Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing.
Harnessing accurate non-homologous end joining for efficient precise deletion in CRISPR/Cas9-mediated genome editing.
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利用准确的非同源末端连接在 CRISPR/Cas9 介导的基因组编辑中实现高效精确删除
DOI：
10.1186/s13059-018-1518-x
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发表时间：
2018-10-19
期刊：
影响因子：
12.3
通讯作者：
Xie AY
中科院分区：
文献类型：
--
作者：
Guo T;Feng YL;Xiao JJ;Liu Q;Sun XN;Xiang JF;Kong N;Liu SC;Chen GQ;Wang Y;Dong MM;Cai Z;Lin H;Cai XJ;Xie AY
背景 CRISPR/Cas9 介导的基因组编辑的许多应用需要 Cas9 诱导的非同源末端连接 (NHEJ)，这被认为容易出错。然而，由于末端可直接连接，Cas9诱导的DNA双链断裂可能会优先被精确的NHEJ修复。结果在配对Cas9-gRNA在71个基因组位点诱导的两个相邻双链断裂的修复中，精确的NHEJ约占NHEJ事件的50%。由于频繁的 + 1 模板化插入，这种配对 Cas9-gRNA 方法低估了准确 NHEJ 的水平，这可以通过预定义的原型间隔子相邻基序 (PAM) 的 Watson/Crick 方向来避免。配对的 Cas9-gRNA 策略还提供了一种灵活的、无报告基因的方法，用于分析细胞和体内的准确 NHEJ 和突变性 NHEJ，并且已在 XRCC4 缺陷细胞和小鼠肝脏中得到验证。由于定义的“3n”-、“3n + 1”-或“3n + 2”-bp长度的精确删除频率很高，因此使用精确的NHEJ来提高基因敲除和靶向框内删除的效率和同质性。与“3n + 1”-bp相比，“3n + 2”-bp可以克服+ 1模板化插入，从而增加框外突变的频率。通过应用配对的 Cas9-gRNA 编辑 MDC1 和关键 53BP1 结构域，我们能够生成预测的、精确的缺失以进行功能分析。最后，Plk3 抑制剂促进 NHEJ，并偏向于准确的 NHEJ，从而提供了一种化学方法来改进需要精确删除的基因组编辑。结论 NHEJ 在修复 Cas9 诱导的 DNA 双链断裂方面本质上是准确的，并且可用于改进需要精确删除指定长度的 CRISPR/Cas9 基因组编辑。
BackgroundMany applications of CRISPR/Cas9-mediated genome editing require Cas9-induced non-homologous end joining (NHEJ), which was thought to be error prone. However, with directly ligatable ends, Cas9-induced DNA double strand breaks may be repaired preferentially by accurate NHEJ.ResultsIn the repair of two adjacent double strand breaks induced by paired Cas9-gRNAs at 71 genome sites, accurate NHEJ accounts for about 50% of NHEJ events. This paired Cas9-gRNA approach underestimates the level of accurate NHEJ due to frequent + 1 templated insertions, which can be avoided by the predefined Watson/Crick orientation of protospacer adjacent motifs (PAMs). The paired Cas9-gRNA strategy also provides a flexible, reporter-less approach for analyzing both accurate and mutagenic NHEJ in cells and in vivo, and it has been validated in cells deficient forXRCC4and in mouse liver. Due to high frequencies of precise deletions of defined “3n”-, “3n + 1”-, or “3n + 2”-bp length, accurate NHEJ is used to improve the efficiency and homogeneity of gene knockouts and targeted in-frame deletions. Compared to “3n + 1”-bp, “3n + 2”-bp can overcome + 1 templated insertions to increase the frequency of out-of-frame mutations. By applying paired Cas9-gRNAs to edit MDC1 and key 53BP1 domains, we are able to generate predicted, precise deletions for functional analysis. Lastly, a Plk3 inhibitor promotes NHEJ with bias towards accurate NHEJ, providing a chemical approach to improve genome editing requiring precise deletions.ConclusionsNHEJ is inherently accurate in repair of Cas9-induced DNA double strand breaks and can be harnessed to improve CRISPR/Cas9 genome editing requiring precise deletion of a defined length.
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影响因子：
4
作者：
Gallagher DN;Haber JE
通讯作者：
Haber JE
影响因子：
64.5
作者：
Hsu PD;Lander ES;Zhang F
通讯作者：
Zhang F
DOI：
10.1083/jcb.201401146
发表时间：
2014-09-29
期刊：
The Journal of cell biology
影响因子：
--
作者：
Barton O;Naumann SC;Diemer-Biehs R;Künzel J;Steinlage M;Conrad S;Makharashvili N;Wang J;Feng L;Lopez BS;Paull TT;Chen J;Jeggo PA;Löbrich M
通讯作者：
Löbrich M
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
7
作者：
Lee, Hyung Joo;Kim, Eunji;Kim, Jin-Soo
通讯作者：
Kim, Jin-Soo