Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription

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.

1 - 155 . (canceled) 
     
     
         156 . A method of cleaving or editing a target DNA molecule or modulating transcription of at least one gene encoded thereon, the method comprising
 contacting a target DNA molecule having a target sequence with 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 Cas9 protein; and 
 (b) a DNA-targeting RNA comprising:
 (i) a targeter-RNA that hybridizes with the target sequence, and 
 (ii) an activator-RNA that hybridizes 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, 
 wherein said contacting takes place outside of a bacterial cell and outside an archaeal cell, 
 wherein the DNA-targeting RNA forms a complex with the Cas9 protein, whereby said target DNA molecule is cleaved or edited or transcription of at least one gene encoded by the target DNA molecule is modulated, 
 wherein said contacting occurs in a eukaryotic cell. 
   
     
     
         157 . The method of  claim 156 , wherein, prior to the contacting step, the method comprises:
 introducing into the eukaryotic cell containing the target DNA molecule:
 1) the DNA-targeting RNA, or one or more DNA molecules comprising one or more nucleotide sequences that (i) encode the DNA-targeting RNA and (ii) are operably linked to regulatory elements operable in said eukaryotic cell; and 
 2) the Cas9 protein, an RNA molecule comprising a nucleotide sequence encoding the Cas9 protein, or a DNA molecule comprising a nucleotide sequence that (i) encodes the Cas9 protein and (ii) is operably linked to a regulatory element operable in said eukaryotic cell. 
   
     
     
         158 . The method of  157 , wherein the eukaryotic cell's genome comprises the target DNA molecule. 
     
     
         159 . The method of  157 , wherein the eukaryotic cell is a mammalian cell or a human cell. 
     
     
         160 . The method of  claim 157 , 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. 
     
     
         161 . The method of  claim 157 , wherein the targeter-RNA and/or the activator-RNA are transcribed from one or more vectors comprising nucleotide sequences encoding the targeter-RNA and/or the activator-RNA, 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. 
     
     
         162 . The method of  claim 157 , wherein the DNA molecule comprising the nucleotide sequence that encodes the Cas9 protein is a vector selected from the group consisting of plasmids, cosmids, minicircles, phage, viral, retroviral, lentiviral, adenoviral, adeno-associated, and herpes simplex virus vectors. 
     
     
         163 . The method of  claim 157 , wherein the CRISPR-Cas system comprises two or more different DNA-targeting RNAs, or one or more DNA molecules comprising two or more nucleotide sequences encoding different DNA-targeting RNAs. 
     
     
         164 . The method of  claim 157 , wherein the method comprises creation of a double strand break in the target DNA molecule which is repaired by a non-homologous end joining (NHEJ) repair mechanism, thereby editing the target DNA molecule. 
     
     
         165 . The method of  claim 157 , wherein the method comprises creation of a double strand break in the target DNA molecule which is repaired by a homology-directed repair mechanism which incorporates a sequence of a donor polynucleotide into the target DNA molecule, thereby editing the target DNA molecule. 
     
     
         166 . The method of  claim 157 , wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s)) that aid in traversal of an organelle membrane. 
     
     
         167 . The method of  claim 166 , wherein the one or more PTD(s) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:268 and 269. 
     
     
         168 . The method of  claim 157 , wherein the Cas9 protein comprises an amino acid sequence that is modified, relative to naturally occurring Cas9, to include one or more mutations in a RuvC domain and/or a HNH domain. 
     
     
         169 . The method of  claim 157 , wherein the activator-RNA comprises the 88 nucleotide tracrRNA sequence set forth in SEQ ID NO:433. 
     
     
         170 . The method of  claim 157 , wherein the method comprises editing the target DNA molecule by insertion of a sequence of a donor polynucleotide into the cleaved strand of the target DNA molecule. 
     
     
         171 . The method of  claim 157 , wherein said Cas9 protein cleaves only one strand of DNA and comprises an amino acid sequence that is modified, relative to naturally occurring Cas9, to include one or more mutations in a RuvC domain and/or a HNH domain. 
     
     
         172 . The method of  claim 171 , wherein the targeter-RNA and/or the activator-RNA are transcribed from one or more vectors comprising nucleotide sequences encoding the targeter-RNA and/or the activator-RNA, 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. 
     
     
         173 . The method of  claim 171 , wherein the DNA molecule comprising the nucleotide sequence that encodes the Cas9 protein is a vector selected from the group consisting of plasmids, cosmids, minicircles, phage, viral, retroviral, lentiviral, adenoviral, adeno-associated, and herpes simplex virus vectors. 
     
     
         174 . The method of  claim 171 , wherein the CRISPR-Cas system comprises two or more different DNA-targeting RNAs, or one or more DNA molecules comprising two or more nucleotide sequences encoding different DNA-targeting RNAs. 
     
     
         175 . The method of  claim 171 , wherein the method comprises editing the target DNA molecule by insertion of a sequence of a donor polynucleotide into the cleaved strand of the target DNA molecule. 
     
     
         176 . The method of  claim 171 , wherein the method comprises editing the target DNA molecule by a homology-directed repair mechanism. 
     
     
         177 . The method of  claim 171 , wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s)) that aid in traversal of an organelle membrane. 
     
     
         178 . The method of  claim 177 , wherein the one or more PTD(s) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:268 and 269. 
     
     
         179 . The method of  claim 157 , wherein the eukaryotic cell is a plant or animal cell in vivo, and wherein the method comprises introducing into the eukaryotic cell, by administering to an individual comprising the eukaryotic cell:
 (1) the DNA-targeting RNA, or one or more DNA molecules comprising nucleotide sequences that (i) encode the DNA-targeting RNA and (ii) are operably linked to a regulatory element operable in said eukaryotic cell; and   (2) the Cas9 protein, an RNA molecule comprising a nucleotide sequence encoding the Cas9 protein, or a DNA molecule comprising a nucleotide sequence that (i) encodes the Cas9 protein and (ii) is operably linked to a regulatory element operable in said eukaryotic cell,   wherein the method results in editing of the target DNA.   
     
     
         180 . The method of  claim 179 , wherein the method is performed to treat a disease, to produce a gene knock-out, to produce a gene knock-in, or to produce a genetically modified organism for agriculture. 
     
     
         181 . The method of  claim 179 , wherein the activator-RNA comprises the 75 nucleotide tracrRNA sequence set forth in SEQ ID NO: 478. 
     
     
         182 . The method of  claim 179 , wherein the Cas9 protein comprises an amino acid sequence that is modified, relative to naturally occurring Cas9, to include one or more mutations in a RuvC domain and/or a HNH domain. 
     
     
         183 . The method of  claim 179 , wherein: the one or more DNA molecules comprising the nucleotide sequences that encode the DNA-targeting RNA, and/or the DNA molecule comprising the nucleotide sequence that encodes the Cas9 protein, are vectors selected from the group consisting of plasmids, cosmids, minicircles, phage, and viral vectors. 
     
     
         184 . The method of  claim 179 , wherein said CRISPR-Cas system comprises two or more DNA-targeting RNAs, or one or more DNA molecules comprising two or more nucleotide sequences encoding DNA-targeting RNAs. 
     
     
         185 . The method of  claim 179 , wherein (1) and (2) are administered as part of a pharmaceutically acceptable vehicle, wherein the pharmaceutically acceptable vehicle is a lipid.