Extended Data Fig. 1: Genome-wide KO screen and analysis.
From: Genome-wide CRISPR screen identifies Menin and SUZ12 as regulators of human developmental timing
a, Schematic of PAX6::H2B-GFP iCas9 cell line. iCas9 was inserted at the AAVS1 locus through TALEN-mediated gene targeting in the PAX6::H2B-GFP cell line. TRE, tetracycline response element; M2rtTA, reverse tetracycline transactivator sequence and protein. b, Karyotypic analysis of the PAX6::H2B-GFP iCas9 H9 hESC clonal cell line used for the study. c, Expression of Cas9 RNA in the PAX6::H2B-GFP iCas9 cell line after one day of doxycycline (dox) treatment compared to no dox treatment (n = 3 independent experiments). Data are the mean ± s.d. d, Representative Western Blot analysis for Cas9 protein in the PAX6::H2B-GFP iCas9 cell line (iCas9) after two days of dox treatment compared to no dox treatment. H9 and H9 carrying a constitutive Cas9 expression (H9 Cas9) were used as negative and positive control respectively. e, Flow cytometry analysis of GFP expression during neuroectoderm differentiation in iCas9 cell line and its parental line (n = 4 independent experiments for iCas9 line, n = 3 independent experiments for the parental line). Data are mean ± s.d. f, Workflow of screen analysis. g, Waterfall plots of the top 100 GSEA enriched pathways from the ranked gene list. Chromatin-related and mitochondrial metabolism-related terms are highlighted in separate plots. h, GSEA plots for two of the chromatin-related GO terms, ATAC complex and SET1C/COMPASS complex, that positively correlate with PAX6-GFPhigh population. i, GSEA plot for one of the mitochondrial metabolism-related GO terms, Tricarboxylic acid cycle, that positively correlate with PAX6-GFPhigh population.
