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Cell. 2013 Sep 12;154(6):1380-9. doi: 10.1016/j.cell.2013.08.021. Epub 2013 Aug 29.

Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.

Author information

1
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.

Erratum in

  • Cell. 2013 Oct 10;155(2):479-80.

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.

PMID:
23992846
PMCID:
PMC3856256
DOI:
10.1016/j.cell.2013.08.021
[Indexed for MEDLINE]
Free PMC Article
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