Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription
First Claim
1. A method of targeting and binding a target DNA, modifying a target DNA, or modulating transcription from a target DNA, the method comprising:
- contacting a target DNA with a complex that comprises;
(a) a Cas9 protein; and
(b) a single molecule DNA-targeting RNA that comprises, in 5′
to 3′
order;
(i) a targeter-RNA that 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 of a protein-binding segment, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 8 to 15 base pairs,wherein (i) and (ii) are covalently linked by intervening nucleotides,and wherein;
the complex binds to the target DNA, the target DNA is modified, the target DNA is cleaved, the target DNA is edited, and/or transcription from the target DNA is modulated.
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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.
218 Citations
29 Claims
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1. A method of targeting and binding a target DNA, modifying a target DNA, or modulating transcription from a target DNA, the method comprising:
-
contacting a target DNA with a complex that comprises; (a) a Cas9 protein; and (b) a single molecule DNA-targeting RNA that comprises, in 5′
to 3′
order;(i) a targeter-RNA that 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 of a protein-binding segment, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 8 to 15 base pairs, wherein (i) and (ii) are covalently linked by intervening nucleotides, and wherein;
the complex binds to the target DNA, the target DNA is modified, the target DNA is cleaved, the target DNA is edited, and/or transcription from the target DNA is modulated. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
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9. A method of targeting and binding a target DNA, modifying a target DNA, or modulating transcription from a target DNA, the method comprising:
-
contacting a target DNA with a complex that comprises; (a) a Cas9 protein; and (b) a single molecule DNA-targeting RNA that comprises, in 5′
to 3′
order;(i) a targeter-RNA that 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 of a protein-binding segment, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 15 to 18 base pairs, wherein (i) and (ii) are covalently linked by intervening nucleotides, and wherein;
the complex binds to the target DNA, the target DNA is modified, the target DNA is cleaved, the target DNA is edited, and/or transcription from the target DNA is modulated. - View Dependent Claims (10, 11, 12, 13, 14, 15, 16)
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17. A composition comprising a single molecule DNA-targeting RNA or a nucleic acid encoding the single molecule DNA-targeting RNA, wherein the single molecule DNA-targeting RNA comprises, in 5′
- to 3′
order;(i) a targeter-RNA comprising a nucleotide sequence that is complementary to a target sequence of a target DNA, and (ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 8 to 15 base pairs, 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 hybridization of the targeter-RNA to the target sequence is capable of targeting the Cas9 protein to the target DNA. - View Dependent Claims (18, 19, 20, 21, 22, 23)
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20. The composition of claim 17, wherein the activator-RNA comprises the 26 nucleotide tracrRNA sequence UAGCAAGUUAAAAUAAGGCUAGUCCG (SEQ ID NO:
- 441).
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21. The composition of claim 17, comprising the single molecule DNA-targeting RNA, wherein the single molecule DNA-targeting RNA comprises one or more of:
- a non-natural internucleoside linkage, a nucleic acid mimetic, a modified sugar moiety, and a modified nucleobase.
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22. The composition of claim 17, comprising the single molecule DNA-targeting RNA, wherein the single molecule DNA-targeting RNA comprises one or more of:
- a phosphorothioate, an inverted polarity linkage, an abasic nucleoside linkage, a locked nucleic acid (LNA), 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′
-dirnethylaminooxyethoxy modified sugar moiety, a 2′
-dimethylaminoethoxyethoxy modified sugar moiety, a peptide nucleic acid (PNA), a morpholino nucleic acid, and a cyclohexenyl nucleic acid (CeNA).
- a phosphorothioate, an inverted polarity linkage, an abasic nucleoside linkage, a locked nucleic acid (LNA), a 2′
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23. The composition of claim 17, wherein the single molecule DNA-targeting RNA, or the nucleic acid encoding the single molecule DNA-targeting RNA, is conjugated to one or more of:
- a polyamine;
a polyamide;
a polyethylene glycol;
a polyether;
a cholesterol moiety;
a cholic acid;
a thioether;
a thiocholesterol;
an aliphatic chain;
a phospholipid;
an adamantane acetic acid;
a palmityl moiety;
an octadecylamine moiety;
a hexylamino-carbonyl-oxycholesterol moiety;
a biotin;
a phenazine;
a folate;
a phenanthridine;
an anthraquinone;
an acridine;
a fluorescein;
a rhodamine;
a dye; and
a coumarin.
- a polyamine;
- to 3′
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24. A composition comprising a single molecule DNA-targeting RNA, or a nucleic acid encoding the single molecule DNA-targeting RNA, wherein the single molecule DNA-targeting RNA comprises, in 5′
- to 3′
order;(i) a targeter-RNA comprising a nucleotide sequence that is complementary to a target sequence of a target DNA, and (ii) an activator-RNA that hybridizes with the targeter-RNA to form a double-stranded RNA duplex of a protein-binding segment, wherein the activator-RNA hybridizes with the targeter-RNA to form a total of 15 to 18 base pairs, 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 hybridization of the targeter-RNA to the target sequence is capable of targeting the Cas9 protein to the target DNA. - View Dependent Claims (25, 26, 27, 28, 29)
- to 3′
Specification