ESSENTIAL GENES FOR DIFFERENTIATION INTO ALL THREE GERM LAYERS DEFINED BY CRISPR/CAS9 SCREENING IN HAPLOID HUMAN EMBRYONIC STEM CELLS

Pluripotent stem cells can differentiate into all three embryonic germ layers, a process that is orchestrated by the dissolution of pluripotency network and activation of pro-differentiation pathways. Yet, the genes essential for these cell fate transitions in human are still elusive. Recently, we have derived haploid human pluripotent stem cells (hPSCs) and generated a loss-of-function library in these cells targeting 18,000 coding genes utilizing CRISPR/Cas9 technology. This library enabled us to define the genes essential for the normal growth and survival of undifferentiated hPSCs. Here, we set out to map the essential genes for the differentiation of hPSCs into all three germ layers, by using the loss-of-function library in haploid hPSCs. Through the analysis of essential genes for the differentiation of hPSCs into ectoderm, mesoderm and endoderm, we defined the essentialome of each germ layer separately. Furthermore, we identified the genes that are common for the transition from pluripotency stage into all three embryonic germ layer fates. Interestingly, this group was enriched by genes that are localized to the Golgi apparatus and the endoplasmic reticulum and regulate membrane and secreted molecules, highlighting the key role of signaling events during these dynamic cell state transitions. Overall, our work sheds light on the gene networks regulating early gastrulation events in human by defining essential drivers of specific embryonic germ layer fates and essential genes for the exit from pluripotency.

The initial part of this study was recently published:

Yilmaz et al. Nature Cell Biology (2018)

Yilmaz and Benvenisty Cell Stem Cell (2019)