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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
AuthorsRan, FA, Hsu, PD, Lin, CY, Gootenberg, JS, Konermann, S, Trevino, AE, Scott, DA, Inoue, A, Matoba, S, Zhang, Y, Zhang, F
Date Published2013/09/12

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.