A scalable CRISPR/Cas9-based fluorescent reporter assay to study DNA double-strand break repair choice
A scalable CRISPR/Cas9-based fluorescent reporter assay to study DNA double-strand break repair choice
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DOI：
10.1038/s41467-020-17962-3
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发表时间：
2020-08-14
影响因子：
16.6
通讯作者：
Mardin, Balca R.
中科院分区：
文献类型：
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作者：
Roidos, Paris;Sungalee, Stephanie;Mardin, Balca R.
双链断裂（DSB）是最具毒性的DNA损伤类型。细胞使用末端保护或末端切除偶联机制修复这些损伤。为了研究DSB修复的选择，我们提出了颜色分析跟踪修复（CAT-R），以同时量化DSB修复通过末端保护和末端切除途径。CAT-R使用串联荧光报告基因中的CRISPR/Cas9引入DSB，其修复将小插入/缺失与大缺失区分开来。我们展示了CAT-R在化学和遗传筛选中的应用。首先，我们评估了目前正在临床试验中的21种针对DNA损伤反应的化合物。其次，我们研究了417个因素参与DNA损伤反应如何影响末端保护和末端切除之间的选择。最后，我们发现削弱核苷酸切除修复有利于无错误修复，为改善基于CRISPR/Cas9的敲入提供了另一种方法。CAT-R是一种高通量，多功能的检测方法，用于评估DSB修复选择，这有助于DNA修复和药物效率测试的全面研究。细胞采用不同的修复途径来修复DNA双链断裂。在这里，作者开发了一种依赖于CRISPR/Cas9的方法来研究DNA修复的选择，称为颜色分析跟踪修复（CAT-R），它通过末端保护和末端切除途径同时测量DSB修复的结果。
Double-strand breaks (DSBs) are the most toxic type of DNA lesions. Cells repair these lesions using either end protection- or end resection-coupled mechanisms. To study DSB repair choice, we present the Color Assay Tracing-Repair (CAT-R) to simultaneously quantify DSB repair via end protection and end resection pathways. CAT-R introduces DSBs using CRISPR/Cas9 in a tandem fluorescent reporter, whose repair distinguishes small insertions/deletions from large deletions. We demonstrate CAT-R applications in chemical and genetic screens. First, we evaluate 21 compounds currently in clinical trials which target the DNA damage response. Second, we examine how 417 factors involved in DNA damage response influence the choice between end protection and end resection. Finally, we show that impairing nucleotide excision repair favors error-free repair, providing an alternative way for improving CRISPR/Cas9-based knock-ins. CAT-R is a high-throughput, versatile assay to assess DSB repair choice, which facilitates comprehensive studies of DNA repair and drug efficiency testing. Cells employ different repair pathways to repair DNA double strand breaks. Here, the authors develop a CRISPR/Cas9-dependent method to study choices in DNA repair called the Color Assay Tracing-Repair (CAT-R) which simultaneously measure outcomes of DSB repair via end-protection and end-resection pathways.