Ge Weikai, Zhao Xiaozhu, Gou Shixue, Jin Qin, Chen Fangbing, Ouyang Zhen, Lai Chengdan, Cui Tao, Mai Baoyi, Lu Sijia, Zhong Kexin, Liang Yanhui, Chen Tao, Wu Han, Li Nan, Ye Yinghua, Lai Liangxue, Wang Kepin
China-New Zealand Joint Laboratory on Biomedicine and Health, CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Centre for Regenerative Medicine and Health, Hong Kong Institute of Science and Innovation, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou 510530, China.
Mol Ther Nucleic Acids. 2023 Sep 20;34:102035. doi: 10.1016/j.omtn.2023.102035. eCollection 2023 Dec 12.
Cas9 protein without sgRNAs can induce genomic damage at the cellular level . However, whether the detrimental effects occur in embryos after Cas9 treatment remains unknown. Here, using pig embryos as subjects, we observed that Cas9 protein transcribed from injected Cas9 mRNA can persist until at least the blastocyst stage. Cas9 protein alone can induce genome damage in preimplantation embryos, represented by the increased number of phosphorylated histone H2AX foci on the chromatin fiber, which led to apoptosis and decreased cell number of blastocysts. In addition, single-blastocyst RNA sequencing confirmed that Cas9 protein without sgRNAs can cause changes in the blastocyst transcriptome, depressing embryo development signal pathways, such as cell cycle, metabolism, and cellular communication-related signal pathways, while activating apoptosis and necroptosis signal pathways, which together resulted in impaired preimplantation embryonic development. These results indicated that attention should be given to the detrimental effects caused by the Cas9 protein when using CRISPR-Cas9 for germline genome editing, especially for the targeted correction of human pathological mutations using germline gene therapy.
没有sgRNA的Cas9蛋白可在细胞水平上诱导基因组损伤。然而，Cas9处理后胚胎中是否会出现有害影响仍不清楚。在这里，我们以猪胚胎为研究对象，观察到注射的Cas9 mRNA转录的Cas9蛋白至少可持续到囊胚阶段。单独的Cas9蛋白可诱导植入前胚胎的基因组损伤，表现为染色质纤维上磷酸化组蛋白H2AX病灶数量增加，这导致了细胞凋亡和囊胚细胞数量减少。此外，单细胞囊胚RNA测序证实，没有sgRNA的Cas9蛋白可导致囊胚转录组发生变化，抑制胚胎发育信号通路，如细胞周期、代谢和细胞通讯相关信号通路，同时激活细胞凋亡和坏死性凋亡信号通路，这些共同导致植入前胚胎发育受损。这些结果表明，在使用CRISPR-Cas9进行种系基因组编辑时，尤其是在使用种系基因疗法对人类病理突变进行靶向校正时，应注意Cas9蛋白所造成的有害影响。
