A self-transmissible conjugative plasmid delivering CRISPR interference with catalytically inactive Cas9 (dCas9) achieves stable transcriptional silencing of colibactin biosynthetic genes in pks+ E. coli, eliminating genotoxicity without resistance mutations, and demonstrates in vivo efficacy by reducing DNA damage, suppressing tumorigenesis in a mouse colorectal cancer model, and decreasing pathogenic bacterial colonization while preserving commensal microbiome diversity—outperforming pharmacologic inhibition and establishing a generalizable platform for programmable live biotherapeutics targeting disease-associated microbial metabolites.

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.