that is complementary to a target sequence of a target DNA, and a second nucleotide sequence that hybridizes with an activator-RNA, wherein the first and second nucleotide sequences are heterologous to one another; and
(ii) the activator-RNA, which comprises a duplex-forming segment that comprises a nucleotide sequence that hybridizes with the targeter-RNA to form a double-stranded duplex,
wherein (i) and (ii) are covalently linked by intervening nucleotides, and
wherein the single molecule DNA-targeting RNA is capable of forming a complex with a Cas9 protein and guiding the complex to the target sequence to cleave the target DNA,
wherein the nucleotide sequence encoding the single molecule DNA-targeting RNA is operably linked to a heterologous promoter.
2. The nucleic acid of claim 1 , wherein said nucleotide sequence of the duplex-forming segment of the activator-RNA comprises a sequence having greater than 80% identity to the tracrRNA sequence UAGCAAGUUAAAAU (SEQ ID NO: 562).
3. The nucleic acid of claim 1 , wherein the activator-RNA comprises the 26 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCG (SEQ ID NO: 441).
4. The nucleic acid of claim 1 , wherein the activator-RNA comprises the 31 nucleotide tracrRNA sequence: UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC (SEQ ID NO: 396).
5. The nucleic acid of claim 1 , wherein the activator-RNA comprises the 67 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 432).
6. The nucleic acid of claim 4 , wherein the activator-RNA comprises a nucleotide sequence having greater than 80% nucleotide sequence identity with any one of the following tracrRNA sequences:
(SEQ ID NO: 478)
AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAA
AGUGGCACCGAGUCGGUGCUUUUUUU;
(SEQ ID NO: 1425)
AAAUAACAUAGCAAGUUAAAAUAAGGCUUUGUCCGUUAUCAACUUUUAA
UUAAGUAGCGCCGUUUCGGCGCUUUUUU;
(SEQ ID NO: 1426)
AAACAACACAGCAAGUUAAAUAAGGCAGUGAUUUUUAAUCCAGUCCGUA
CACAACUUGAAAAAGUGCGCACCGAUUCGGUGCUUUUUU;
(SEQ ID NO: 1427)
AAACAACACAGCGAGUUAAAUAAGGCUUAGUCCGUACUCAACUUGAAAA
GGUGGCACCGAUUCGGUGUUUUUU;
(SEQ ID NO: 1428)
AAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUCAACACC
CUGUCAUUUUAUGGCAGGGUGUUUUCGUUAUUU;
(SEQ ID NO: 1429)
AAAUGAGAACCGUUGCUACAAUAAGGCCGUCUGAAAAGAUGUGCCGCAA
CGCUCUGCCCCUUAAAGCUUCUGCUUUAAGGGGCAUCGUUUAUUUC;
and
(SEQ ID NO: 1430)
AAAUGAGAGAGUUGCUACAAUAAGGCUUCUGAAAAGAAUGACCGUAACG
CUCUGCCCCUUGUGAUUCUUAAUUGCAAGGGGCAUCGUUUU.
7. The nucleic acid of claim 1 , wherein the activator-RNA comprises a nucleotide sequence having greater than 90% nucleotide sequence identity with any one of the following tracrRNA sequences:
(SEQ ID NO: 397)
UAGCAAGUUAAAAUAAGGCUAGUCCG;
(SEQ ID NO: 1431)
UAGCAAGUUAAAAUAAGGCUUUGUCCG;
(SEQ ID NO: 1432)
CAGCGAGUUAAAUAAGGCUUAGUCCG;
(SEQ ID NO: 1433)
AGAAGCUACAAAGAUAAGGCUUCAUGCCG;
(SEQ ID NO: 1434)
GUUGCUACAAUAAGGCCGUCUGAAAAGAUGUGCCG; and
(SEQ ID NO: 1435)
AGAGUUGCUACAAUAAGGCUUCUGAAAAGAAUGACCG.
