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 method of targeting and binding a target DNA, the method comprising: contacting the target DNA with a complex that comprises:
(a) a Cas9 protein; and
(b) a DNA-targeting RNA comprising:
(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 activator-RNA hybridizes with the targeter-RNA to form a total of 8 to 15 base pairs, and wherein the activator-RNA comprises one or more of: a non-natural internucleoside linkage, a nucleic acid mimetic, a modified sugar moiety, and a modified nucleobase
wherein the complex binds to the target DNA.
2. The method of claim 1 , wherein the nucleotide sequence that is complementary to the target sequence of the target DNA is 15-25 nucleotides (nt) long.
3. The method of claim 1 , wherein the Cas9 protein is fused to a heterologous polypeptide.
4. The method of claim 1 , wherein the activator-RNA comprises the non-natural internucleoside linkage and the modified sugar moiety.
5. The method of claim 1 , wherein the activator-RNA comprises one or more of:
(1) the non-natural internucleoside linkage, which comprises a phosphorothioate, an inverted polarity linkage, or an abasic nucleoside linkage,
(2) the modified sugar moiety, which comprises a locked nucleic acid (LNA) sugar moiety, a 2′-substituted sugar moiety, a 2′-O-methoxyethyl modified sugar moiety, a 2′-methyl modified sugar moiety, a 2′-O-(2-methoxyethyl) modified sugar moiety, a 2′-fluoro modified sugar moiety, a 2′-dimethylaminooxyethoxy modified sugar moiety, or a 2′-dimethylaminoethoxyethoxy modified sugar moiety, and
(3) the nucleic acid mimetic, which comprises a peptide nucleic acid (PNA), a morpholino nucleic acid, or a cyclohexenyl nucleic acid (CeNA).
6. The method of claim 1 , wherein the activator-RNA comprises:
(1) the non-natural internucleoside linkage, which comprises a phosphorothioate, a phosphoramidate, a non-phosphodiester, a heteroatom, a chiral phosphorothioate, a phosphorodithioate, a phosphotriester, an aminoalkylphosphotriester, a 3′-alkylene phosphonates, a 5′-alkylene phosphonate, a chiral phosphonate, a phosphinate, a, a 3′-amino phosphoramidate, an aminoalkylphosphorarnidate, phosphorodiamidate, a thionophosphoramidate, a thionoalkylphosphonate, a thionoalkylphosphotriester, a selenophosphate, or a boranophosphate; and/or
(2) the modified nucleobase, which that comprises a 5-methylcytosine; a 5-hydroxymethyl cytosine; a xanthine; a hypoxanthine; a 2-aminoadenine; a 6-methyl derivative of adenine; a 6-methyl derivative of guanine; a 2-propyl derivative of adenine; a 2-propyl derivative of guanine; a 2-thiouracil; a 2-thiothymine; a 2-thiocytosine; a 5-propynyl uracil; a 5-propynyl cytosine; a 6-azo uracil; a 6-azo cytosine; a 6-azo thymine; a pseudouracil; a 4-thiouracil; an 8-haloadenin; an 8-aminoadenin; an 8-thioladenin; an 8-thioalkyladenin; an 8-hydroxyladenin; an 8-haloguanin; an 8-aminoguanin; an 8-thiolguanin; an 8-thioalkylguanin; an 8-hydroxylguanin; a 5-halouracii; a 5-bromouracil; a 5-trifluoromethyluracil; a 5-halocytosine; a 5-bromocytosine; a 5-trifluoromethylcytosine; a 5-substituted uracil; a 5-substituted cytosine; a 7-methylguanine; a 7-methyladenine; a 2-F-adenine; a 2-amino-adenine; an 8-azaguanine; an 8-azaadenine; a 7-deazaguanine; a 7-deazaadenine; a 3-deazaguanine; a 3-deazaadenine; a tricyclic pyrimidine; a phenoxazine cytidine; a phenothiazine cytidine; a substituted phenoxazine cytidine; a carbazole cytidine; a pyridoindole cytidine; a 7-deazaguanosine; a 2-aminopyridine; a 2-pyridone; a 5-substituted pyrimidine; a 6-azapyrimidine; an N-2, N-6 or O-6 substituted purine; a 2-aminopropyladenine; a 5-propynyluracil; or a 5-propynylcytosine.
