Ligase IV inhibitor SCR7 enhances gene editing directed by CRISPR-Cas9 and ssODN in human cancer cells.
Ligase IV inhibitor SCR7 enhances gene editing directed by CRISPR-Cas9 and ssODN in human cancer cells.
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连接酶 IV 抑制剂 SCR7 增强人类癌细胞中 CRISPR-Cas9 和 ssODN 指导的基因编辑
DOI：
10.1186/s13578-018-0200-z
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发表时间：
2018
影响因子：
7.5
通讯作者：
Zhu YS
中科院分区：
文献类型：
--
作者：
Hu Z;Shi Z;Guo X;Jiang B;Wang G;Luo D;Chen Y;Zhu YS
精确的基因组编辑对于基础研究和转化研究都至关重要。最近开发的CRISPR/Cas9系统可以特异性地切割靶基因的指定位点以产生DNA双链断裂，其触发不准确的非同源末端连接或位点特异性同源重组的细胞DNA修复机制。不幸的是，同源定向修复（HDR）是具有挑战性的，由于其非常低的效率。在本文中，我们专注于在人类癌细胞中使用CRISPR/Cas9、eGFP、DNA连接酶IV抑制剂SCR 7和单链寡脱氧核苷酸（ssODN）的组合来提高HDR的效率。当Cas9、gRNA和eGFP组装到共表达载体中时，与那些未分选的细胞相比，在转染细胞中GFP阳性细胞分选后，破坏率增加一倍以上。使用ssODNs作为模板，与没有SCR 7处理的细胞相比，SCR 7处理使转染细胞中的靶向插入效率增加了三倍。此外，这种组合方法大大提高了GFP沉默突变和β-连环蛋白Ser 45缺失突变细胞中HDR和靶向基因突变校正的效率。本研究的数据表明，共表达载体、ssODN和连接酶IV抑制剂的组合可以显著改善CRISPR/Cas9指导的基因编辑，这将在靶向基因编辑和遗传疾病治疗中具有重要应用。本文的在线版本（10.1186/s13578-018-0200-z）包含补充材料，可供授权用户使用。
Precise genome editing is essential for both basic and translational research. The recently developed CRISPR/Cas9 system can specifically cleave a designated site of target gene to create a DNA double-strand break, which triggers cellular DNA repair mechanism of either inaccurate non-homologous end joining, or site-specific homologous recombination. Unfortunately, homology-directed repair (HDR) is challenging due to its very low efficiency. Herein, we focused on improving the efficiency of HDR using a combination of CRISPR/Cas9, eGFP, DNA ligase IV inhibitor SCR7, and single-stranded oligodeoxynucleotides (ssODN) in human cancer cells. When Cas9, gRNA and eGFP were assembled into a co-expression vector, the disruption rate more than doubled following GFP-positive cell sorting in transfected cells compared to those unsorted cells. Using ssODNs as templates, SCR7 treatment increased targeted insertion efficiency threefold in transfected cells compared to those without SCR7 treatment. Moreover, this combinatorial approach greatly improved the efficiency of HDR and targeted gene mutation correction at both the GFP-silent mutation and the β-catenin Ser45 deletion mutation cells. The data of this study suggests that a combination of co-expression vector, ssODN, and ligase IV inhibitor can markedly improve the CRISPR/Cas9-directed gene editing, which should have significant application in targeted gene editing and genetic disease therapy. The online version of this article (10.1186/s13578-018-0200-z) contains supplementary material, which is available to authorized users.
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影响因子：
46.9
作者：
Cho, Seung Woo;Kim, Sojung;Kim, Jin-Soo
通讯作者：
Kim, Jin-Soo
影响因子：
7
作者：
Maresca M;Lin VG;Guo N;Yang Y
通讯作者：
Yang Y
影响因子：
46.9
作者：
通讯作者：
--
影响因子：
20.1
作者：
Abrahimi P;Chang WG;Kluger MS;Qyang Y;Tellides G;Saltzman WM;Pober JS
通讯作者：
Pober JS
DOI：
10.1126/science.1232033
发表时间：
2013-02-15
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Mali P;Yang L;Esvelt KM;Aach J;Guell M;DiCarlo JE;Norville JE;Church GM
通讯作者：
Church GM