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 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) a Cas9 protein, or a nucleic acid comprising a nucleotide sequence encoding said Cas9 protein; and   b) 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 RNA duplex of a protein-binding segment, 
 
       wherein the activator-RNA and the targeter-RNA are covalently linked to one another with intervening nucleotides; and 
       wherein the single molecule 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. 
 
     
     
         157 . The eukaryotic cell of  claim 156 , wherein the genome of said eukaryotic cell comprises the target DNA molecule. 
     
     
         158 . The eukaryotic cell of  claim 156 , wherein the eukaryotic cell is a mammalian cell or a human cell. 
     
     
         159 . The eukaryotic cell of  claim 156 , wherein one or both of the nucleic acids of a) and b) are one or more vectors, wherein the nucleotide sequence encoding said Cas9 protein and/or the nucleotide sequence encoding said single molecule DNA-targeting RNA is operably linked to a control element operable in said eukaryotic cell. 
     
     
         160 . The eukaryotic cell of  claim 159 , wherein the one or more vectors are selected from the group consisting of plasmids, cosmids, minicircles, phage, and viral vectors. 
     
     
         161 . The eukaryotic cell of  claim 160 , wherein the viral vector is selected from the group consisting of retroviral, lentiviral, adenoviral, adeno-associated, and herpes simplex virus vectors. 
     
     
         162 . The eukaryotic cell of  claim 159 , wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s)). 
     
     
         163 . The eukaryotic cell of  claim 162 , wherein the one or more PTD(s) aid in traversal of an organelle membrane 
     
     
         164 . The eukaryotic cell of  claim 162 , wherein the one or more PTD(s) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:268 and 269. 
     
     
         165 . The eukaryotic cell of  claim 162 , wherein said Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain and wherein the activator-RNA comprises the 88 nucleotide tracrRNA sequence set forth in SEQ ID NO:433. 
     
     
         166 . The eukaryotic cell 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 eukaryotic cell of  claim 159 , wherein the Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain. 
     
     
         168 . The eukaryotic cell of  claim 156 , comprising two or more single molecule DNA-targeting RNAs, or one or more nucleic acids comprising two or more nucleotide sequences encoding single molecule DNA-targeting RNAs. 
     
     
         169 . The eukaryotic cell of  claim 156 , 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. 
     
     
         170 . The eukaryotic cell of  claim 156 , wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s)). 
     
     
         171 . The eukaryotic cell of  claim 170 , wherein the one or more PTD(s) aid in traversal of an organelle membrane. 
     
     
         172 . The eukaryotic cell of  claim 170 , wherein the one or more PTD(s) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:268 and 269. 
     
     
         173 . The eukaryotic cell of  claim 170 , 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. 
     
     
         174 . The eukaryotic cell of  claim 173 , wherein the one or more PTD(s) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:268 and 269. 
     
     
         175 . The eukaryotic cell of  claim 171 , wherein said Cas9 protein comprises one or more mutations in a RuvC domain or a HNH domain and is capable of cleaving only one strand of DNA. 
     
     
         176 . The eukaryotic cell of  claim 175 , wherein the one or more PTD(s) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:268 and 269. 
     
     
         177 . The eukaryotic cell of  claim 156 , wherein the Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain. 
     
     
         178 . The eukaryotic cell of  claim 156 , 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. 
     
     
         179 . The eukaryotic cell of  claim 156 , wherein the activator-RNA comprises the 88 nucleotide tracrRNA sequence set forth in SEQ ID NO:433. 
     
     
         180 . The eukaryotic cell of  claim 156 , wherein said Cas9 protein is capable of cleaving only one strand of DNA and comprises one or more mutations in a RuvC domain and/or a HNH domain. 
     
     
         181 . The eukaryotic cell of  claim 180 , comprising two or more single molecule DNA-targeting RNAs, or one or more nucleic acids comprising two or more nucleotide sequences encoding single molecule DNA-targeting RNAs. 
     
     
         182 . The eukaryotic cell of  claim 180 , wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s)). 
     
     
         183 . The eukaryotic cell of  claim 182 , wherein the one or more PTD(s) aid in traversal of an organelle membrane. 
     
     
         184 . The eukaryotic cell of  claim 182 , wherein the one or more PTD(s) comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:268 and 269. 
     
     
         185 . A non-human plant or animal comprising the eukaryotic cell of  claim 156 .