Defining essential genes for human pluripotent stem cells by CRISPR-Cas9 screening in haploid cells

The maintenance of pluripotency requires coordinated expression of a set of essential genes. Using our recently established haploid human pluripotent stem cells (hPSCs), we generated a genome-wide loss-of-function library targeting 18,166 protein-coding genes to define the essential genes in hPSCs. With this we could allude to an intrinsic bias of essentiality across cellular compartments, uncover two opposing roles for tumour suppressor genes and link autosomal-recessive disorders with growth-retardation phenotypes to early embryogenesis. hPSC-enriched essential genes mainly encode transcription factors and proteins related to cell-cycle and DNA-repair, revealing that a quarter of the nuclear factors are essential for normal growth. Our screen also led to the identification of growth-restricting genes whose loss of function provides a growth advantage to hPSCs, highlighting the role of the P53-mTOR pathway in this context. Overall, we have constructed an atlas of essential and growth-restricting genes in hPSCs, revealing key aspects of cellular essentiality and providing a reference for future studies on human pluripotency.