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US 9,970,030 B2Granted
Methods for increasing CAS9-mediated engineering efficiency
AssigneeCaribou Biosciences, Inc.
InventorsPeter Sean Cameron, Rachel E. Haurwitz, Andrew P. May, Christopher H. Nye, Megan van Overbeek
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Abstract
Methods for use with Type II CRISPR-Cas9 systems for increasing Cas9-mediated genome engineering efficiency are disclosed. The methods can be used to decrease the number of off-target nucleic acid double-stranded breaks and/or to enhance homology-directed repair of a cleaved target nucleic acid.
Claims19
A method for reducing binding and/or cleavage of an off-target nucleic acid by a complex comprising a catalytically active Cas9 protein and a guide polynucleotide, said method comprising:
- contacting a first complex with a selected target nucleic acid, wherein said first complex comprises:
- (i) a catalytically active Cas9 protein and (ii) a first guide polynucleotide that comprises a spacer adapted to bind to said selected target nucleic acid;
- and
- contacting a second complex with an off-target nucleic acid, wherein said second complex comprises (i) a catalytically inactive Cas9 protein (dCas9 protein) that does not cleave the off-target nucleic acid and (ii) a second guide polynucleotide that comprises a spacer adapted to bind to said off-target nucleic acid,
- thereby reducing binding and/or cleavage by said first complex of said off-target nucleic acid.
2 more elements
Dependencies are inferred from the claim text.