Fig. 1: Domain-focused CRISPR/Cas9 screen identifies potential regulators of epigenetic processes contributing to self-renewal of ground-state mESCs in 2i/LIF.

a Schematic representation of the CRISPR/Cas9 screening strategy. All sgRNAs were cloned into the pU6-sgRNA-SFFV-Puro-P2A-EGFP vector. mESCs were transduced at a multiplicity of infection (MOI) of ~0.3. Cells were passaged every second day, pellets were collected at day 1, day 7 (10 population doublings) and day 11 (15 population doublings) of the screen and the abundance of the individual sgRNAs was determined. b Scatterplot comparing day 7 and day 11, respectively, to day 1 log2 median-normalized read counts of the individual sgRNAs. c Overlap analysis of mouse homologues of common essential genes in 739 human cancer cell lines (Project Achilles, Depmap, 20Q1²³), 1680 and 1664 genes identified as essential for mESCs grown in serum/LIF²¹ or 2i/LIF²², respectively using genome-wide sgRNA screens, and the 146 genes encoding proteins with potential epigenetic function identified in this study. d Ranked comparison of median CERES scores (Project Achilles, Depmap, 20Q1²³) for human genes and for the genes identified in this study. CERES scores provide estimates of gene dependency with values lower than −0.5 representing noteworthy depletion across the 739 cancer cell lines tested and hence the gene is classified as common essential²³. e–g Competition-based proliferation assays in indicated Cas9-expressing cell lines. GFP was monitored over a time course of more than ten population doublings. sgRNAs targeting the core essential genes Nat10 and Pcna served as positive controls while a non-targeting sgRNA served as a negative control (NegControl). The percentage of GFP+ cells is normalized to the day 0 measurement and the measurement of the NegControl at the respective day (n = 1). h Western blot analysis of MPP8 and ßACTIN (loading control) in the indicated cell lines (n = 1). mESCs = mouse embryonic stem cells; NSCs = neural stem cells; MEFs = mouse embryonic fibroblasts. Source data are provided as a Source Data file.