7. The method of claim 1 , wherein the targeter-RNA and the activator-RNA are not covalently linked to one another by intervening nucleotides.
8. The method of claim 1 , wherein
the targeter-RNA comprises a heterologous moiety; and/or
the activator-RNA further comprises a heterologous moiety in addition to said one or more of: the non-natural internucleoside linkage, the nucleic acid mimetic, the modified sugar moiety, and the modified nucleobase.
9. The method of claim 1 , wherein the targeter-RNA and/or the activator-RNA comprises one or more of the following heterologous moieties: a polyamine; a polyamide; a polyethylene glycol; a polyether; a cholesterol moiety; a cholic acid; a thioester; a thiocholesterol; a lipid; an aliphatic chain; a phospholipid; an adamantane acetic acid; a palmityl moiety; an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety; a biotin; a phenazine; a folate; a phenanthridine; an anthraquinone; an acridine; a fluorescein; a rhodamine; a coumarin; and a dye.
10. The method of claim 1 , wherein the Cas9 protein comprises a mutation in a RuvC and/or an HNH domain.
11. The method of claim 1 , wherein the method comprises contacting the target DNA with two or more DNA-targeting RNAs, wherein the two or more DNA-targeting RNAs hybridize to different target sequences within the same or different target DNA molecules.
12. The method of claim 1 , wherein the target DNA is inside of a cell and said contacting comprises introducing the Cas9 protein into the cell as protein.
13. The method of claim 1 , wherein the target DNA is inside of a cell and said contacting comprises introducing a nucleic acid that encodes the Cas9 protein into the cell.
14. The method of claim 1 , wherein the target DNA is inside of a cell and said contacting comprises introducing a nucleic acid encoding the Cas9 protein into the cell, wherein the nucleic acid encoding the Cas9 protein comprises a nucleotide sequence modification that replaces one or more codons of a wild-type Cas9-encoding nucleotide sequence with one or more different codons encoding the same amino acid.
15. A method of modifying a target DNA, the method comprising: contacting the target DNA with a complex that comprises:
(a) a Cas9 protein; and
(b) a DNA-targeting RNA comprising:
(i) 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 activator-RNA hybridizes with the targeter-RNA to form a total of 8 to 15 base pairs, and wherein the activator-RNA comprises one or more of: a non-natural internucleoside linkage, a nucleic acid mimetic, a modified sugar moiety, and a modified nucleobase,
wherein said contacting results in modification of the target DNA.
16. The method of claim 15 , wherein said modification is cleavage of the target DNA.
17. The method of claim 16 , wherein the target DNA is inside of a cell and the method further comprises introducing a donor polynucleotide into the cell, wherein a nucleotide sequence of the donor polynucleotide inserts into the target DNA, producing a genetically modified cell.
18. The method of claim 15 , wherein the nucleotide sequence that is complementary to the target sequence of the target DNA is 15-25 nucleotides (nt) long.
19. The method of claim 15 , wherein the Cas9 protein is fused to a heterologous polypeptide.
20. The method of claim 15 , wherein the activator-RNA comprises Fail the non-natural internucleoside linkage and the modified sugar moiety.
21. The method of claim 15 , wherein the activator-RNA comprises one or more of:
(1) the non-natural internucleoside linkage, which comprises a phosphorothioate, an inverted polarity linkage, or an abasic nucleoside linkage,
(2) the modified sugar moiety, which comprises a locked nucleic acid (LNA) sugar moiety, a 2′-substituted sugar moiety, a 2′-O-methoxyethyl modified sugar moiety, a 2′-O-methyl modified sugar moiety, a 2′-O-(2-methoxyethyl) modified sugar moiety, a 2′-fluoro modified sugar moiety, a 2′-dimethylaminooxyethoxy modified sugar moiety, or a 2′-dimethylaminoethoxyethoxy modified sugar moiety, and
(3) the nucleic acid mimetic, which comprises a peptide nucleic acid (PNA), a morpholino nucleic acid, or a cyclohexenyl nucleic acid (CeNA).
