CRMA: A robust and versatile toolkit for rapid identification of regulators for germ cell fate induction from human pluripotent stem cells

Primordial germ cells (PGC) are the precursors that establish the germline for reproduction. The induction of human PGC-like cells (hPGCLCs) from human pluripotent cells (hPSCs) is critical for uncovering the molecular mechanisms governing the specification of germ cell fate. Current methods for generating hPGCLCs are suffering from achieving both high biological relevance and high throughput, with significant batch effects hindering functional studies. Here, we developed an improved three-dimensional (3D) differentiation method for robust generation of hPGCLCs with high yield within 3D aggregates. Incorporating reporters for labelling hPGCLCs, we employed mosaic analysis by mixing hESCs of control and fluorescence-tagged knock-down or over-expression by CRISPR interference (CRISPRi) or CRISPR activation (CRISPRa), respectively, for hPGCLC induction, minimizing the batch effect. We termed this rapid and reliable system as CRISPR-and reporter-based mosaic analysis (CRMA), and verified it by CRIPSRi of SOX17 and TFAP2C, and CRISPRa of SOX2 and SOX17. This methodology offers a powerful tool for functional investigation of candidate genes during hESC differentiation towards germ cell fate, and could be applied to other differentiation lineages.