A comprehensive CRISPR/Cas9-targeted base mutagenesis identifies an amino acid substitution separating functions of SPEN in X chromosome inactivation from development

While genetic screens have facilitated the dissection of protein function in animal development, advances in systematic point mutagenesis open new opportunities for forward genetics in mammalian cells. Here, we develop a CRISPR/Cas9-mediated base editing screen that allows to generate extensive maps of single amino acid substitutions of endogenous proteins. We demonstrate the application on the X-chromosomal Hprt and autosomal Msh2 gene in diploid male and haploid mouse embryonic stem cells, respectively. Finally, we use this methodology to generate a sequence-function map of the transcriptional co-repressor SPEN in X chromosome inactivation. We demonstrate that the substitution of the SPEN RRM4-residue W522 abrogates X-linked gene repression by Xist RNA and impairs H3K27-deacetylation, but does not affect Polycomb recruitment and H3K27me3 deposition. Our results demonstrate that screening in haploid cells allow the efficient identification of separation-of-function mutations that would be recessive in diploid cells suggesting applications to a wide range of areas