22. The method of claim 15 , wherein the activator-RNA comprises:
(1) the non-natural internucleoside linkage, which comprises a phosphorothioate, a phosphoramidate, a non-phosphodiester, a heteroatom, a chiral phosphorothioate, a phosphorodithioate, a phosphotriester, an aminoalkylphosphotriester, a 3′-alkylene phosphonates, a 5′-alkylene phosphonate, a chiral phosphonate, a phosphinate, a, a 3′-amino phosphoramidate, an aminoalkylphosphoramidate, a phosphorodiamidate, a thionophosphoramidate, a thionoalkylphosphonate, a thionoalkylphosphotriester, a selenophosphate, or a boranophosphate; and/or
(2) the modified nucleobase, which that comprises a 5-methylcytosine; a 5-hydroxymethyl cytosine; a xanthine; a hypoxanthine; a 2-aminoadenine; a 6-methyl derivative of adenine; a 6-methyl derivative of guanine; a 2-propyl derivative of adenine; a 2-propyl derivative of guanine; a 2-thiouracil; a 2-thiothymine; a 2-thiocytosine; a 5-propynyl uracil; a 5-propynyl cytosine; a 6-azo uracil; a 6-azo cytosine; a 6-azo thymine; a pseudouracil; a 4-thiouracil; an 8-haloadenin; an 8-aminoadenin; an 8-thioladenin; an 8-thioalkyladenin; an 8-hydroxyladenin; an 8-haloguanin; an 8-aminoguanin; an 8-thiolguanin; an 8-thioalkylguanin; an 8-hydroxylguanin; a 5-halouracil; a 5-bromouracil; a 5-trifluoromethyluracil; a 5-halocytosine; a 5-bromocytosine; a 5-trifluoromethylcytosine; a 5-substituted uracil; a 5-substituted cytosine; a 7-methylguanine; a 7-methyladenine; a 2-F-adenine; a 2-amino-adenine; an 8-azaguanine; an 8-azaadenine; a 7-deazaguanine; a 7-deazaadenine; a 3-deazaguanine; a 3-deazaadenine; a tricyclic pyrimidine; a phenoxazine cytidine; a phenothiazine cytidine; a substituted phenoxazine cytidine; a carbazole cytidine; a pyridoindole cytidine; a 7-deazaguanosine; a 2-aminopyridine; a 2-pyridone; a 5-substituted pyrimidine; a 6-azapyrimidine; an N-2, N-6 or O-6 substituted purine; a 2-aminopropyladenine; a 5-propynyluracil; or a 5-propynylcytosine.
23. The method of claim 15 , wherein the targeter-RNA and the activator-RNA are not covalently linked to one another by intervening nucleotides.
24. The method of claim 15 , wherein
the targeter-RNA comprises a heterologous moiety; and/or
the activator-RNA further comprises a heterologous moiety in addition to said one or more of; the non-natural internucleoside linkage, the nucleic acid mimetic, the modified sugar moiety, and the modified nucleobase.
25. The method of claim 15 , wherein the targeter-RNA and/or the activator-RNA comprises one or more of the following heterologous moieties: a polyamine; a polyamide; a polyethylene glycol; a polyether; a cholesterol moiety; a cholic acid; a thioester; a thiocholesterol; a lipid; an aliphatic chain; a phospholipid; an adamantine acetic acid; a palmityl moiety; an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety; a biotin; a phenazine; a folate; a phenanthridine; an anthraquinone; an acridine; a fluorescein; a rhodamine; a coumarin; and a dye.
26. The method of claim 15 , wherein the Cas9 protein comprises a mutation in a RuvC and/or an HNH domain.
27. The method of claim 15 , wherein the method comprises contacting the target DNA with two or more DNA-targeting RNAs, wherein the two or more DNA-targeting RNAs hybridize to different target sequences within the same or different target DNA molecules.
