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

A method of targeting and binding a nucleic acid 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 proteins
• wherein the DNA-targeting RNA forms a complex with the Cas9 protein,
• whereby the DNA-targeting RNA targets the target sequence, and the Cas9 protein binds the target DNA molecule;
• and
• wherein said contacting occurs in a eukaryotic cell.

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 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,
• 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, 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.

A method of cleaving or editing a target DNA molecule, 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 the DNA-targeting RNA forms a complex with the Cas9 protein, whereby said target DNA molecule is cleaved or edited,
• wherein said contacting occurs in a eukaryotic cell.