Details:

Alternative Title:
Cell
Publisher's site:
http://dx.doi.org/10.1016/j.cell.2013.08.021
Personal Author:
Ran, F. Ann ; Hsu, Patrick D. ; Lin, Chie-Yu ; Gootenberg, Jonathan S. ; Konermann, Silvana ; ... More +
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 Less -
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:
[+]
Animals
Article
Base Sequence
DNA Breaks, Double-Stranded
Gene Targeting
Genome
Mice
Molecular Sequence Data
RNA, Guide
Streptococcus Pyogenes
Zygote

Source:
Cell. 2013; 154(6):1380-1389.
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 +
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 Less -
Collection(s):
CDC Public Access
Main Document Checksum:
[+]
urn:sha256:be6087c53535211cb83f21eafef653119f438d0588178c869db1bea34bffa42f
File Type:

Supporting Files

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