28. The method of claim 15 , wherein the target DNA is inside of a cell and said contacting comprises introducing the Cas9 protein into the cell as protein.
29. The method of claim 15 , wherein the target DNA is inside of a cell and said contacting comprises introducing a nucleic acid that encodes the Cas9 protein into the cell.
30. The method of claim 15 , wherein the target DNA is inside of a cell and said contacting comprises introducing a nucleic acid encoding the Cas9 protein into the cell, wherein the nucleic acid encoding the Cas9 protein comprises a nucleotide sequence modification that replaces one or more codons of a wild-type Cas9-encoding nucleotide sequence with one or more different codons encoding the same amino acid.
31. A method of targeting and binding a target DNA, the method comprising: contacting the target DNA with a complex that comprises:
(a) a Cas9 protein that comprises a mutation in a RuvC and/or an HNH domain; and
(b) a DNA-targeting RNA comprising:
(i) a targeter-RNA comprising: a first nucleotide sequence that is complementary to, and hybridizes with, a target sequence of the 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 that hybridizes with the targeter-RNA to form a double-stranded RNA duplex, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 8 to 15 base pairs,
wherein the complex binds to the target DNA.
32. The method of claim 31 , wherein the nucleotide sequence that is complementary to the target sequence of the target DNA is 15-25 nucleotides (nt) long.
33. The method of claim 31 , wherein the Cas9 protein is fused to a heterologous polypeptide.
34. The method of claim 31 , wherein the Cas9 protein is fused to a heterologous polypeptide that comprises one or more of: a protein tag, an endosomolytic domain, an influenza HA domain, an IF2 domain, a GST domain, a GRPE domain, a 6×His tag, a hemagglutinin (FIA) tag, and green fluorescent protein.
35. The method of claim 31 , wherein the targeter-RNA and/or the activator-RNA comprises one or more of: a non-natural internucleoside linkage, a nucleic acid mimetic, a modified sugar moiety, and a modified nucleobase.
36. The method of claim 35 , wherein the targeter-RNA and/or the activator-RNA comprises said non-natural internucleoside linkage and/or said modified sugar moiety.
37. The method of claim 31 , wherein the targeter-RNA and/or the activator-RNA comprises one or more of: a phosphorothioate, an inverted polarity linkage, an abasic nucleoside linkage, a locked nucleic acid (LNA), a 2′-substituted sugar moiety, a 2′-O-methoxyethyl modified sugar moiety, a 2′-O-methyl modified sugar moiety, a 2′-O-(2-methoxyethyl) modified sugar moiety, a 2′-fluoro modified sugar moiety, a 2′-dimethylaminooxyethoxy modified sugar moiety, a 2′-dimethylaminoethoxyethoxy modified sugar moiety, a peptide nucleic acid (PNA), a morpholino nucleic acid, and a cyclohexenyl nucleic acid (CeNA).
38. The method of claim 31 , wherein the targeter-RNA and the activator-RNA are not covalently linked to one another by intervening nucleotides.
39. The method of claim 31 , wherein the targeter-RNA and/or the activator-RNA comprises a heterologous moiety.
40. The method of claim 39 , wherein the heterologous moiety comprises: a polyamine; a polyamide; a polyethylene glycol; a polyether; a cholesterol moiety; a cholic acid; a thioester; a thiocholesterol; a lipid; an aliphatic chain; a phospholipid; an adamantane acetic acid; a palmityl moiety; an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety; a biotin; a phenazine; a folate; a phenanthridine; an anthraquinone; an acridine; a fluorescein; a rhodamine; a coumarin; or a dye.
41. The method of claim 31 , wherein the targeter-RNA and the activator-RNA are conjugated to a heterologous moiety.
42. The method of claim 31 , wherein the method comprises contacting the target DNA with two or more DNA-targeting RNAs, wherein the two or more DNA-targeting RNAs hybridize to different target sequences within the same or different target DNA molecules.
