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 DNA-targeting RNA comprising:
(i) a first segment comprising a nucleotide sequence that is complementary to a sequence in a target DNA; and
(ii) a second segment that interacts with a site-directed modifying polypeptide.
2 . The DNA-targeting RNA of claim 1 , wherein the first segment comprises 8 nucleotides that have 100% complementarity to a sequence in the target DNA.
3 . The DNA-targeting RNA of claim 1 , wherein the second segment comprises a nucleotide sequence with at least 60% identity over a stretch of at least 8 contiguous nucleotides to any one of the nucleotide sequences set forth in SEQ ID NOs:563-682, or a complement thereof.
4 . The DNA-targeting RNA of claim 1 , wherein the second segment comprises a nucleotide sequence with at least 60% identity over a stretch of at least 8 contiguous nucleotides to any one of the nucleotide sequences set forth in SEQ ID NOs:431-562, or a complement thereof.
5 . The DNA-targeting RNA of claim 1 , wherein the site-directed modifying polypeptide comprises an amino acid sequence having at least about 75% amino acid sequence identity to amino acids 7-166 or 731-1003 of the Cas9/Csn1 amino acid sequence depicted in FIG. 3 , or to the corresponding portions in any of the amino acid sequences set forth as SEQ ID NOs:1-256 and 795-1346.
6 . A DNA polynucleotide comprising a nucleotide sequence that encodes the DNA-targeting RNA of claim 1 .
7 . A recombinant expression vector comprising the DNA polynucleotide of claim 6 .
8 . The recombinant expression vector of claim 7 , wherein the nucleotide sequence encoding the DNA-targeting RNA is operably linked to a promoter.
9 . The recombinant expression vector of claim 8 , wherein the promoter is an inducible promoter.
10 . The recombinant expression vector of claim 7 , wherein the nucleotide sequence encoding the DNA-targeting RNA of claim 1 further comprises a multiple cloning site.
11 . An in vitro genetically modified host cell comprising the DNA polynucleotide of claim 6 .
12 . A recombinant expression vector comprising:
(i) a nucleotide sequence encoding a DNA-targeting RNA, wherein the DNA-targeting RNA comprises:
(a) a first segment comprising a nucleotide sequence that is complementary to a sequence in a target DNA; and
(b) a second segment that interacts with a site-directed modifying polypeptide; and
(ii) a nucleotide sequence encoding the site-directed modifying polypeptide comprising:
(a) an RNA-binding portion that interacts with the DNA-targeting RNA; and
(b) an activity portion that exhibits site-directed enzymatic activity, wherein the site of enzymatic activity is determined by the DNA-targeting RNA.
13 . A recombinant expression vector comprising:
(i) a nucleotide sequence encoding a DNA-targeting RNA, wherein the DNA-targeting RNA comprises:
(a) a first segment comprising a nucleotide sequence that is complementary to a sequence in a target DNA; and
(b) a second segment that interacts with a site-directed modifying polypeptide; and
(ii) a nucleotide sequence encoding the site-directed modifying polypeptide, wherein the site-directed modifying polypeptide comprises:
(a) an RNA-binding portion that interacts with the DNA-targeting RNA; and
(b) an activity portion that modulates transcription within the target DNA, wherein the site of modulated transcription within the target DNA is determined by the DNA-targeting RNA.
14 .- 155 . (canceled)