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 Cas9 protein or a nucleic acid comprising a nucleotide sequence encoding the 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 (II) 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. 
 
     
     
         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 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. 
     
     
         160 . The eukaryotic cell of  claim 159 , comprising two or more different targeter-RNAs, or one or more nucleic acids comprising two or more nucleotide sequences encoding different targeter-RNAs. 
     
     
         161 . The eukaryotic cell of  claim 156 , wherein the system comprises a nucleic acid molecule comprising a nucleotide sequence encoding the activator-RNA, and wherein said nucleic acid molecule comprising a nucleotide sequence encoding the activator-RNA does not comprise a nucleotide sequence encoding said Cas9 protein. 
     
     
         162 . The eukaryotic cell of  claim 156 , wherein the system comprises a double stranded DNA molecule comprising nucleotide sequences encoding both the activator-RNA and the targeter-RNA, wherein the nucleotide sequences encoding the targeter-RNA and the activator-RNA are on the same strand. 
     
     
         163 . The eukaryotic cell of  claim 156 , wherein one or more of the nucleic acids of a) and b) 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 nucleotide sequence encoding said single molecule DNA-targeting RNA is operably linked to a control element operable in said eukaryotic cell. 
     
     
         164 . The eukaryotic cell of  claim 156 , wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s)). 
     
     
         165 . The eukaryotic cell of  claim 164 , wherein the one or more PTD(s) aid in traversal of an organelle membrane. 
     
     
         166 . The eukaryotic cell of  claim 164 , 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 156 , 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 different DNA-targeting RNAs, or one or more nucleic acids comprising two or more nucleotide sequences encoding different 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 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. 
     
     
         171 . The eukaryotic cell of  claim 156 , wherein said Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain. 
     
     
         172 . The eukaryotic cell of  claim 156 , wherein the activator-RNA comprises the 88 nucleotide tracrRNA sequence set forth in SEQ ID NO:433. 
     
     
         173 . The eukaryotic cell of  claim 156 , 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. 
     
     
         174 . The eukaryotic cell of  claim 173 , 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. 
     
     
         175 . The eukaryotic cell of  claim 173 , comprising two or more different DNA-targeting RNAs, or one or more nucleic acids comprising two or more nucleotide sequences encoding different DNA-targeting RNAs. 
     
     
         176 . The eukaryotic cell of  claim 173 , 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 and wherein the system comprises two or more different targeter-RNAs or one or more nucleic acids comprising two or more nucleotide sequence encoding different targeter-RNAs. 
     
     
         177 . The eukaryotic cell of  claim 173 , wherein the Cas9 protein comprises one or more Protein Transduction Domain(s) (PTD(s)). 
     
     
         178 . The eukaryotic cell 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 eukaryotic cell of  claim 173 , wherein the Cas9 protein comprises one or more mutations in a RuvC domain and/or a HNH domain. 
     
     
         180 . 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 Cas9 protein, or a nucleic acid comprising a nucleotide sequence encoding said 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. 
 
     
     
         181 . The eukaryotic cell of  claim 180 , wherein one or more of the nucleic acids of a) and b) 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; wherein the nucleotide sequence encoding said Cas9 protein and/or the one or more nucleotide sequence encoding said DNA-targeting RNA are operably linked to control elements operable in said eukaryotic cell. 
     
     
         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 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. 
     
     
         184 . The eukaryotic cell of  claim 183 , 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 .