methods for modifying specific site of target dna molecule, to obtain complex in genetically modified microorganism, to cleave polynucleotide and nucleotide sequence, to cut specific site of polynucleotide and nucleotide sequences, in vitro to generate complexes, to reprogram specificity of cas9-crrna complex and to selectively modify site-specific target DNA molecule in vivo, rna-protein and cas9-crrna complexes, cas9-isolated protein and isolated crrna

摘要

Isolation orin vitroassembly of the Cas9-crRNA complex of theStreptococcus thermophilusCRISPR3/Cas system and use for cleavage of DNA bearing a nucleotide sequence complementary to the crRNA and a proto-spacer adjacent motif. Methods for site-specific modification of a target DNA molecule in vitro or in vivo using an RNA-guided DNA endonuclease comprising RNA sequences and at least one of an RuvC active site motif and an HNH active site motif; for conversion of Cas9 polypeptide into a nickase cleaving one strand of double-stranded DNA by inactivating one of the active sites (RuvC or HNH) in the polypeptide by at least one point mutation; for assembly of active polypeptide-polyribonucleotides complexin vivoorin vitro; and for re-programming a Cas9-crRNA complex specificityin vitroand using a cassette containing a single repeat-spacer-repeat unit.