CRISPR Knockout screens for the identification of X-linked genes that drive sex differences in mouse embryonic stem cells

Dosage imbalance for X-chromosomal genes contributes to sex differences, in particular during early development, when both X chromosomes are active in females. X-encoded inhibitors of the differentiation-promoting MAP kinase (MAPK) signalling pathway slow down development, increase levels of naive pluripotency factors, and decrease MAPK target gene expression. Through a hierarchical CRISPR screening approach in murine embryonic stem cells(mESC) we have comprehensively identified X-linked genes that modulate MAPK signalling, pluripotency factor expression, and differentiation. Overall design: 1. Primary knockout screen for the identification of genes that modulate MAPK target genes expression using an sgRNA library targeting all X-linked genes (SRE-Elk screen, GeCKOx sgRNA library). 2. Secondary knockout screens on top hits from the primary screen (GeCKOxs sgRNA library) for the identification of genes modulating pluripotency factor expression (Nanog screen), differentiation kinetics (Esrrb screen) and Mek phosphorylation (pMek screen).