DNAseq analysis of a genome-wide loss-of-function CRISPR/Cas9 library established in haploid human embryonic stem cells (hESCs)

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. We could allude to an intrinsic bias of essentiality across cellular-compartments, uncover two opposing roles for tumor-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 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. Haploid hESCs were transduced with a lentiviral pooled gRNA library carrying 181,131 gRNAs at a multiplicity of infection of 0.3 to enrich for single integrations/mutations per cell. Transduced haploid hESCs were selected with puromycin and cultured for 30 days. Mutant hESCs were passaged every 4-5 days and DNA samples were collected from the population at multiple time points during the passaging. gRNA sequences were amplified from the genomic DNA and analyzed by high-throughput deep sequencing.