Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells

被引：911

作者：

Van Trung Chu ^{[1
]}

Weber, Timm ^{[1
]}

Wefers, Benedikt ^{[2
,3
]}

Wurst, Wolfgang ^{[2
,3
,4
]}

Sander, Sandrine ^{[1
]}

Rajewsky, Klaus ^{[1
]}

Kuehn, Ralf ^{[1
,2
,5
]}

机构：

[1] Max Delbruck Ctr Mol Med, Berlin, Germany

[2] Helmholtz Zentrum Munchen, German Res Ctr Environm Hlth, Neuherberg, Germany

[3] Deutsch Zentrum Neurodegenerat Erkrankungen eV, Munich, Germany

[4] Univ Munich, Munich Cluster Syst Neurol SyNergy, Munich, Germany

[5] Berlin Inst Hlth, Berlin, Germany

来源：

NATURE BIOTECHNOLOGY | 2015年 / 33卷 / 05期

基金：

欧洲研究理事会;

关键词：

DAMAGE RESPONSE; BREAK REPAIR; DNA; RECEPTOR; PROTEIN; KINASE; KU70;

D O I：

10.1038/nbt.3198

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

The insertion of precise genetic modifications by genome editing tools such as CRISPR-Cas9 is limited by the relatively low efficiency of homology-directed repair (HDR) compared with the higher efficiency of the nonhomologous end-joining (NHEJ) pathway. To enhance HDR, enabling the insertion of precise genetic modifications, we suppressed the NHEJ key molecules KU70, KU80 or DNA ligase IV by gene silencing, the ligase IV inhibitor SCR7 or the coexpression of adenovirus 4 E1B55K and E4orf6 proteins in a 'traffic light' and other reporter systems. Suppression of KU70 and DNA ligase IV promotes the efficiency of HDR 4-5-fold. When co-expressed with the Cas9 system, E1B55K and E4orf6 improved the efficiency of HDR up to eightfold and essentially abolished NHEJ activity in both human and mouse cell lines. Our findings provide useful tools to improve the frequency of precise gene modifications in mammalian cells.

引用

页码：543 / U160

页数：8

共 29 条

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