CRISPR–Cas9 generates double-stranded DNA breaks (DSBs) to activate cellular DNA repair pathways for genome editing. The repair of DSBs leads to small insertions or deletions (indels) and other complex byproducts, including large deletions and chromosomal translocations. Indels are well understood to disrupt target genes, while the other deleterious byproducts remain elusive. We developed a new in silico analysis pipeline for the previously described primer-extension-mediated sequencing assay to comprehensively characterize CRISPR–Cas9-induced DSB repair outcomes in human or mouse cells. We identified tremendous deleterious DSB repair byproducts of CRISPR–Cas9 editing, including large deletions, vector integrations, and chromosomal translocations. We further elucidated the important roles of microhomology, chromosomal interaction, recurrent DSBs, and DSB repair pathways in the generation of these byproducts. Our findings provide an extra dimension for genome editing safety besides off-targets. And caution should be exercised to avoid not only off-target damages but also deleterious DSB repair byproducts during genome editing.

中文翻译：

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全球检测 CRISPR–Cas9 诱导的 DNA 修复结果

CRISPR–Cas9 产生双链 DNA 断裂 (DSB) 以激活用于基因组编辑的细胞 DNA 修复途径。DSB 的修复会导致小的插入或缺失（indels）和其他复杂的副产品，包括大的缺失和染色体易位。众所周知，插入缺失会破坏靶基因，而其他有害的副产物仍然难以捉摸。我们为先前描述的引物延伸介导的测序测定开发了一种新的计算机分析流程，以全面表征人类或小鼠细胞中 CRISPR-Cas9 诱导的 DSB 修复结果。我们发现了 CRISPR–Cas9 编辑的巨大有害 DSB 修复副产物，包括大缺失、载体整合和染色体易位。我们进一步阐明了微同源性、染色体相互作用、复发性 DSB、和 DSB 修复途径在这些副产品的产生。我们的研究结果为基因组编辑安全提供了一个额外的维度，除了脱靶。在基因组编辑过程中，不仅要避免脱靶损伤，还要避免有害的 DSB 修复副产物。