A primordial germ cell-like-cell platform enables CRISPRi screen for epigenetic fertility modifiers.

Primordial germ cells (PGCs) are the precursors of gametes, and the ability to derive PGC-like cells (PGCLCs) from pluripotent stem cells has transformed germline research. A key limitation remains producing PGCLCs in sufficient numbers for large-scale applications. Here, we show that overexpression of Nanog plus three PGC master regulators - Prdm1, Prdm14, and Tfap2c - in mouse epiblast-like cells and formative embryonic stem cells yields abundant and highly enriched PGCLCs without costly recombinant cytokines. Nanog enhances the PGC regulatory network, suppresses somatic differentiation, and stabilizes PGCLC fate. Transcriptomically, these PGCLCs are developmentally more advanced than cytokine-induced counterparts and can be sustained long-term or differentiated into spermatogonia-like cells. Using this platform, we conduct a CRISPRi screen of 701 epigenetic genes to identify those needed for PGCLC formation. Downregulation of Ncor2, a histone deacetylase (HDAC) recruiter, has the greatest impact. Additionally, the HDAC inhibitors valproic acid and sodium butyrate suppress PGCLC formation and sperm counts of in utero-exposed animals. This work establishes a scalable system for functional screening of genes that influence germline development.