Abstract：
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 Puf60, U2af2, Wdr75, and Usp16 as novel positive regulators in mESC self-renewal. Meanwhile, further analysis showed the relevance between Puf60 expression and tumor. In conclusion, our study screened key genes maintaining ESC self-renewal and successfully identified Puf60, U2af2, Wdr75, and Usp16 as novel positive regulators in mESC self-renewal, which provided theoretical basis and research clues for a better understanding of ESC self-renewal regulation.
摘要：
胚胎干细胞（ESCs）的自我更新机制在干细胞生物学和再生医学研究的临床应用中具有重要价值。为了研究ESC自我更新的机制，通过全基因组CRISPR-Cas9敲除病毒文库筛选和鉴定维持ESC自我更新的关键基因。用CRISPR-Cas9敲除病毒文库感染小鼠ESCR1并培养14天。通过高通量测序分析单向导RNA(sgRNA)比例的变异,然后进行生物信息学分析以描述改变的基因。我们的结果表明，发现1375个sgRNA比例增加的基因主要参与信号转导，细胞分化，和细胞凋亡;2929个sgRNA比例降低的基因主要参与细胞周期调控,RNA剪接,和生物代谢过程。我们通过将Puf60，U2af2，Wdr75和Usp16鉴定为mESC自我更新的新型正调节因子，进一步证实了我们的筛选特异性。同时,进一步分析显示Puf60表达与肿瘤的相关性。总之,我们的研究筛选了维持ESC自我更新的关键基因，并成功地将Puf60，U2af2，Wdr75和Usp16鉴定为mESC自我更新的新型正调节因子，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。
