Application of the clustered regularly interspaced short palindromic repeats associated 9 (CRISPR-Cas9) technology has revolutionized biology by greatly enhancing the ability to introduce mutations into DNA for research and prospective therapeutic purposes. However, the understanding of Cas9 editing outcomes is still limited. Previously, it was considered that Cas9 introduces stochastic insertions or deletions (indels) at the target site. In the current study, we performed in vivo multiplex editing, deep sequencing, and comprehensive analysis of its editing outcomes in Bombyx mori (B. mori). A total of 31161 editing events from 9 single-guide RNA (sgRNA) sites in 16 individuals were generated and analyzed, and we found that Cas9 introduces mutations with some regularity rather than via stochastic indels. The editing efficiency varies with sgRNA sequences, individuals, and orientation. Small deletions account for the vast majority of mutated sequences, followed by a small fraction of substitutions and insertions. The most likely mutations are deletions between two microhomologous sequences or single-base deletions at the cleavage site in the absence of microhomologous pairs. Insertions are formed by diverse mechanisms, including direct acquisition of free genomic fragments, duplication of broken ends, replication of adjacent sequences, or random addition of free nucleotides. The above results indicate that the Cas9 editing spectrum is reproducible and predictable. Thus, our findings enable a deeper understanding of Cas9-mediated mutagenesis and better design of genome editing experiments, as well as elucidate the DNA double-strand break repair processes in B. mori.

中文翻译：

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深度测序揭示了家蚕的全面 CRISPR-Cas9 编辑谱


成簇规则间隔短回文重复序列相关 9 (CRISPR-Cas9) 技术的应用极大地增强了将突变引入 DNA 以用于研究和前瞻性治疗目的的能力，从而彻底改变了生物学。然而，对 Cas9 编辑结果的了解仍然有限。此前，人们认为 Cas9 在目标位点引入随机插入或删除 (indel)。在本研究中，我们对家蚕（B. mori）进行了体内多重编辑、深度测序并对其编辑结果进行了综合分析。生成并分析了来自 16 个个体的 9 个单引导 RNA (sgRNA) 位点的总共 31161 个编辑事件，我们发现 Cas9 引入了具有一定规律性的突变，而不是通过随机插入缺失。编辑效率因 sgRNA 序列、个体和方向而异。小缺失占突变序列的绝大多数，其次是一小部分替换和插入。最可能的突变是两个微同源序列之间的缺失或在不存在微同源对的情况下在切割位点处的单碱基缺失。插入通过多种机制形成，包括直接获取游离基因组片段、断端复制、相邻序列复制或随机添加游离核苷酸。上述结果表明Cas9编辑谱是可重复且可预测的。因此，我们的研究结果使人们能够更深入地了解 Cas9 介导的诱变和更好地设计基因组编辑实验，并阐明家蚕中的 DNA 双链断裂修复过程。