43. A method of modifying a target DNA, the method comprising: contacting the target DNA with a complex that comprises:
(a) a Cas9 protein that comprises a mutation in a RuvC and/or an HNH domain; and
(b) a DNA-targeting RNA comprising:
(i) a targeter-RNA comprising: a first nucleotide sequence that is complementary to, and hybridizes with, a target sequence of the 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 that hybridizes with the targeter-RNA to form a double-stranded RNA duplex, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 8 to 15 base pairs,
wherein said contacting results in modification of the target DNA.
44. The method of claim 43 , wherein said modification is cleavage of the target DNA.
45. The method of claim 44 , wherein a nucleotide sequence of a donor polynucleotide is integrated into the target DNA.
46. The method of claim 43 , wherein the nucleotide sequence that is complementary to the target sequence of the target DNA is 15-25 nucleotides (nt) long.
47. The method of claim 43 , wherein the Cas9 protein is fused to a heterologous polypeptide.
48. The method of claim 43 , wherein the Cas9 protein is fused to a heterologous polypeptide that comprises one or more of: a protein tag, an endosomolytic domain, an influenza HA domain, an IF2 domain, a GST domain, a GRPE domain, a 6×His tag, a hemagglutinin (HA) tag, and green fluorescent protein.
49. The method of claim 43 , wherein the targeter-RNA and/or the activator-RNA comprises one or more of: a non-natural internucleoside linkage, a nucleic acid mimetic, a modified sugar moiety, and a modified nucleobase.
50. The method of claim 49 , wherein the targeter-RNA and/or the activator-RNA comprises said non-natural internucleoside linkage and/or said modified sugar moiety.
51. The method of claim 43 , wherein the targeter-RNA and/or the activator-RNA comprises one or more of: a phosphorothioate, an inverted polarity linkage, an abasic nucleoside linkage, a locked nucleic acid (LNA), a 2′-substituted sugar moiety, a 2′-O-methoxyethyl modified sugar moiety, a 2′-O-methyl modified sugar moiety, a 2′-O-(2-methoxyethyl) modified sugar moiety, a 2′-fluoro modified sugar moiety, a 2′-dimethylaminooxyethoxy modified sugar moiety, a 2′-dimethylaminoethoxyethoxy modified sugar moiety, a peptide nucleic acid (PNA), a morpholino nucleic acid, and a cyclohexenyl nucleic acid (CeNA).
52. The method of claim 43 , wherein the targeter-RNA and the activator-RNA are not covalently linked to one another by intervening nucleotides.
53. The method of claim 43 , wherein the targeter-RNA and/or the activator-RNA comprises a heterologous moiety.
54. The method of claim 53 , wherein the heterologous moiety comprises: a polyamine; a polyamide; a polyethylene glycol; a polyether; a cholesterol moiety; a cholic acid; a thioester; a thiocholesterol; a lipid; an aliphatic chain; a phospholipid; an adamantane acetic acid; a palmityl moiety; an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety; a biotin; a phenazine; a folate; a phenanthridine; an anthraquinone; an acridine; a fluorescein; a rhodamine; a coumarin; or a dye.
55. The method of claim 43 , wherein the targeter-RNA and the activator-RNA are conjugated to a heterologous moiety.
56. The method of claim 43 , wherein the method comprises contacting the target DNA with two or more DNA-targeting RNAs, wherein the two or more DNA-targeting RNAs hybridize to different target sequences within the same or different target DNA molecules.
57. The method of claim 43 , wherein the target DNA is inside of a cell and said contacting comprises introducing the Cas9 protein into the cell as protein.
58. The method of claim 43 , wherein the target DNA is inside of a cell and said contacting comprises introducing a nucleic acid that encodes the Cas9 protein into the cell.
59. The method of claim 43 , wherein the target DNA is inside of a cell and said contacting comprises introducing a nucleic acid encoding the Cas9 protein into the cell, wherein the nucleic acid encoding the Cas9 protein comprises a nucleotide sequence modification that replaces one or more codons of a wild-type Cas9-encoding nucleotide sequence with one or more different codons encoding the same amino acid.