8. The nucleic acid of claim 1 , wherein the activator-RNA comprises any one of the following tracrRNA sequences:
(SEQ ID NO: 478)
AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAA
AGUGGCACCGAGUCGGUGCUUUUUUU;
(SEQ ID NO: 1425)
AAAUAACAUAGCAAGUUAAAAUAAGGCUUUGUCCGUUAUCAACUUUUAA
UUAAGUAGCGCCGUUUCGGCGCUUUUUU;
(SEQ ID NO: 1426)
AAACAACACAGCAAGUUAAAUAAGGCAGUGAUUUUUAAUCCAGUCCGUA
CACAACUUGAAAAAGUGCGCACCGAUUCGGUGCUUUUUU;
(SEQ ID NO: 1427)
AAACAACACAGCGAGUUAAAUAAGGCUUAGUCCGUACUCAACUUGAAAA
GGUGGCACCGAUUCGGUGUUUUUU;
(SEQ ID NO: 1428)
AAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUCAACACC
CUGUCAUUUUAUGGCAGGGUGUUUUCGUUAUUU;
(SEQ ID NO: 1429)
AAAUGAGAACCGUUGCUACAAUAAGGCCGUCUGAAAAGAUGUGCCGCAA
CGCUCUGCCCCUUAAAGCUUCUGCUUUAAGGGGCAUCGUUUAUUUC;
and
(SEQ ID NO: 1430)
AAAUGAGAGAGUUGCUACAAUAAGGCUUCUGAAAAGAAUGACCGUAACG
CUCUGCCCCUUGUGAUUCUUAAUUGCAAGGGGCAUCGUUUU.
9. The nucleic acid of claim 1 , wherein the activator-RNA comprises any one of the following tracrRNA sequences:
(SEQ ID NO: 397)
UAGCAAGUUAAAAUAAGGCUAGUCCG;
(SEQ ID NO: 1431)
UAGCAAGUUAAAAUAAGGCUUUGUCCG;
(SEQ ID NO: 1432)
CAGCGAGUUAAAUAAGGCUUAGUCCG;
(SEQ ID NO: 1433)
AGAAGCUACAAAGAUAAGGCUUCAUGCCG;
(SEQ ID NO: 1434)
GUUGCUACAAUAAGGCCGUCUGAAAAGAUGUGCCG; and
(SEQ ID NO: 1435)
AGAGUUGCUACAAUAAGGCUUCUGAAAAGAAUGACCG.
10. The nucleic acid of claim 1 , wherein said nucleotide sequence of the duplex-forming segment of the activator-RNA comprises any one of the following tracrRNA sequences:
(SEQ ID NO: 395)
AAACAGCAUAGCAAGUUAAAAU;
(SEQ ID NO: 1436)
AAAUAACAUAGCAAGUUAAAAU;
(SEQ ID NO: 1437)
AAACAACACAGCAAGUUAAA;
(SEQ ID NO: 1438)
AAACAACACAGCGAGUUAAA;
(SEQ ID NO: 1439)
AAAUCUUGCAGAAGCUACAAAGAU;
(SEQ ID NO: 1440)
AAAUGAGAACCGUUGCUACAAU; and
(SEQ ID NO: 1441)
AAAUGAGAGAGUUGCUACAAU.
11. The nucleic acid of claim 1 , wherein said nucleotide sequence of the duplex-forming segment of the activator-RNA comprises any one of the following tracrRNA sequences:
(SEQ ID NO: 562)
UAGCAAGUUAAAAU;
(SEQ ID NO: 1442)
CAGCGAGUUAAA;
(SEQ ID NO: 1443)
AGAAGCUACAAAGAU;
(SEQ ID NO: 1444)
GUUGCUACAAU; and
(SEQ ID NO: 1445)
AGAGUUGCUACAAU.
12. The nucleic acid of claim 1 , wherein the activator-RNA comprises a nucleotide sequence having 90% nucleotide sequence identity with the tracrRNA sequence set forth in any one of SEQ ID NOs.: 478 and 1425-1430 over a stretch of at least 25 nucleotides.
13. The nucleic acid of claim 1 , wherein the activator-RNA comprises a nucleotide sequence having 90% nucleotide sequence identity with the tracrRNA sequence set forth in any one of SEQ ID NOs.: 397 and 1431-1435 over a stretch of at least 25 nucleotides.
14. The nucleic acid of claim 1 , wherein the activator-RNA comprises a nucleotide sequence having 100% nucleotide sequence identity with the tracrRNA sequence set forth in any one of SEQ ID NOs.: 478 and 1425-1430 over a stretch of at least 25 nucleotides.
15. The nucleic acid of claim 1 , wherein the activator-RNA comprises a nucleotide sequence having 100% nucleotide sequence identity with the tracrRNA sequence set forth in any one of SEQ ID NOs.: 397 and 1431-1435 over a stretch of at least 25 nucleotides.
