Programmable conjugative CRISPR interference targeting genotoxin in the gut.

Area of Science:

• Microbiology
• Genetics
• Oncology

Background:

• Colibactin, a metabolite from *pks*⁺ *Escherichia coli*, is linked to early-onset colorectal cancer.
• No current therapeutics directly inhibit colibactin production.

Purpose of the Study:

• To engineer a novel system for targeted gene silencing of colibactin biosynthesis.
• To evaluate the efficacy of this system in reducing *pks*⁺ *E. coli* genotoxicity and associated tumorigenesis.

Main Methods:

• Engineered a self-transmissible conjugative plasmid to deliver CRISPR interference (CRISPRi).
• Utilized CRISPRi to silence colibactin biosynthetic genes in *pks*⁺ *E. coli* strains.
• Assessed genotoxicity, colonization, DNA damage, and tumorigenesis in mouse models.

Main Results:

• The CRISPRi system effectively silenced colibactin production and abolished *pks*⁺ *E. coli* genotoxicity without inducing resistance mutations.
• In mice, conjugation-mediated CRISPRi reduced DNA damage, *pks*⁺ *E. coli* colonization, and tumorigenesis.
• The platform was extended to silence a second pathogenic metabolite, demonstrating broad applicability.

Conclusions:

• Conjugation-mediated CRISPRi is a promising strategy to neutralize colibactin and other microbial metabolites.
• This approach offers a programmable live biotherapeutic strategy for gut health and disease prevention.
• The developed system outperforms existing pharmacologic inhibitors in preclinical models.