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

Colibactin is a genotoxic secondary metabolite produced by certain Enterobacteriaceae strains that populate the intestine and produces a specific mutational signature in human colonocytes. However, the host pathways involved in colibactin response remain unclear. To address this gap, we performed genome-wide CRISPR/Cas9 knockout screens and RNA sequencing utilizing live pks bacteria and a synthetic colibactin analog. We identified 20 enriched genes with a MAGeCK score of >2.0 in both screens, including proteasomal subunits (e.g., PSMG4 and PSMD4), RNA processing factors (e.g., SF1 and PRPF8), and RNA polymerase III (e.g., CRCP), and validated the role of PSMD4 in colibactin sensitization. PSMD4 knockout in HEK293T and HT-29 cells promoted cell viability and ameliorated G2-M cell cycle arrest but did not affect the amount of phosphorylated H2AX foci after exposure to synthetic colibactin 742. Consistent with these observations, PSMD4^-/- cells had a significantly higher colony formation rate and bigger 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 PSMD4 deficiency may continue to replicate despite DNA damage, potentially increasing the risk of malignant transformation.

Colibactin has been implicated as a causative agent of colorectal cancer. However, colibactin-producing bacteria are also present in many healthy individuals, leading to the hypothesis that some aspects of colibactin regulation or host response dictate the molecule's carcinogenic potential. Elucidating the host-response pathways involved in dictating cell fate after colibactin intoxication has been difficult, partially due to an inability to isolate the molecule. This study provides the first high-throughput CRISPR/Cas9 screening to identify genes conferring colibactin sensitivity. Here, we utilize both bacterial infection and a synthetic colibactin analog to identify genes directly involved in colibactin response. These findings provide insight into how differences in gene expression may render certain individuals more vulnerable to colibactin-initiated tumor formation after DNA damage.