Measuring nonhomologous end-joining, homologous recombination and alternative end-joining simultaneously at an endogenous locus in any transfectable human cell.
Measuring nonhomologous end-joining, homologous recombination and alternative end-joining simultaneously at an endogenous locus in any transfectable human cell.
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DOI：
10.1093/nar/gkab262
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发表时间：
2021-07-21
影响因子：
14.9
通讯作者：
Higginson DS
中科院分区：
文献类型：
--
作者：
Hussain SS;Majumdar R;Moore GM;Narang H;Buechelmaier ES;Bazil MJ;Ravindran PT;Leeman JE;Li Y;Jalan M;Anderson KS;Farina A;Soni R;Mohibullah N;Hamzic E;Rong-Mullins X;Sifuentes C;Damerla RR;Viale A;Powell SN;Higginson DS
双链断裂（DSB）修复主要通过3种途径进行：非同源末端连接（NHEJ）、选择性末端连接（Alt-EJ）和同源重组（HR）。测量通路使用的典型方法包括集成盒式报告分析或DNA损伤诱导核病灶的可视化。现在已经很清楚，cas9诱导断裂的修复也涉及NHEJ、Alt-EJ和HR通路，这为测量通路的使用提供了一种新的格式。在这里，我们开发了一个简单的基于cas9的系统，其经过验证的修复结果准确地代表了每个途径，然后将其转换为液滴数字PCR （ddPCR）读数，从而避免了下一代测序和生物信息学分析的需要，目标是使基于cas9的系统可用于更多的实验室。该分析系统再现了几个重要的见解。首先，关键的Alt-EJ因子Pol θ的缺失只抵消了总Alt-EJ的50%。其次，单链模板修复（SSTR）需要BRCA1和MRE11活性，但不需要BRCA2活性，这表明基因组编辑中常用的SSTR不是传统的HR。第三，与BRCA2相比，BRCA1促进双端dsb中Alt-EJ的使用。该试验可用于任何系统，允许Cas9传递，重要的是，允许在等基因细胞系对中快速进行基因型-表型关联。
Double strand break (DSB) repair primarily occurs through 3 pathways: non-homologous end-joining (NHEJ), alternative end-joining (Alt-EJ), and homologous recombination (HR). Typical methods to measure pathway usage include integrated cassette reporter assays or visualization of DNA damage induced nuclear foci. It is now well understood that repair of Cas9-induced breaks also involves NHEJ, Alt-EJ, and HR pathways, providing a new format to measure pathway usage. Here, we have developed a simple Cas9-based system with validated repair outcomes that accurately represent each pathway and then converted it to a droplet digital PCR (ddPCR) readout, thus obviating the need for Next Generation Sequencing and bioinformatic analysis with the goal to make Cas9-based system accessible to more laboratories. The assay system has reproduced several important insights. First, absence of the key Alt-EJ factor Pol θ only abrogates ∼50% of total Alt-EJ. Second, single-strand templated repair (SSTR) requires BRCA1 and MRE11 activity, but not BRCA2, establishing that SSTR commonly used in genome editing is not conventional HR. Third, BRCA1 promotes Alt-EJ usage at two-ended DSBs in contrast to BRCA2. This assay can be used in any system, which permits Cas9 delivery and, importantly, allows rapid genotype-to-phenotype correlation in isogenic cell line pairs.
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影响因子：
6.2
作者：
Delaney, G;Jacob, S;Barton, M
通讯作者：
Barton, M
影响因子：
14.9
作者：
Ahrabi S;Sarkar S;Pfister SX;Pirovano G;Higgins GS;Porter AC;Humphrey TC
通讯作者：
Humphrey TC
影响因子：
4
作者：
Chapman, J. Ross;Sossick, Alex J.;Jackson, Stephen P.
通讯作者：
Jackson, Stephen P.
影响因子：
8.8
作者：
Cruz-Garcia, Andres;Lopez-Saavedra, Ana;Huertas, Pablo
通讯作者：
Huertas, Pablo
影响因子：
16.6
作者：
Bothmer A;Phadke T;Barrera LA;Margulies CM;Lee CS;Buquicchio F;Moss S;Abdulkerim HS;Selleck W;Jayaram H;Myer VE;Cotta-Ramusino C
通讯作者：
Cotta-Ramusino C