RNA-directed DNA cleavage by the Cas9-crRNA complex

A composition comprising:

• a first plasmid for preparing a recombinant Cas9-crRNA complex, the first plasmid comprising:
• a polynucleotide sequence encoding a tracrRNA;
• and
• a polynucleotide sequence consisting essentially of one spacer flanked on either side by a repeat, wherein the sequence encodes an engineered crRNA
• reprogrammed to guide the recombinant Cas9-crRNA complex to a region comprising a site in a target DNA molecule;
• a second plasmid comprising a polynucleotide sequence encoding a Cas9 protein, wherein the Cas9 protein comprises a fusion polypeptide comprising at least one additional amino acid sequence;
• and
• the target DNA molecule, wherein the region of the target DNA molecule comprises at least 20 nucleotides complementary to the engineered crRNA;
• wherein the first plasmid does not encode a Cas9 protein wherein the recombinant Cas9-crRNA complex is assembled in vitro.

A composition comprising:

• a first plasmid for preparing a recombinant Cas9-crRNA complex, the first plasmid comprising:
• a polynucleotide sequence encoding a tracrRNA;
• and
• a polynucleotide sequence consisting essentially of one spacer flanked on either side by a repeat, wherein the sequence encodes an engineered crRNA reprogrammed to guide the recombinant Cas9-crRNA complex to a region comprising a site in a target DNA molecule;
• wherein the first plasmid does not encode a Cas9 protein;
• a second plasmid for preparing the recombinant Cas9-crRNA complex, the second plasmid comprising a polynucleotide sequence encoding a Cas9 protein, wherein the Cas9 protein comprises a fusion polypeptide comprising at least one additional amino acid sequence;
• and
• the target DNA molecule, wherein the region of the target DNA molecule comprises at least 20 nucleotides complementary to the engineered crRNA;
• wherein the recombinant Cas9-crRNA complex is assembled in vitro;
• and
• wherein the recombinant Cas9-crRNA complex is capable of cleaving the site of the target DNA molecule.