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

Abstract

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.

Genes essential for embryonic stem cells are associated with neurodevelopmental disorders

“…Genes essential specifically in mESCs are associated with the early pluripotent state Essential genes in mESCs are enriched for the most basic cellular functions, similar to findings in other cell types (Yilmaz et al 2018;Wang et al 2017). We were interested to discover if there are genes that are essential specifically in mESCs.…”

USP9X deubiquitinase couples the pluripotency network and cell metabolism to regulate ESC differentiation potential

“…Using a pvalue cut-off of 0.05 and a fold change of 1.2, we identified 311 genes differentially expressed between Usp9X KD and control cells, including 170 genes down-regulated and 141 genes upregulated in Usp9X KD cells ( Figure 3A and Supplementary Table 4A). Although the amplitude of gene expression changes was quite modest, it is reminiscent of what has been observed upon depletion of numerous transcription factors or deubiquitinases in murine and human ESC [9,26,42]. Very little correlation also existed between the proteomic and transcriptomic changes in Usp9x KD ESC (Supplementary Figure 4A-B).…”

“…USP9X is very highly expressed in several stem cell populations compared to their differentiated counterparts, as well as in induced pluripotent cells, thus indicating USP9X expression as a marker of stemness [4,40,41]. It associates with the stem cell transcription factors SALL4 and SOX2 in co-immunoprecipitation assays and it was identified in a couple of high-throughput genetic screens aimed at identifying regulators of murine ESC biology and essential genes in human haploid ESC [26,[42][43][44]. However, despite compelling evidences suggesting that USP9X regulates ESC biology, its function has not been properly studied in ESC.…”

Ubiquitin Dynamics in Stem Cell Biology: Current Challenges and Perspectives

“…A number of genetic screens and analyses were conducted to identify and characterize DUBs and E3 Ub ligases essential for stem cell self‐renewal and cell fate choice . This has been facilitated by the production of haploid human embryonic stem cells and the development of sophisticated genetic screening assays based on the CRISPR/Cas9 technology . The increasing number of assembled genomes from diverse invertebrate and vertebrate species also allows the study of the conservation of these pathways and enzymes during evolution.…”

“…[20,[24][25][26][27][28][29][30][31] This has been facilitated by the production of haploid human embryonic stem cells and the development of sophisticated genetic screening assays based on the CRISPR/Cas9 technology. [31] The increasing number of assembled genomes from diverse invertebrate and vertebrate species also allows the study of the conservation of these pathways and enzymes during evolution. For instance, the regeneration of complete worms from very small body fragments in planarians is believed to involve adult stem cells that share a number of characteristics of mammalian stem cells.…”