Genome editing using cas9 nickases
AssigneesThe Broad Institute, Inc., Massachusetts Institute of Technology, President and Fellows of Harvard College
InventorsFeng ZHANG, Fei RAN
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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.
Claims20
An engineered CRISPR-Cas system comprising:
- a S. aureus Cas9 protein or a polynucleotide encoding the S. aureus Cas9 protein, wherein the S. aureus Cas9 protein comprises a mutation at N580 and is a nickase,
- a CRISPR-Cas system chimeric RNA or a polynucleotide encoding the chimeric RNA, wherein the chimeric RNA comprises a guide sequence capable of hybridizing to a target sequence adjacent to a protospacer adjacent motif (PAM) in a genomic locus of interest of a eukaryotic cell, a tracr-mate sequence, and a tracr sequence capable of hybridizing with the tracr-mate sequence,
- wherein the chimeric RNA is capable of forming a CRISPR complex with the Cas9 protein and directing sequence-specific binding of the CRISPR complex to the target sequence in the eukaryotic cell.
Dependencies are inferred from the claim text.