Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity
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Zhang_Double nicking.pdf
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Author(s)
Ran, F. Ann
Hsu, Patrick D.
Lin, Chie Yu
Gootenberg, Jonathan S.
Konermann, Silvana M.
Trevino, Alexandro E.
Scott, David A.
Inoue, Azusa
Matoba, Shogo
Zhang, Yi
Date Issued
September 2013
Journal
Cell
Publisher
Cell Press/Elsevier
Citation
Ran, F. Ann, Patrick D. Hsu, Chie-Yu Lin, Jonathan S. Gootenberg, Silvana Konermann, Alexandro E. Trevino, David A. Scott, et al. “Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity.” Cell 154, no. 6 (September 2013): 1380–1389.
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Author's final manuscript
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.
MIT Department
Institute for Medical Engineering and Science
Massachusetts Institute of Technology. Department of Biological Engineering
McGovern Institute for Brain Research at MIT
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Creative Commons Attribution-NonCommercial-NoDerivs License
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DOI of Published Version
https://doi.org/10.1016/j.cell.2013.08.021
