RNA-DIRECTED DNA CLEAVAGE BY THE Cas9-crRNA COMPLEX

Patent metadata

Field	Value
Publication number	US-2015291961-A1
Application number	US-201514743764-A
Country	US
Kind code	A1
Filing date	Jun 18, 2015
Priority date	Mar 20, 2012
Publication date	Oct 15, 2015
Grant date	—

Abstract

Isolation or in vitro assembly of the Cas9-crRNA complex of the Streptococcus thermophilus CRISPR3/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 using an RNA-guided DNA endonuclease comprising at least one RNA sequence 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 pint mutation; for assembly of active polypeptide-polyribonucleotides complex in vivo or in vitro; and for re-programming a Cas9-crRNA complex specificity in vitro or using a cassette containing a single repeat-spacer-repeat unit.

First claim

1 .- 41 . (canceled)

42 . An engineered Cas9-crRNA complex comprising a Cas9 protein and at least one crRNA engineered to guide the Cas9-crRNA complex to a target double-stranded DNA molecule having a proto-spacer sequence and a proto-spacer adjacent (PAM) sequence, the Cas9-crRNA complex functioning to cleave the target sequence under suitable conditions.

43 . The complex of claim 42 wherein the suitable conditions are in vitro.

44 . The complex of claim 42 further comprising a tracrRNA.

45 . The complex of claim 42 , wherein the crRNA comprises an approximately 20 nucleotide fragment complementary to a proto-spacer sequence in the target double-stranded DNA molecule.

46 . The Cas9-crRNA complex of claim 42 , wherein the target DNA sequence is a plasmid DNA.

47 . The Cas9-crRNA complex of claim 42 , wherein the target DNA sequence is a human genomic DNA.

48 . The Cas9-crRNA complex of claim 42 , wherein the Cas9 protein comprises a fusion polypeptide comprising at least one additional amino acid sequence.

49 . The Cas9-crRNA complex of claim 48 , wherein the additional amino acid sequence is used for purification of the fusion polypeptide by affinity chromatography.

50 . The Cas9-crRNA complex of claim 42 , wherein the Cas9 protein contains a point mutation in the RuvC active site motif.

51 . The Cas9-crRNA complex of claim 50 , wherein the point mutation is D31A.

52 . The Cas9-crRNA complex of claim 42 , wherein the Cas9 protein contains a point mutation in the HNH active site motif.

53 . The Cas9-crRNA complex of claim 52 , wherein the point mutation is N891A.

Assignees

• Univ Vilnius

Inventors

• Siksnys Virginijus
• Gasiunas Giedrius
• Karvelis Tautvydas

Classifications

• C12N15/902

  using homologous recombination · CPC title

• C12N15/113 Primary

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title

• C12N2310/14

  interfering nucleic acids [NA] · CPC title

• C12N2310/12

  catalytic nucleic acids, e.g. ribozymes · CPC title

• C12Q1/6811

  Selection methods for production or design of target specific oligonucleotides or binding molecules · CPC title