CRISPR Genome-scale CRISPR/Cas9 screening reveals the role of PSMD4 in colibactin-mediated cell cycle arrest

Colibactin is a genotoxic secondary metabolite produced by selective Enterobacteriaceae strains that populate the mammalian intestine and produces specific mutational signature in human colonocytes. However, the host pathways involved in colibactin response remain unclear. To address this gap, we performed global transcriptomics and genome wide CRISPR/Cas9 knockout screens and RNA sequencing utilizing live pks+ bacteria and synthetic colibactins. From our genome-wide screens we identified 20 enriched genes with a MaGECK score > 2.0 in both screens, including proteasomal subunits (e.g., PSMG4, PSMD4), RNA processing factors (e.g., SF1, PRPF8), and RNA polymerase III (e.g., CRCP), and validated the role of PSMD4 in colibactin sensitization. PSMD4 knockout ameliorated reductions in cell viability and G2/M cell cycle arrest after 742 exposure. Conversely, PSMD4 knockout did not affect the amount of gammaH2AX foci after 742 exposure. Consistent with these observations, PSMD4-/- cells had a significantly higher colony formation rate and reduced colony size than control cells after 742 exposure. These findings suggest that PSMD4 regulates cell-cycle arrest following colibactin-induced DNA damage, and that cells with deficiencies in these pathways may continue to replicate despite DNA damage, potentially increasing the risk of malignant transformation.