16. An expression vector comprising a nucleotide sequence encoding a single molecule DNA-targeting RNA, wherein the single molecule DNA-targeting RNA comprises, in 5′ to 3′ order:
(i) a targeter-RNA comprising a first nucleotide sequence that is complementary to a target sequence of a target DNA, and a second nucleotide sequence that hybridizes with an activator-RNA, wherein the first and second nucleotide sequences are heterologous to one another; and
(ii) the activator-RNA, which comprises a duplex-forming segment that comprises a nucleotide sequence that hybridizes with the targeter-RNA to form a double-stranded duplex,
wherein (i) and (ii) are covalently linked by intervening nucleotides, and
wherein the single molecule DNA-targeting RNA is capable of forming a complex with a Cas9 protein and guiding the complex to the target sequence to cleave the target DNA,
wherein the nucleotide sequence encoding the single molecule DNA-targeting RNA is operably linked to a heterologous promoter.
17. The expression vector of claim 16 , wherein said nucleotide sequence of the duplex-forming segment of the activator-RNA comprises a sequence having greater than 80% identity to the tracrRNA sequence UAGCAAGUUAAAAU (SEQ ID NO: 562).
18. The expression vector of claim 16 , wherein the activator-RNA comprises the 26 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCG (SEQ ID NO: 441).
19. The expression vector of claim 16 , wherein the activator-RNA comprises the 31 nucleotide tracrRNA sequence: UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC (SEQ ID NO: 396).
20. The expression vector of claim 16 , wherein the activator-RNA comprises the 67 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 432).
21. The expression vector of claim 16 , wherein the activator-RNA comprises a nucleotide sequence having greater than 80% nucleotide sequence identity with any one of the following tracrRNA sequences:
(SEQ ID NO: 478)
AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAA
AGUGGCACCGAGUCGGUGCUUUUUUU;
(SEQ ID NO: 1425)
AAAUAACAUAGCAAGUUAAAAUAAGGCUUUGUCCGUUAUCAACUUUUAA
UUAAGUAGCGCCGUUUCGGCGCUUUUUU;
(SEQ ID NO: 1426)
AAACAACACAGCAAGUUAAAUAAGGCAGUGAUUUUUAAUCCAGUCCGUA
CACAACUUGAAAAAGUGCGCACCGAUUCGGUGCUUUUUU;
(SEQ ID NO: 1427)
AAACAACACAGCGAGUUAAAUAAGGCUUAGUCCGUACUCAACUUGAAAA
GGUGGCACCGAUUCGGUGUUUUUU;
(SEQ ID NO: 1428)
AAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUCAACACC
CUGUCAUUUUAUGGCAGGGUGUUUUCGUUAUUU;
(SEQ ID NO: 1429)
AAAUGAGAACCGUUGCUACAAUAAGGCCGUCUGAAAAGAUGUGCCGCAA
CGCUCUGCCCCUUAAAGCUUCUGCUUUAAGGGGCAUCGUUUAUUUC;
and
(SEQ ID NO: 1430)
AAAUGAGAGAGUUGCUACAAUAAGGCUUCUGAAAAGAAUGACCGUAACG
CUCUGCCCCUUGUGAUUCUUAAUUGCAAGGGGCAUCGUUUU.
22. The expression vector of claim 16 , wherein the activator-RNA comprises a nucleotide sequence having greater than 90% nucleotide sequence identity with any one of the following tracrRNA sequences:
(SEQ ID NO: 397)
UAGCAAGUUAAAAUAAGGCUAGUCCG;
(SEQ ID NO: 1431)
UAGCAAGUUAAAAUAAGGCUUUGUCCG;
(SEQ ID NO: 1432)
CAGCGAGUUAAAUAAGGCUUAGUCCG;
(SEQ ID NO: 1433)
AGAAGCUACAAAGAUAAGGCUUCAUGCCG;
(SEQ ID NO: 1434)
GUUGCUACAAUAAGGCCGUCUGAAAAGAUGUGCCG; and
(SEQ ID NO: 1435)
AGAGUUGCUACAAUAAGGCUUCUGAAAAGAAUGACCG.
