CRISPR loss of function screening to identify genes involved in human primordial germ cell-like cell development

Despite our increasing knowledge of molecular mechanisms guiding various aspects of human reproduction, those underlying human primordial germ cell (PGC) development remain largely unknown. Here, we conducted custom CRISPR screening in an in vitro system of hPGC-like cells (hPGCLCs) to identify genes required for acquisition and maintenance of PGC fate in humans. Amongst our candidates, we identified TCL1A, an AKT coactivator. Functional assessment in our in vitro hPGCLCs system revealed that TCL1A played a critical role in later stages of hPGCLC development. Moreover, we found that TCL1A loss reduced AKT-mTOR signaling, downregulated expression of genes related to translational control, and subsequently led to a reduction in global protein synthesis and proliferation. Together, our study highlights the utility of CRISPR screening for human in vitro-derived germ cells and identifies novel translational regulators critical for hPGCLC development. Proper development of the germline lineage into sperm and oocytes is required for propagation of inherited genetic information across generations. Accordingly, its abnormal development in humans results in a variety of medical conditions including infertility and congenital diseases. Despite its importance to human health, a comprehensive understanding of the mechanisms regulating human germ cell development is limited by both technical challenges and ethical concerns. The germline is established early in development in primordial germ cells (PGCs), the common precursor for both spermatozoa and oocytes. Notably, recent generation of PGC-like cells (hPGCLCs) derived from pluripotent stem cells allows for an unprecedented investigation of molecular mechanisms driving early germline development in humans. Using CRISPR loss-of-function screening, we now provide functional genomics resources critical for understanding hPGCLCs development. Using this screening technique, we have identified TCL1A as a novel gene that is critical for maintaining hPGCLCs. Functional validation analyses revealed that TCL1A promotes global protein synthesis and cell proliferation through AKT-mTOR signaling pathways in hPGCLCs. This study highlights the utility of CRISPR screening in deciphering the genetic basis of human germ cell development.