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.
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