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 single molecule 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 the targeter-RNA and the activator-RNA are covalently linked to one another with intervening nucleotides; and 
 b) a Cas9 protein, 
   wherein the single molecule DNA-targeting RNA forms a complex with the Cas9 protein, thereby targeting the Cas9 protein to the target DNA molecule,   whereby said target DNA molecule is cleaved or edited or transcription of at least one gene encoded by the target DNA molecule is modulated, and   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 single molecule DNA-targeting RNA, or a DNA molecule comprising a nucleotide sequence that (i) encodes the single molecule DNA-targeting RNA and (ii) is 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.   
     
     
         158 . The method of  claim 157 , wherein the eukaryotic cell's genome comprises the target DNA molecule. 
     
     
         159 . The method of  claim 157 , wherein the eukaryotic cell is a mammalian cell or a human cell. 
     
     
         160 . The method of  claim 157 , wherein the DNA molecule comprising the nucleotide sequence that encodes the single molecule DNA-targeting RNA is a vector selected from the group consisting of: plasmids, cosmids, minicircles, phage, viral, retroviral, lentiviral, adenoviral, adeno-associated, and herpes simplex virus vectors. 
     
     
         161 . 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. 
     
     
         162 . The method of  claim 157 , wherein the CRISPR-Cas system comprises two or more single molecule DNA-targeting RNAs, or one or more DNA molecules comprising two or more nucleotide sequences encoding single molecule DNA-targeting RNAs. 
     
     
         163 . 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. 
     
     
         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 homology-directed repair mechanism which incorporates a sequence of a donor polynucleotide into the target DNA molecule, thereby editing the target DNA molecule. 
     
     
         165 . 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. 
     
     
         166 . The method of  claim 165 , wherein the one or more PTD(s) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:268 and 269. 
     
     
         167 . The method of  claim 157 , wherein the Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain. 
     
     
         168 . The method of  claim 157 , wherein the activator-RNA comprises the 88 nucleotide tracrRNA sequence set forth in SEQ ID NO:433. 
     
     
         169 . 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. 
     
     
         170 . The method of  claim 157 , wherein said Cas9 protein cleaves only one strand of DNA and comprises one or more mutations in a RuvC domain and/or a HNH domain. 
     
     
         171 . The method of  claim 170 , wherein the DNA molecule comprising the nucleotide sequence that encodes the single molecule DNA-targeting RNA is a vector selected from the group consisting of: plasmids, cosmids, minicircles, phage, viral, retroviral, lentiviral, adenoviral, adeno-associated, and herpes simplex virus vectors. 
     
     
         172 . The method of  claim 170 , 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. 
     
     
         173 . The method of  claim 170 , wherein the CRISPR-Cas system comprises two or more single molecule DNA-targeting RNAs, or one or more DNA molecules comprising two or more nucleotide sequences encoding single molecule DNA-targeting RNAs. 
     
     
         174 . The method of  claim 170 , 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. 
     
     
         175 . The method of  claim 170 , wherein the method comprises editing the target DNA molecule by a homology-directed repair mechanism. 
     
     
         176 . The method of  claim 170 , wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s)) that aid in traversal of an organelle membrane. 
     
     
         177 . The method of  claim 176 , wherein the one or more PTD(s) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:268 and 269. 
     
     
         178 . 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 single molecule DNA-targeting RNA, or a DNA molecule comprising a nucleotide sequence that
 (i) encodes the single molecule DNA-targeting RNA and (ii) is 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.   
     
     
         179 . The method of  claim 178 , 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. 
     
     
         180 . The method of  claim 178 , wherein the activator-RNA comprises the 75 nucleotide tracrRNA sequence set forth in SEQ ID NO: 478. 
     
     
         181 . The method of  claim 178 , wherein the Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain. 
     
     
         182 . The method of  claim 178 , wherein:
 the DNA molecule comprising the nucleotide sequence that encodes the single molecule DNA-targeting RNA, and/or   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, and viral vectors.   
     
     
         183 . The method of  claim 178 , wherein the method comprises introducing into the eukaryotic cell two or more single molecule DNA-targeting RNAs, or one or more DNA molecules comprising two or more nucleotide sequences encoding single molecule DNA-targeting RNAs. 
     
     
         184 . The method of  claim 178 , wherein (1) and (2) are administered as part of a pharmaceutically acceptable vehicle, wherein the pharmaceutically acceptable vehicle is a lipid. 
     
     
         185 . A method of cleaving or editing a target DNA molecule or modulating transcription of a gene encoded by the target DNA molecule, the method comprising introducing a Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)—CRISPR associated (Cas) (CRISPR-Cas) system comprising
 (1) a single molecule DNA-targeting RNA, or a nucleic acid comprising a nucleotide sequence encoding said single molecule DNA-targeting RNA, 
 wherein the single molecule 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 duplex of a protein-binding segment, 
 
 wherein i) and ii) are arranged in a 5′ to 3′ orientation and are covalently linked to one another with intervening nucleotides; 
 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 molecule, and 
 (2) the Cas9 protein, or a nucleic acid comprising a nucleotide sequence encoding the Cas9 protein, into a eukaryotic cell containing the target DNA molecule having the target sequence.