Programmable conjugative CRISPR interference targeting genotoxin in the gut

Abstract

Among microbially derived metabolites that influence host disease, colibactin garners increasing attention for its roles in the rising incidence of early-onset colorectal cancer. Produced by pks Escherichia coli, colibactin is a potent genotoxin, yet no approved therapeutics directly suppress it. Here, we engineered a self-transmissible conjugative plasmid to deliver CRISPR interference (CRISPRi) into multiple pks strains. This system silences transcription of colibactin biosynthetic genes and abolishes pks E. coli genotoxicity without the resistance mutations associated with wild-type Cas9-mediated bacterial inhibition. In mice, conjugation-mediated CRISPRi reduces DNA damage and pks E. coli colonization while preserving commensal diversity. Importantly, the system also lowers tumorigenesis driven by pks E. coli and outperforms a pharmacologic inhibitor in a mouse colorectal cancer model. Finally, we extend this platform to silence a second pathogenic metabolite, establishing a translational strategy to neutralize diverse microbial metabolites and expanding the toolkit for programmable live biotherapeutics in the gut.