alexa Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.
Genetics & Molecular Biology

Genetics & Molecular Biology

Gene Technology

Author(s): Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S,

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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. Copyright © 2013 Elsevier Inc. All rights reserved.
This article was published in Cell and referenced in Gene Technology

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