Fig. 5: Inducible Cas9 whole genome CRISPR KO screens using dFLASH-HIF.

a HEK293T (upper panel) or U2OS (lower panel) mcdFLASH-HIF/iCas9-BFP cells were used to validate CRISPR KO loss of function screening by short term (3 days) inducible (dox) knockout of HIF-1α or gain of function with knockout of VHL. Data is representative of n = 2 independent experiments. b Schematic of the whole genome KO screening strategy. Briefly, clonal dFLASH-HRE reporter lines were transduced with a dox inducible Cas9-TagBFP virus and selected with blasticidin (BlastS) and for homogeneous Cas9-BFP induction. The Brunello whole genome KO library, ~77,000 guide RNAs targeting 19,114 genes, was then introduced and cells selected with puromycin (Puro) prior to a short-term (5 days) Cas9 induction with dox and FACS isolation of low activity pools. Created in BioRender. Peet, D. (2025) https://BioRender.com/m13q226c dFLASH-HIF benchmarking against another published pooled HIF CRISPR screen. Loss of function CRISPR screen performed with Hela 3xHRE-mCherry^ODD (Ortmann et al. 2021) vs HEK293T or U2OS dFLASH-HIF CRISPR screens. The number of enriched MAGeck CRISPR hits (p < 0.05, one-tailed) were quantified and plotted. d Volcano plots of dFLASH-HRE CRISPR screens in HEK293T (left panel) or U2OS (right panel) demonstrating the strong selection of obligate regulators HIF-1α and ARNT. Differentially regulated genes from DEseq2 (generalised linear model with negative binomial distribution) with a p-value < 0.001 (grey dotted horizontal line, two-sided Wald test) and >0.6 LFC (grey dotted vertical line) are marked as red dots. Data presented is from n = 3 independent CRISPR screens. Source data are provided as a Source Data file.