Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair.
Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair.
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DOI：
10.1093/nar/gkw064
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发表时间：
2016-05-19
影响因子：
14.9
通讯作者：
Feng B
中科院分区：
文献类型：
--
作者：
He X;Tan C;Wang F;Wang Y;Zhou R;Cui D;You W;Zhao H;Ren J;Feng B
CRISPR/Cas9诱导的位点特异性DNA双链断裂（DSB）可以通过同源定向修复（HDR）或非同源末端连接（NHEJ）途径修复。已经进行了广泛的努力来将外源DNA敲入人类细胞中的选定基因组基因座;然而，其集中于基于HDR的策略并且被证明是低效的。在这里，我们报告了NHEJ途径介导CRISPR/Cas9诱导的DNA DSB后基因组和质粒的有效重新连接，并促进各种人类细胞类型中的高效DNA整合。利用这种同源非依赖性敲入策略，将4.6 kb无启动子的ires-eGFP片段整合到GAPDH基因座中，在体细胞LO 2细胞中产生了高达20%的GFP+细胞，在人胚胎干细胞（ESC）中产生了1.70%的GFP+细胞。定量比较进一步证明，在所检查的所有人类细胞类型中，基于NHEJ的敲入比HDR介导的基因靶向更有效。这些数据支持CRISPR/Cas9诱导的NHEJ为人类ESC和体细胞中的有效基因组编辑提供了有价值的新途径。
CRISPR/Cas9-induced site-specific DNA double-strand breaks (DSBs) can be repaired by homology-directed repair (HDR) or non-homologous end joining (NHEJ) pathways. Extensive efforts have been made to knock-in exogenous DNA to a selected genomic locus in human cells; which, however, has focused on HDR-based strategies and was proven inefficient. Here, we report that NHEJ pathway mediates efficient rejoining of genome and plasmids following CRISPR/Cas9-induced DNA DSBs, and promotes high-efficiency DNA integration in various human cell types. With this homology-independent knock-in strategy, integration of a 4.6 kb promoterless ires-eGFP fragment into the GAPDH locus yielded up to 20% GFP+ cells in somatic LO2 cells, and 1.70% GFP+ cells in human embryonic stem cells (ESCs). Quantitative comparison further demonstrated that the NHEJ-based knock-in is more efficient than HDR-mediated gene targeting in all human cell types examined. These data support that CRISPR/Cas9-induced NHEJ provides a valuable new path for efficient genome editing in human ESCs and somatic cells.