Identification of X-chromosomal genes that drive sex differences in embryonic stem cells through a hierarchical CRISPR screening approach

Background X-chromosomal genes contribute to sex differences, in particular during early development, when both X chromosomes are active in females. Double X-dosage shifts female pluripotent cells towards the naive stem cell state by increasing pluripotency factor expression, inhibiting the differentiation-promoting MAP kinase (MAPK) signaling pathway, and delaying differentiation. Results To identify the genetic basis of these sex differences, we use a two-step CRISPR screening approach to comprehensively identify X-linked genes that cause the female pluripotency phenotype in murine embryonic stem cells. A primary chromosome-wide CRISPR knockout screen and three secondary screens assaying for different aspects of the female pluripotency phenotype allow us to uncover multiple genes that act in concert and to disentangle their relative roles. Among them, we identify Dusp9 and Klhl13 as two central players. While Dusp9 mainly affects MAPK pathway intermediates, Klhl13 promotes pluripotency factor expression and delays differentiation, with both factors jointly repressing MAPK target gene expression. Conclusions Here, we elucidate the mechanisms that drive sex-induced differences in pluripotent cells and our approach serves as a blueprint to discover the genetic basis of the phenotypic consequences of other chromosomal effects.