State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, Yunnan 650500, China.
State Key Laboratory of Primate Biomedical Research, Institute of Primate Translational Medicine, Kunming University of Science and Technology, Kunming, Yunnan 650500, China; Yunnan Key Laboratory of Primate Biomedical Research, Kunming, Yunnan 650500, China.
Genomics. 2024 May;116(3):110836. doi: 10.1016/j.ygeno.2024.110836. Epub 2024 Mar 25.
The CRISPR/Cas9 system can induce off-target effects in programmed gene editing, but there have been few reports on cleavage detection and their affection in embryo development. To study these events, sgRNAs with different off-target rates were designed and compared after micro-injected into mouse zygotes, and γH2AX was used for DNA cleavage sites analysis by immunostaining and CUT&Tag. Although the low off-target sgRNA were usually selected for production gene editing animals, γH2AX immunofluorescence indicated that there was a relative DSB peak at 15 h after Cas9 system injection, and the number of γH2AX foci at the peak was significantly higher in the low off-target sgRNA-injected group than in the control group. Further, the result of CUT&Tag sequencing analysis showed more double-strand breaks (DSBs) related sequences were detected in low off-target sgRNA-injected group than control and the distribution of DSB related sequences had no chromosome specificity. Gene Ontology (GO) annotation analysis of the DSB related sequences showed that these sequences were mainly concentrated at genes associated with some important biological processes, molecular functions, and cell components. In a conclusion, there are many sgRNA-sequence-independent DSBs in early mouse embryos when the Cas9 system is used for gene editing and the DSB related sequence could be detected and characterized in the genome. These results and method should also be considered in using or optimizing the Cas9 system.
CRISPR/Cas9 系统可在基因编辑中引起脱靶效应，但关于其在胚胎发育中的检测和影响的报道较少。为了研究这些事件，设计了不同脱靶率的 sgRNA，并在微注射到小鼠受精卵后进行了比较，并通过免疫染色和 CUT&Tag 分析γH2AX 用于 DNA 断裂检测。虽然在生产基因编辑动物时通常会选择低脱靶 sgRNA，但γH2AX 免疫荧光表明 Cas9 系统注射后 15 小时存在相对 DSB 峰，并且低脱靶 sgRNA 注射组的γH2AX 焦点数量明显高于对照组。此外，CUT&Tag 测序分析的结果表明，低脱靶 sgRNA 注射组检测到的与双链断裂（DSB）相关的序列更多，而 DSB 相关序列的分布没有染色体特异性。对 DSB 相关序列的基因本体（GO）注释分析表明，这些序列主要集中在与一些重要生物学过程、分子功能和细胞成分相关的基因上。总之，当 Cas9 系统用于基因编辑时，早期小鼠胚胎中存在许多 sgRNA 序列独立的 DSB，并且可以在基因组中检测和表征与 DSB 相关的序列。在使用或优化 Cas9 系统时，也应该考虑这些结果和方法。
