High throughput CRISPR perturbation screens identify epigenetic regulators impacting primordial germ cell development [CRISPRi]

There is concern that environmental factors underlie reported declines in fertility and reproductive parameters such as the number and quality of sperm and eggs. One possible mechanism is the perturbation of epigenetic landscapes in the germline. To explore this possibility, we utilized a highly scalable in vitro platform for the differentiation of embryonic stem cells (ESCs) into primordial germ cell-like cells (PGCLCs) to conduct a CRISPRi screen of epigenetic-related genes to identify those that specifically perturb the efficiency of PGCLC formation. This platform leveraged an ESC line containing 4 inducible transcription factors (Prdm1, Prdm14, Tfap2c, and Nanog) and a PGC reporter (Stella-eGFP) that enabled highly efficient, cytokine-free differentiation and quantification en masse. A total of 701 genes with distinct sgRNAs were screened, and 53 genes were identified that decreased the efficiency of PGCLC formation. NCOR2, a transcriptional repressor that acts via recruitment of Class I and Class IIa histone deacetylases (HDACs) to gene targets, was particularly potent in suppressing PGCLC differentiation. Consistent with evidence that histone deacetylation is crucial for germline differentiation, we found that HDAC inhibitors (HDACi), including the anti-convulsive drug valproic acid (VPA) and widely-available dietary supplement sodium butyrate (SB), also suppressed ESC>PGCLC differentiation. Transcriptome analyses of HDACi-treated, differentiating ESC>PGCLC cultures revealed suppression of germline-associated pathways and enhancement of somatic pathways. Additionally, exposure of developing mouse embryos to SB or VPA caused hypospermatogenesis. This work demonstrates the feasibility of conducting large scale functional screens of genes, chemical agents and possibly genetic variants that impact germline development and epigenome. Overall design: Using a CRISPR inhibition (CRISPRi) screen to identify essential epigenetic regulators of PGC development.