Zhang Yue, Wang Jiaqi, Ruan Yan, Yang Yi, Cheng Yuda, Wang Fengsheng, Zhang Chen, Xu Yixiao, Liu Lianlian, Yu Meng, Ren Bangqi, Wang Jiangjun, Zhao Binyu, Yang Ran, Xiong Jiaxiang, Wang Jiali, Zhang Junlei, Jian Rui, Liu Yong, Tian Yanping
Southwest Hospital/Southwest Eye Hospital, Third Military Medical University (Army Medical University), Chongqing, China.
Laboratory of Stem Cell and Developmental Biology, Department of Histology and Embryology, Army Medical University, Chongqing, China.
Stem Cells Dev. 2022 Mar;31(5-6):132-142. doi: 10.1089/scd.2021.0309. Epub 2022 Feb 14.
The mechanisms underlying self-renewal of embryonic stem cells (ESCs) hold great value in the clinical translation of stem cell biology and regenerative medicine research. To study the mechanisms in ESC self-renewal, screening and identification of key genes maintaining ESC self-renewal were performed by a genome-wide CRISPR-Cas9 knockout virus library. The mouse ESC R1 were infected with CRISPR-Cas9 knockout virus library and cultured for 14 days. The variation of single guide RNA (sgRNA) ratio was analyzed by high-throughput sequencing, followed by bioinformatics analysis to profile the altered genes. Our results showed 1375 genes with increased sgRNA ratio were found to be mainly involved in signal transduction, cell differentiation, and cell apoptosis; 2929 genes with decreased sgRNA ratio were mainly involved in cell cycle regulation, RNA splicing, and biological metabolic processes. We further confirmed our screen specificity by identifying , and as novel positive regulators in mESC self-renewal. Meanwhile, further analysis showed the relevance between expression and tumor. In conclusion, our study screened key genes maintaining ESC self-renewal and successfully identified , and as novel positive regulators in mESC self-renewal, which provided theoretical basis and research clues for a better understanding of ESC self-renewal regulation.
胚胎干细胞（ESC）自我更新的机制在干细胞生物学和再生医学研究的临床转化中具有重要价值。为了研究ESC自我更新的机制，通过全基因组CRISPR-Cas9敲除病毒文库对维持ESC自我更新的关键基因进行筛选和鉴定。将小鼠ESC R1用CRISPR-Cas9敲除病毒文库感染并培养14天。通过高通量测序分析单导向RNA（sgRNA）比例的变化，随后进行生物信息学分析以描绘改变的基因。我们的结果显示，sgRNA比例增加的1375个基因主要参与信号转导、细胞分化和细胞凋亡；sgRNA比例降低的2929个基因主要参与细胞周期调控、RNA剪接和生物代谢过程。我们通过鉴定 、 和 作为mESC自我更新的新型正调控因子进一步证实了我们筛选的特异性。同时，进一步分析显示 表达与肿瘤之间的相关性。总之，我们的研究筛选了维持ESC自我更新的关键基因，并成功鉴定了 、 和 作为mESC自我更新的新型正调控因子，为更好地理解ESC自我更新调控提供了理论基础和研究线索。
