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.
Claims68
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:
- (a) 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
- (b) 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.
A method for cleaving a single strand of a target nucleic acid comprising contacting first and second complexes at spaced-apart locations on the same strand of a nucleic acid molecule, wherein:
- (a) said first complex comprises (i) a first Cas9 protein with a mutation in an endonuclease domain thereof to render the Cas9 protein a nickase;
- and (ii) a first guide polynucleotide that comprises a spacer adapted to bind to a first target nucleic acid;
- and
- (b) said second complex comprises (i) a second Cas9 protein with a mutation in an endonuclease domain thereof, to render the Cas9 protein a nickase;
- and (ii) a second guide polynucleotide that comprises a spacer adapted to bind to a second target nucleic;
- wherein said first and second Cas9 proteins cleave a single strand of said nucleic acid molecule at the spaced-apart locations on the same strand, to render a single-stranded break.
A method for directing homology-directed repair (HDR) in a target nucleic acid comprising:
- (a) contacting a first complex with a first 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 first target nucleic acid, wherein said first complex cleaves the first target nucleic acid;
- and
- (b) contacting a second complex with a second target nucleic acid, wherein said second complex comprises:
- (i) a first catalytically inactive Cas9 protein (dCas9 protein) that comprises at least one mutation in one or more endonuclease domains to render the dCas9 protein catalytically inactive such that the dCas9 protein does not cleave the second target nucleic acid, and (ii) a second guide polynucleotide that comprises a spacer adapted to bind to said second target nucleic acid, wherein the second complex comprises one end of a polynucleotide donor associated therewith and configured in proximity to the cleaved first target nucleic acid;
- wherein at least a portion of the polynucleotide donor is inserted into the first target nucleic acid via HDR.
Dependencies are inferred from the claim text.