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

Ran, F. Ann; Hsu, Patrick D.; Lin, Chie-Yu; Gootenberg, Jonathan S.; Konermann, Silvana; Trevino, Alexandro; Scott, David A.; Inoue, Azusa; Matoba, Shogo; Zhang, Yi; Zhang, Feng

doi:10.1016/j.cell.2013.08.021

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

Published Date:

Aug 29 2013

Publisher's site:

http://dx.doi.org/10.1016/j.cell.2013.08.021 New Site Icon

Source:

Cell. 2013; 154(6):1380-1389.

[PDF-1.36 MB]

Details:

Alternative Title:

Cell

Personal Author:

Ran, F. Ann ; Hsu, Patrick D. ; Lin, Chie-Yu ; Gootenberg, Jonathan S. ; Konermann, Silvana ; ... More ▼

Description:

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.

Subject:

[+]

Pubmed ID:

23992846

Pubmed Central ID:

PMC3856256

Document Type:

Journal Article

Funding:

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 ; ... More ▼

Collection(s):

CDC Public Access

Main Document Checksum:

[+]

Supporting Files:

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