23992846

PMC3856256

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.

CDC Public Access

1DP1-MH100706/DP/NCCDPHP CDC HHS/United States
1R01-DK097768/DK/NIDDK NIH HHS/United States
DP1 MH100706/MH/NIMH NIH HHS/United States
GM68804/GM/NIGMS NIH HHS/United States
R01 DK097768/DK/NIDDK NIH HHS/United States
T32 GM007753/GM/NIGMS NIH HHS/United States
T32GM007753/GM/NIGMS NIH HHS/United States
U01DK089565/DK/NIDDK NIH HHS/United States
Howard Hughes Medical Institute/United States