Fig. 2

Secondary CRISPR screens profiling pluripotency factor expression, differentiation kinetics and Mek phosphorylation. a Composition of the GeCKOxs sgRNA library, targeting hits from the primary MAPK screen and positive control genes with 3 sgRNAs per gene. b Schematic representation of the C-terminal tagging of the Nanog and Esrrb genes with the mCherry fluorescent protein through Cas9-mediated homologous recombination and subsequent Cre-mediated excision of the puromycin resistance cassette. Nanog/Esrrb and mCherry are linked through a P2A self-cleaving peptide. c–e Schematic depiction of the three secondary screens to profile effects on pluripotency factor expression (c), differentiation (d), and Mek phosphorylation (e). Female mESCs, carrying mCherry-tagged Esrrb/Nanog loci, as indicated, expressing the Cas9 endonuclease, were transduced with the sgRNA library in a. c In the Nanog screen, the 25% cells with the weakest mCherry fluorescence were enriched in two consecutive sorts (day 7 and day 9 after transduction). d For the Esrrb screen, cells were differentiated via LIF withdrawal for 3 days and the 10% cells with the lowest mCherry fluorescence were FACS sorted. e In the pMek screen, cells were stained intracellularly with a pMek-specific antibody and the 25% cells with the lowest signal were sorted. Three replicates were generated for the Esrrb and pMek reporter screens and two for the Nanog screen. f Volcano plots of the most enriched and depleted genes in the Nanog, Esrrb, and pMek screens. Genes with an FDR < 0.05 are highlighted as indicated. g Heatmap summarizing the results from all 4 screens. Enrichment of all X-linked (left) and control genes (right) that were significantly enriched or depleted in at least 2 screens is shown. *FDR < 0.05 (MAGeCK), n.d non-determined. h Expression levels for a subset of X-linked genes shown in g in 1.8XX and 1.8XO mESCs assessed by RNA sequencing