We developed a CRISPR/Cas9-mediated base-editing screen to functionally screen endogenous proteins. We screened SPEN, a key factor in X chromosome inactivation, for loss-of-function mutations. Some of the screen hits were mutations of serine residues that could be potential phosphorylation sites (by prediction) and could serve as regulatory sites for the function and/or structure of SPEN. To follow up on our observations, we analyzed phosphorylation sites of SPEN in mouse embryonic stem cells, in which both X chromosomes are active (“noDox” sample), and in cells in which X chromosome inactivation was artificially induced by Xist expression (“Dox” sample).