Methods and compositions for RNA-directed target DNA modification and for RNA-directed modulation of transcription

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 single molecule DNA-targeting RNA or a nucleic acid comprising a nucleotide sequence encoding the 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 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 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,
• whereby hybridization of the targeter-RNA to the target sequence is capable of targeting the Cas9 protein to the target DNA molecule.

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.

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 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 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.

A Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system comprising:

• a Cas9 protein;
• and
• 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 a 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 the 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
• wherein the single molecule DNA-targeting RNA comprises one or more sequence modifications compared to a sequence of a corresponding wild type tracrRNA and/or crRNA.