RNA-DIRECTED DNA CLEAVAGE BY THE Cas9-crRNA COMPLEX

• US 20150240261A1
• Filed: 04/10/2015
• Published: 08/27/2015
• Est. Priority Date: 03/20/2012
• Status: Abandoned Application

• Current Assignee: Vilnius University
• Original Assignee: Vilnius University
• Inventors: SIKSNYS, Virginijus, GASINAS, Giedrius, KARVELIS, Tautvydas
• Application Number:
  US14/683,443
• Publication Number:
  US 20150240261A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/113 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 Class Codes: C12N 15/902 using homologous recombination
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2310/12 catalytic nucleic acids, e.g. ribozymes
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/20 involving clustered regularly interspaced short palindromic repeats [CRISPRs]
• CPC Class Codes: C12N 2310/3513 Protein; Peptide
• CPC Class Codes: C12N 2310/531 Stem-loop; Hairpin
• CPC Class Codes: C12N 2320/00 Applications; Uses
• CPC Class Codes: C12N 2800/80 Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2525/121 incorporating both deoxyribonucleotides and ribonucleotides
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides

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 point 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.

Claims

1-78. -78. (canceled)

79. A Cas9-crRNA complex comprising:

a. a Cas9 protein;

b. a tracrRNA transcript; and

c. a CRISPR RNA transcript that is engineered to guide the Cas9-crRNA complex to a target DNA sequence for site specific modification of the target DNA sequence,

wherein (a), (b), and (c) are assembled in vitro.

114. The Cas9-crRNA complex of claim 79, wherein the crRNA comprises an approximately 20 nucleotide fragment complementary to a proto-spacer sequence in the target DNA sequence.

115. The Cas9-crRNA complex of claim 79, wherein the target DNA sequence is a plasmid DNA.

116. The Cas9-crRNA complex of claim 79, wherein the target DNA sequence is a human genomic DNA.

117. The Cas9-crRNA complex of claim 79, wherein the Cas9 protein comprises a fusion polypeptide comprising at least one additional amino acid sequence.

118. The Cas9-crRNA complex of claim 117, wherein the additional amino acid sequence is used for purification of the fusion polypeptide by affinity chromatography.

119. The Cas9-crRNA complex of claim 79, wherein the Cas9 protein contains a point mutation in the RuvC active site motif.

120. The Cas9-crRNA complex of claim 119, wherein the point mutation is D31A.

121. The Cas9-crRNA complex of claim 79, wherein the Cas9 protein contains a point mutation in the HNH active site motif.

122. The Cas9-crRNA complex of claim 121, wherein the point mutation is N891A.

80-113. -113. (canceled)