Infertility is a common reproductive health problem, affecting ~15% of couples worldwide. Studies over the past decades have identified hundreds of causative genes, yet the genetic causes of most cases still remain unexplained. Due to the highly polygenic and heterogeneous nature of the disease, genome-wide association studies (GWAS) have had limited success. Risk variants identified so far mostly reside in non-coding regulatory regions, suggesting that defects in gene regulation may underlie many infertility cases. However, the regulatory landscape governing germline development, particularly in primordial germ cells (PGCs), remains poorly characterized. In this proposal I seek to identify the essential genes and non-coding enhancer elements that safeguard PGC development.

Leveraging a high-efficiency, four transcription factors–based in vitro differentiation system that produces up to 80% Stella⁺ primordial germ cell-like cells (PGCLCs), I have already generated nascent RNA sequencing data to identify active enhancers across PGCLC differentiation. For this proposal, I will integrate CRISPR-based functional screening for essential PGC genes with multi-omic enhancer–gene interaction mapping. Aim 1 will employ a dualsgRNA CRISPR interference (CRISPRi) screen targeting 1,746 candidate genes to identify those essential for PGCLC fate and proliferation. Aim 2 will generate a genome-wide enhancer–gene regulatory atlas by integrating PRO-cap, ATAC-seq, and Hi-C datasets across ESC, EpiLC, and PGCLC stages, applying the Activity-By-Contact (ABC) model to predict enhancer–promoter interactions. Aim 3 will functionally validate high-confidence enhancers by CRISPR-mediated disruption and quantify effects on target gene expression and PGCLC differentiation efficiency.

All proposed assays, including PGCLC differentiation, pooled CRISPRi screening, and enhancer perturbation are well established and supported by preliminary data. Within one year, this work will define essential genes of PGC development, establish a comprehensive enhancer–gene interaction map, and identify functional non-coding elements critical for germline specification. These findings will provide a foundation for interpreting infertility associated non-coding variants and advance understanding of mammalian germ cell biology.