title: Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA.
abstract: Targeted genomic manipulation by Cas9 can efficiently generate knockout cells and organisms via error-prone nonhomologous end joining (NHEJ), but the efficiency of precise sequence replacement by homology-directed repair (HDR) is substantially lower. Here we investigate the interaction of Cas9 with target DNA and use our findings to improve HDR efficiency. We show that dissociation of Cas9 from double-stranded DNA (dsDNA) substrates is slow (lifetime ∼6 h) but that, before complete dissociation, Cas9 asymmetrically releases the 3' end of the cleaved DNA strand that is not complementary to the sgRNA (nontarget strand). By rationally designing single-stranded DNA (ssDNA) donors of the optimal length complementary to the strand that is released first, we increase the rate of HDR in human cells when using Cas9 or nickase variants to up to 60%. We also demonstrate HDR rates of up to 0.7% using a catalytically inactive Cas9 mutant (dCas9), which binds DNA without cleaving it.
authors.affiliation: Innovative Genomics Initiative, University of California, Berkeley, Berkeley, California, USA.|Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Richardson
authors.affiliation: Innovative Genomics Initiative, University of California, Berkeley, Berkeley, California, USA.|Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Graham J
authors.initials: GJ
authors.lastname: Ray
authors.affiliation: Innovative Genomics Initiative, University of California, Berkeley, Berkeley, California, USA.|Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Mark A
authors.initials: MA
authors.lastname: DeWitt
authors.affiliation: Innovative Genomics Initiative, University of California, Berkeley, Berkeley, California, USA.|Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Gemma L
authors.initials: GL
authors.lastname: Curie
authors.affiliation: Innovative Genomics Initiative, University of California, Berkeley, Berkeley, California, USA.|Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
authors.forename: Jacob E
authors.identifier: http://orcid.org/0000-0002-7798-5309
authors.initials: JE
authors.lastname: Corn
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics*; D002460:Cell Line; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair / Q000235:genetics; D004277:DNA, Single-Stranded / Q000235:genetics; D005818:Genetic Engineering; D016678:Genome; D006801:Humans; D017393:RNA Editing / Q000235:genetics*; D059767:Recombinational DNA Repair / Q000235:genetics*
chemical_list: D004277:DNA, Single-Stranded