23. The expression vector of claim 16 , wherein the activator-RNA comprises any one of the following tracrRNA sequences:
(SEQ ID NO: 478)
AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAA
AGUGGCACCGAGUCGGUGCUUUUUUU;
(SEQ ID NO: 1425)
AAAUAACAUAGCAAGUUAAAAUAAGGCUUUGUCCGUUAUCAACUUUUAA
UUAAGUAGCGCCGUUUCGGCGCUUUUUU;
(SEQ ID NO: 1426)
AAACAACACAGCAAGUUAAAUAAGGCAGUGAUUUUUAAUCCAGUCCGUA
CACAACUUGAAAAAGUGCGCACCGAUUCGGUGCUUUUUU;
(SEQ ID NO: 1427)
AAACAACACAGCGAGUUAAAUAAGGCUUAGUCCGUACUCAACUUGAAAA
GGUGGCACCGAUUCGGUGUUUUUU;
(SEQ ID NO: 1428)
AAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUCAACACC
CUGUCAUUUUAUGGCAGGGUGUUUUCGUUAUUU;
(SEQ ID NO: 1429)
AAAUGAGAACCGUUGCUACAAUAAGGCCGUCUGAAAAGAUGUGCCGCAA
CGCUCUGCCCCUUAAAGCUUCUGCUUUAAGGGGCAUCGUUUAUUUC;
and
(SEQ ID NO: 1430)
AAAUGAGAGAGUUGCUACAAUAAGGCUUCUGAAAAGAAUGACCGUAACG
CUCUGCCCCUUGUGAUUCUUAAUUGCAAGGGGCAUCGUUUU.
24. The expression vector of claim 16 , wherein the activator-RNA comprises any one of the following tracrRNA sequences:
(SEQ ID NO: 397)
UAGCAAGUUAAAAUAAGGCUAGUCCG;
(SEQ ID NO: 1431)
UAGCAAGUUAAAAUAAGGCUUUGUCCG;
(SEQ ID NO: 1432)
CAGCGAGUUAAAUAAGGCUUAGUCCG;
(SEQ ID NO: 1433)
AGAAGCUACAAAGAUAAGGCUUCAUGCCG;
(SEQ ID NO: 1434)
GUUGCUACAAUAAGGCCGUCUGAAAAGAUGUGCCG; and
(SEQ ID NO: 1435)
AGAGUUGCUACAAUAAGGCUUCUGAAAAGAAUGACCG.
25. The expression vector of claim 16 , wherein said nucleotide sequence of the duplex-forming segment of the activator-RNA comprises any one of the following tracrRNA sequences:
(SEQ ID NO: 395)
AAACAGCAUAGCAAGUUAAAAU;
(SEQ ID NO: 1436)
AAAUAACAUAGCAAGUUAAAAU;
(SEQ ID NO: 1437)
AAACAACACAGCAAGUUAAA;
(SEQ ID NO: 1438)
AAACAACACAGCGAGUUAAA;
(SEQ ID NO: 1439)
AAAUCUUGCAGAAGCUACAAAGAU;
(SEQ ID NO: 1440)
AAAUGAGAACCGUUGCUACAAU; and
(SEQ ID NO: 1441)
AAAUGAGAGAGUUGCUACAAU.
26. The expression vector of claim 16 , wherein said nucleotide sequence of the duplex-forming segment of the activator-RNA comprises any one of the following tracrRNA sequences:
(SEQ ID NO: 562)
UAGCAAGUUAAAAU;
(SEQ ID NO: 1442)
CAGCGAGUUAAA;
(SEQ ID NO: 1443)
AGAAGCUACAAAGAU;
(SEQ ID NO: 1444)
GUUGCUACAAU; and
(SEQ ID NO: 1445)
AGAGUUGCUACAAU.
27. The expression vector of claim 16 , wherein the activator-RNA comprises a nucleotide sequence having 90% nucleotide sequence identity with the tracrRNA sequence set forth in any one of SEQ ID NOs.: 478 and 1425-1430 over a stretch of at least 25 nucleotides.
28. The expression vector of claim 16 , wherein the activator-RNA comprises a nucleotide sequence having 90% nucleotide sequence identity with the tracrRNA sequence set forth in any one of SEQ ID NOs.: 397 and 1431-1435 over a stretch of at least 25 nucleotides.
29. The expression vector of claim 16 , wherein the activator-RNA comprises a nucleotide sequence having 100% nucleotide sequence identity with the tracrRNA sequence set forth in any one of SEQ ID NOs.: 478 and 1425-1430 over a stretch of at least 25 nucleotides.
30. The expression vector of claim 16 , wherein the activator-RNA comprises a nucleotide sequence having 100% nucleotide sequence identity with the tracrRNA sequence set forth in any one of SEQ ID NOs.: 397 and 1431-1435 over a stretch of at least 25 nucleotides.
31. The expression vector of claim 16 , wherein the expression vector is a plasmid.