Fig. 1: Identification of KAT5 as a G0-trap in GSC-0827 cells.

a Schematic of the G0-trap screen. For the screen, GSC-0827 cells containing mCherry-CDT1 (G0/G1) and p27-mVenus (G0) were transduced with a genome-wide CRISPR-Cas9 library, allowed to expand for 10 days, and sorted for double positive cells using the top 20% of p27-mVenus+ cells as a cut off. FACS machine cartoon taken from BioRender. Paddison, P. (2025) https://BioRender.com/o2c4y3l. b Results G0-trap screen from sgRNA-seq of p27^hi vs. total cell population (n = 3; edgeR was used to assess p values and logFC cuttoffs) (Supplementary Data 1). Supporting QC data and gene set enrichment can be found in Supplementary Fig. 1. c FACS-based assessment of total RNA and DNA content, using pyronin Y and DAPI, respectively, in GSC-0827 cells via nucleofection of sgRNA:Cas9 RNPs. Additional retest assays are available in Supplementary Figs. 2, 3. d Quantification of (c) (≥2350 cells per condition from two independent treatments; KS test, p < .0001; median and quartiles shown). e FACS-based assessment of EdU incorporation after KAT5 KO using nucleofection of sgRNA:Cas9 RNPs (5 days post-nucleofection) in GSC-0827 cells. Quantification is shown in Supplementary Fig. 3a. f GSC-0827 cells were engineered to have a doxycycline controllable KAT5 open reading frame and knockout insertion-deletion mutations in the endogenous KAT5 gene. We found one such clone, #13 (C13), to have Dox-dependent KAT5 expression and requirement of Dox+ for continued growth. g Western blot analysis of KAT5 and histones H3 and H4 acetylation and methylation status after Dox withdrawal of 0, 4, 7, and 14 days in C13 cells. *indicates KAT5 targets. h FACS analysis of histone H4 acetylation and p27-mVenus levels after 0, 7, and 14 days of Dox withdrawal in C13 cells. i A growth curve with C13 cells grown in various concentrations of Dox (n = 3; mean ± SEM is shown, significance determined by unpaired, 2 tailed student’s t test). j FACS analysis of p27 induction in C13 cells grown in shown concentrations of Dox for 7 days. k Violin plot of H4-Ac levels in C13 cells grown in shown concentrations of Dox for 5 days. ≥5703 cells plotted for single trial. KS test was used to test significance, p <0.0001. Median and quartiles displayed. Source data with exact p values are provided with this paper as a Source Data file.