Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells
Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells
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DOI：
10.1038/nbt.3198
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发表时间：
2015-05-01
影响因子：
46.9
通讯作者：
Kuehn, Ralf
中科院分区：
文献类型：
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作者：
Van Trung Chu;Weber, Timm;Kuehn, Ralf
与非同源末端连接(NHEJ)途径的高效率相比，同源定向修复(HDR)的效率相对较低，限制了CRISPR-Cas9等基因组编辑工具插入精确的遗传修饰。为了提高HDR，从而能够插入精确的遗传修饰，我们通过基因沉默、连接酶IV抑制剂SCR7或腺病毒4E1B55K和E4orf6蛋白的共表达以及其他报告系统来抑制NHEJ关键分子KU70、KU80或DNA连接酶IV。抑制KU70和DNA连接酶IV可使HDR的效率提高4-5倍。当与Cas9系统共表达时，E1B55K和E4orf6将HDR的效率提高了8倍，并基本上取消了人和小鼠细胞系中NHEJ的活性。我们的发现为提高哺乳动物细胞中精确基因修饰的频率提供了有用的工具。
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.