清脆的
基因组编辑
Ku70型
Ku80型
同源定向修复
Cas9
生物
DNA连接酶
非同源性末端接合
DNA修复
基因靶向
基因
遗传学
计算生物学
细胞生物学
核苷酸切除修复
DNA结合蛋白
转录因子
作者
Van Trung Chu,Timm Weber,Benedikt Wefers,Wolfgang Wurst,Sandrine Sander,Klaus Rajewsky,Ralf Kühn
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.
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