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Cell DOI:10.1016/j.cell.2013.08.021

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

Publication TypeJournal Article
Year of Publication2013
AuthorsF Ran, A, Hsu, PD, Lin, C-Y, Gootenberg, JS, Konermann, S, Trevino, AE, Scott, DA, Inoue, A, Matoba, S, Zhang, Y, Zhang, F
Date Published2013 Sep 12
KeywordsAnimals, Base Sequence, DNA Breaks, Double-Stranded, Gene Targeting, Genome, Mice, Molecular Sequence Data, RNA, Guide, Streptococcus pyogenes, Zygote

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.


Alternate JournalCell
PubMed ID23992846
PubMed Central IDPMC3856256
Grant List1R01-DK097768 / DK / NIDDK NIH HHS / United States
T32 GM007753 / GM / NIGMS NIH HHS / United States
DP1 MH100706 / MH / NIMH NIH HHS / United States
/ / Howard Hughes Medical Institute / United States
R01 DK097768 / DK / NIDDK NIH HHS / United States
U01DK089565 / DK / NIDDK NIH HHS / United States
GM68804 / GM / NIGMS NIH HHS / United States
T32GM007753 / GM / NIGMS NIH HHS / United States
1DP1-MH100706 / DP / NCCDPHP CDC HHS / United States