Optimized CRISPR-Cas double nickase systems, methods and compositions for sequence manipulation
AssigneesThe Broad Institute, Inc., Massachusetts Institute of Technology, President and Fellows of Harvard College
InventorsFeng Zhang, Patrick Hsu, Chie-Yu Lin, Fei Ran
Life of this patent
Estimating the term…
Abstract
The invention provides for delivery, engineering and optimization of systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are vectors and vector systems, some of which encode one or more components of a CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing CRISPR complex formation in prokaryotic and eukaryotic cells to ensure enhanced specificity for target recognition and avoidance of toxicity.
Claims27
A method of modifying a genomic locus of interest in a eukaryotic cell, comprising introducing into the eukaryotic cell an engineered composition comprising or encoding:
- (a) a Cas9, wherein the Cas9 comprises one or more mutations in a RuvC or HNH domain and is a nickase;
- (b) a first CRISPR-Cas system RNA comprising a first guide sequence capable of hybridizing to a first target sequence at the genomic locus of interest, a first tracr-mate sequence, and a first tracr sequence capable of hybridizing to the first tracr-mate sequence;
- and
- (c) a second CRISPR-Cas system RNA comprising a second guide sequence capable of hybridizing to a second target sequence at the genomic locus of interest, a second tracr-mate sequence, and a second tracr sequence capable of hybridizing to the second tracr-mate sequence;
- wherein the first and the second guide sequences direct sequence-specific binding of a first and a second CRISPR complexes to the first and second target sequences, respectively, wherein the first CRISPR complex comprises the Cas9 complexed with the first CRISPR-Cas system RNA, wherein the second CRISPR complex comprises the Cas9 complexed with the second CRISPR-Cas system RNA, wherein the first CRISPR complex cleaves one DNA strand of the genomic locus of interest to produce a first nick, wherein the second CRISPR complex cleaves the opposite DNA strand of the genomic locus of interest to produce a second nick, wherein the first nick is at least 26 nucleotides 5′ of the second nick.
Dependencies are inferred from the claim text.