DNA Repair Profiling Reveals Nonrandom Outcomes at Cas9-Mediated Breaks
DNA Repair Profiling Reveals Nonrandom Outcomes at Cas9-Mediated Breaks
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DOI：
10.1016/j.molcel.2016.06.037
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发表时间：
2016-08-18
期刊：
影响因子：
16
通讯作者：
May, Andrew P.
中科院分区：
文献类型：
--
作者：
van Overbeek, Megan;Capurso, Daniel;May, Andrew P.
由RNA引导的DNA内切核酸酶Cas9产生的位点特异性DNA双链断裂（DSB）的修复结果决定了基因功能如何改变。尽管CRISPR-Cas9技术被广泛采用来诱导DSB用于基因组工程，但由此产生的修复产物尚未被深入研究。在这里，人类基因组中223个位点的DNA修复谱证明，在每个位点处Cas9切割后的DNA修复模式是非随机的，并且在实验重复、细胞系和试剂递送方法中是一致的。此外，修复结果由原型间隔区序列而不是基因组背景决定，表明细胞系中的DNA修复谱可用于预测原代细胞中的修复结果。DNA-PK的化学抑制使得能够将DNA修复谱分解为来自c-NHEJ和MMEJ的贡献。最后，这项工作阐明了一种策略，使用“易错”的DNA修复机制，以产生精确的编辑。
The repair outcomes at site-specific DNA double-strand breaks (DSBs) generated by the RNA-guided DNA endonuclease Cas9 determine how gene function is altered. Despite the widespread adoption of CRISPR-Cas9 technology to induce DSBs for genome engineering, the resulting repair products have not been examined in depth. Here, the DNA repair profiles of 223 sites in the human genome demonstrate that the pattern of DNA repair following Cas9 cutting at each site is nonrandom and consistent across experimental replicates, cell lines, and reagent delivery methods. Furthermore, the repair outcomes are determined by the protospacer sequence rather than genomic context, indicating that DNA repair profiling in cell lines can be used to anticipate repair outcomes in primary cells. Chemical inhibition of DNA-PK enabled dissection of the DNA repair profiles into contributions from c-NHEJ and MMEJ. Finally, this work elucidates a strategy for using "error-prone" DNA-repair machinery to generate precise edits.