Chang Jiasong, Chen Xiaoxu, Zhang Tong, Wang Ruolin, Wang Aoming, Lan Xinhui, Zhou Yuyu, Ma Sanyuan, Xia Qingyou
State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China; Biological Science Research Center, Southwest University, Chongqing 400716, China.
State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China; Biological Science Research Center, Southwest University, Chongqing 400716, China; Chongqing Engineering and Technology Research Center for Novel Silk Materials, Southwest University, Chongqing 400716, China; Chongqing Key Laboratory of Sericulture, Southwest University, Chongqing 400716, China.
Int J Biol Macromol. 2020 Nov 15;163:711-717. doi: 10.1016/j.ijbiomac.2020.07.039. Epub 2020 Jul 8.
The CRISPR/Cas (clustered regularly interspaced short palindromic repeat technology/CRISPR-associated protein) is a widely used and powerful research tool in biosciences and a promising therapeutic agent for treating genetic diseases. Mutations induced by Cas9 are generally considered stochastic and unpredictable, thus hindering its applications where precise genetic alternations are required. Here, through deep sequencing and analysis of genome editing outcomes of multiple sites in four distinct species, we found that Cas9-induced mutations are coincident in mutation types but are significantly different in indel patterns among species. In human and mouse cells, indels were almost evenly distributed at both ends of the cleavage sites. However, the indels mainly appeared at the upstream of cleavage sites in Bombyx mori, while they predominantly occurred downstream of the cleavage sites in the zebrafish Danio rerio. We also found that within a species, indel patterns are sequence dependent, wherein deletions between two adjacent micro-homology sequences were the most frequently observed mutations in the repair spectrum. These results suggested the species differences in DNA repair processes during Cas9-induced gene editing, and the important role of sequence structure at the target site in predicting the gene editing outcome.
CRISPR/Cas（成簇规律间隔短回文重复序列技术/CRISPR相关蛋白）是生物科学中广泛使用且强大的研究工具，也是治疗遗传疾病的一种有前景的治疗剂。Cas9诱导的突变通常被认为是随机且不可预测的，因此阻碍了其在需要精确基因改变的应用中的使用。在此，通过对四个不同物种多个位点的基因组编辑结果进行深度测序和分析，我们发现Cas9诱导的突变在突变类型上是一致的，但在物种间的插入缺失模式上有显著差异。在人类和小鼠细胞中，插入缺失在切割位点的两端几乎均匀分布。然而，在家蚕中，插入缺失主要出现在切割位点的上游，而在斑马鱼中，它们主要发生在切割位点的下游。我们还发现，在一个物种内，插入缺失模式取决于序列，其中两个相邻微同源序列之间的缺失是修复谱中最常观察到的突变。这些结果表明了Cas9诱导基因编辑过程中DNA修复过程的物种差异，以及靶位点序列结构在预测基因编辑结果中的重要作用。
