title: Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.
abstract: Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence, which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Here, we describe an approach that combines a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs is required for double-stranded breaks and extends the number of specifically recognized bases for target cleavage. We demonstrate that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide variety of genome editing applications that require high specificity.
authors.affiliation: Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
authors.forename: F Ann
authors.initials: FA
authors.lastname: Ran
authors.forename: Patrick D
authors.initials: PD
authors.lastname: Hsu
authors.forename: Chie-Yu
authors.initials: CY
authors.lastname: Lin
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Gootenberg
authors.forename: Silvana
authors.initials: S
authors.lastname: Konermann
authors.forename: Alexandro E
authors.initials: AE
authors.lastname: Trevino
authors.forename: David A
authors.initials: DA
authors.lastname: Scott
authors.forename: Azusa
authors.initials: A
authors.lastname: Inoue
authors.forename: Shogo
authors.initials: S
authors.lastname: Matoba
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.forename: Feng
authors.initials: F
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001483:Base Sequence; D053903:DNA Breaks, Double-Stranded; D018390:Gene Targeting / Q000379:methods*; D016678:Genome; D051379:Mice; D008969:Molecular Sequence Data; D013297:Streptococcus pyogenes / Q000201:enzymology / Q000235:genetics; D015053:Zygote / Q000378:metabolism; D058727:RNA, Small Untranslated
chemical_list: D058727:RNA, Small Untranslated