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 eukaryotic cell comprising a target DNA molecule and an engineered and/or non-naturally occurring Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system comprising
a nucleic acid comprising a nucleotide sequence encoding a Cas9 protein, and
a DNA-targeting RNA or one or more nucleic acids comprising one or more nucleotide sequences encoding the DNA-targeting RNA;
wherein the DNA-targeting RNA comprises
i) a targeter-RNA that is capable of hybridizing with a target sequence in a target DNA molecule, and
ii) an activator-RNA that is capable of hybridizing 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 (11) the Cas9 protein comprises an amino acid sequence that is modified relative to naturally occurring Cas9 proteins,
whereby hybridization of the targeter-RNA to the target sequence is capable of targeting the Cas9 protein to the target DNA molecule.
4 . The eukaryotic cell of claim 3 , wherein the genome of said eukaryotic cell comprises the target DNA molecule.
5 . The eukaryotic cell of claim 3 , wherein the eukaryotic cell is a mammalian cell or a human cell.
6 . The eukaryotic cell of claim 3 , wherein the nucleic acid comprising the nucleotide sequence encoding the Cas9 protein is an RNA molecule.
7 . The eukaryotic cell of claim 3 , comprising the DNA-targeting RNA as RNA.
8 . The eukaryotic cell of claim 3 , wherein the nucleic acid comprising the nucleotide sequence encoding the Cas9 protein is an RNA molecule, and the eukaryotic cell comprises the DNA-targeting RNA as RNA.
9 . The eukaryotic cell of claim 8 , wherein the eukaryotic cell is in vivo.
10 . The eukaryotic cell of claim 8 , wherein the eukaryotic cell is a mammalian cell.
11 . The eukaryotic cell of claim 3 , wherein the nucleic acid comprising the nucleotide sequence encoding the DNA-targeting RNA is a plasmid, and the eukaryotic cell comprises said plasmid.
12 . The eukaryotic cell of claim 3 , wherein the nucleic acid comprising the nucleotide sequence encoding the Cas9 protein is a plasmid, and the eukaryotic cell comprises said plasmid.
13 . The eukaryotic cell of claim 3 , comprising:
the nucleic acid comprising the nucleotide sequence encoding the DNA-targeting RNA, and
the nucleic acid comprising the nucleotide sequence encoding the Cas9 protein,
wherein both of said nucleic acids are plasmids.
14 . The eukaryotic cell of claim 13 , wherein the eukaryotic cell is a mammalian cell.
15 . The eukaryotic cell of claim 14 , wherein the Cas9 protein comprises the amino acid sequence set forth in SEQ ID NO: 2.
16 . The eukaryotic cell of claim 13 , wherein the eukaryotic cell is a single-cell organism.
17 . The eukaryotic cell of claim 3 , wherein the nucleic acid comprising the nucleotide sequence encoding the DNA-targeting RNA is a viral vector, and the eukaryotic cell comprises said viral vector.
18 . The eukaryotic cell of claim 3 , wherein the nucleic acid comprising the nucleotide sequence encoding the Cas9 protein is a viral vector, and the eukaryotic cell comprises said viral vector.
19 . The eukaryotic cell of claim 3 , comprising one or more viral vectors, wherein said one or more viral vectors comprises:
the nucleic acid comprising the nucleotide sequence encoding the DNA-targeting RNA, and
the nucleic acid comprising the nucleotide sequence encoding the Cas9 protein.
20 . The eukaryotic cell of claim 19 , wherein the eukaryotic cell is a mammalian cell.
21 . The eukaryotic cell of claim 19 , wherein the eukaryotic cell is a single-cell organism.
22 . The eukaryotic cell of claim 3 , 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.
23 . The eukaryotic cell of claim 3 , wherein one or more of a) the nucleic acid comprising the nucleotide sequence encoding said Cas9 protein and b) the one or more nucleic acids comprising one or more nucleotide sequences encoding said DNA-targeting RNA, are one or more vectors, wherein the one or more vectors are selected from the group consisting of plasmids, cosmids, minicircles, phage, viral, retroviral, lentiviral, adenoviral, adeno-associated, and herpes simplex virus vectors; and wherein the nucleotide sequence encoding said Cas9 protein and/or the one or more nucleotide sequences encoding said DNA-targeting RNA is operably linked to a control element operable in said eukaryotic cell.
24 . The eukaryotic cell of claim 3 , wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s)) that comprise an amino acid sequence selected from the group consisting of SEQ ID NOs:268 and 269.
25 . The eukaryotic cell of claim 3 , wherein the Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain.
26 . The eukaryotic cell of claim 3 , wherein the system comprises a donor polynucleotide and the system is capable of editing the target DNA molecule by inserting a sequence of the donor polynucleotide into a cleaved strand of the target DNA molecule.
27 . The eukaryotic cell of claim 3 , wherein the nucleotide sequence encoding said 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.
28 . The eukaryotic cell of claim 3 , wherein the activator-RNA comprises the 88 nucleotide tracrRNA sequence set forth in SEQ ID NO:433.
29 . The eukaryotic cell of claim 3 , wherein said Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain and is capable of cleaving only one strand of DNA.
30 . A eukaryotic cell comprising a target DNA and 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 nucleic acid comprising a nucleotide sequence encoding a Cas9 protein; and
b) a DNA-targeting RNA, or one or more nucleic acids comprising one or more nucleotide sequences encoding said DNA-targeting RNA;
wherein the DNA-targeting RNA comprises:
i) a targeter-RNA that is capable of hybridizing with a target sequence in the target DNA molecule, and
ii) an activator-RNA that is capable of hybridizing 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 is capable of forming a complex with the Cas9 protein, thereby targeting the Cas9 protein to the target DNA molecule,
whereby said system is capable of cleaving or editing the target DNA molecule or modulating transcription of at least one gene encoded by the target DNA molecule.
31 . The eukaryotic cell of claim 30 , wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s)) that comprise an amino acid sequence selected from the group consisting of SEQ ID NOs:268 and 269.
32 . A non-human plant or animal comprising the eukaryotic cell of claim 3 .