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Programmable conjugative CRISPR interference targeting genotoxin in the gut.

Jiahe Li1, Brian Hamp1, Hania Timek1,2

  • 1Department of Biomedical Engineering, College of Engineering and School of Medicine, University of Michigan, Ann Arbor, MI, 48109, United States.

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Engineered CRISPR interference (CRISPRi) delivered by a plasmid silences colibactin production by Escherichia coli, reducing genotoxicity and colorectal cancer risk in mice. This programmable live biotherapeutic approach offers a novel strategy against microbial metabolites.

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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.