Programmable conjugative CRISPR interference targeting genotoxin in the gut.

Area of Science:

• Microbiology
• Genetics
• Oncology

Background:

• Colibactin, a genotoxin produced by *pks*+ *Escherichia coli*, is increasingly linked to early-onset colorectal cancer.
• No approved therapeutics currently target colibactin production directly.
• Existing methods for bacterial inhibition may lead to resistance mutations.

Purpose of the Study:

• To engineer a novel system for targeted suppression of colibactin biosynthesis in *pks*+ *E. coli*.
• To evaluate the efficacy of this system in reducing bacterial genotoxicity, colonization, and tumorigenesis in a mouse model.
• To establish a versatile platform for programmable live biotherapeutics targeting microbial metabolites.

Main Methods:

• Development of a self-transmissible conjugative plasmid to deliver CRISPR interference (CRISPRi) system.
• CRISPRi-mediated silencing of colibactin biosynthetic genes in *pks*+ *E. coli* strains.
• In vivo testing in mice to assess reduction in DNA damage, bacterial colonization, and tumorigenesis.

Main Results:

• Engineered CRISPRi system successfully silenced colibactin production and abolished *pks*+ *E. coli* genotoxicity without inducing resistance mutations.
• Conjugation-mediated CRISPRi reduced DNA damage and *pks*+ *E. coli* colonization in mice while preserving gut commensal diversity.
• The system demonstrated superior efficacy in lowering tumorigenesis compared to a pharmacologic inhibitor in a mouse colorectal cancer model.

Conclusions:

• A conjugation-mediated CRISPRi system provides a potent strategy to suppress colibactin production and mitigate its associated health risks.
• This programmable live biotherapeutic platform is extendable to neutralize other pathogenic microbial metabolites.
• The developed system expands the toolkit for engineering gut bacteria as therapeutic agents.