Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining.
Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining.
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DOI：
10.1038/nbt.3190
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发表时间：
2015-05
影响因子：
46.9
通讯作者：
Ploegh HL
中科院分区：
文献类型：
--
作者：
Maruyama T;Dougan SK;Truttmann MC;Bilate AM;Ingram JR;Ploegh HL
将定向双链断裂(DSB)引入DNA的方法通过提高外部供应的DNA片段通过同源重组进入基因组的速度，从而实现精确的基因组编辑。这些方法的效率受到非同源末端连接(NHEJ)的限制，NHEJ是与同源定向修复(HDR)竞争的另一种DNA修复途径。为了以牺牲NHEJ为代价促进HDR，我们使用Scr7抑制剂针对NHEJ途径中的关键酶DNA连接酶IV。Scr7治疗将在哺乳动物细胞系和小鼠中使用Cas9进行HDR介导的基因组编辑的效率提高19倍。这种方法应该适用于其他可定制的内切酶，如锌指核酸酶和转录激活物样效应核酸酶，以及具有足够保守的NHEJ和HDR机制的非哺乳动物细胞。
Methods to introduce targeted double-strand breaks (DSBs) into DNA enable precise genome editing by increasing the rate at which externally supplied DNA fragments are incorporated into the genome through homologous recombination. The efficiency of these methods is limited by non-homologous end joining (NHEJ), an alternative DNA repair pathway that competes with homology-directed repair (HDR). To promote HDR at the expense of NHEJ, we targeted DNA ligase IV, a key enzyme in the NHEJ pathway, using the inhibitor Scr7. Scr7 treatment increased the efficiency of HDR-mediated genome editing using Cas9 in mammalian cell lines and in mice for all four genes examined up to 19-fold. This approach should be applicable to other customizable endonucleases, such as zinc finger nucleases and transcription activator like effector nucleases, and to non-mammalian cells with sufficiently conserved mechanisms of NHEJ and HDR.