Screening and identification of genes for exiting naïve pluripotency in embryonic stem cells using the CRISPR-Cas9 knockout system

To systematically identify the key genes regulating the exit from naïve pluripotency in embryonic stem cells（ESCs）in order to provide novel targets and theoretical insights into the mechanisms for pluripotency transition and early cell fate determination.

Nanog-green fluorescent protein（Nanog-GFP）reporter-labeled ESCs were infected with a genome-wide Brie knockout library, and further cultured under leukemia inhibitory factor/serum（LIF/S）conditions for 14 d. Flow cytometry was used to sort Nanog-GFP⁺（naïve-state）and Nanog-GFP^-（primed state）cell populations, followed by genomic DNA extraction and high-throughput sequencing. Model-based Analysis of Genome-wide CRISPR/Cas9 Knockout（MAGeCK）was applied to identify differential genes between GFP^-/Input, GFP⁺/Input, and GFP⁺/GFP^- groups. Metascape and Gene Set Enrichment Analysis（GSEA）were conducted for functional enrichment analysis. Then the obtained candidate genes were employed to construct knockout models, and their roles were assessed through cell morphology observation, Nanog-positive rate detection, colony formation assays, and pluripotency gene expression analysis.

The GFP⁺/Input screening revealed 2921 negatively regulated genes（mainly enriched in basic life processes, such as RNA metabolism and cell cycle）and 1393 positively regulated genes（enriched in the processes of nervous system development, carbohydrate metabolism, and vascular system development）. In the GFP^-/Input screening, 2765 negatively regulated genes（enriched in RNA metabolism, cell cycle, and other fundamental processes）and 1303 positively regulated genes（enriched in neural development, cell survival, and endothelial migration）were identified. The GFP⁺/GFP^- comparison identified 1001 negatively regulated genes [involved in stress response and inhibition of mitogen-activated protein kinase（MAPK）signaling] and 983 positively regulated genes [related to fibroblast growth factor/extracellular signal-regulated kinase（FGF/ERK）signaling pathway and glucose metabolism). These genes, were not only known pluripotency regulators（e. g., Nanog, Nr5a2, Klf2, Klf4）and exit-associated genes（e. g., Gata6, Grb2, Zeb1, Fgfr1）, but also some novel candidates（e. g., Dmrt1, Rxra, Zbtb14 and Tmem41b）. Functional validation showed that transient knockout of Dmrt1, Tmem41b, and Hic2 significantly increased the proportion of Nanog⁺ cells（P<0. 01）, suggesting their role in suppressing ground-state exit. ESCs with stable Dmrt1 knockout exhibited a more naïve-state phenotype, presenting compact, dome-shaped colonies, with increased ratio of undifferentiated colonies（P<0. 01）, up-regulation of ground-state markers（Nanog, Nr5a2, Dppa3, P<0. 01）, and down-regulation of primed-state markers（Fgf5, Lefty1, Dnmt3b, P<0. 01）. Rescue experiments for Dmrt1 expression reversed these above phenotypes.

A candidate gene set regulating exit from naïve pluripotency in ESC is screened out and identified with genome-wide CRISPR. Our findings implicate Dmrt1 plays a critical role in promoting the exit.