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  • CRISPR-Engineered Microbiome as Living Therapeutics for Colorectal Cancer: Targeted Elimination of Oncogenic Fusobacterium nucleatum and Neutralization of Colibactin Genotoxicity

  • Sara Farahzadi,1,* Seyed Mohammad Hoseini,2 Zahra Farzaneh,3 Kianoosh Soltani,4
    1. Faculty of Basic Sciences, Islamic Azad University, Islamshahr Branch
    2. Faculty of Basic Sciences, Islamic Azad University, Karaj Branch
    3. Faculty of Basic Sciences, Islamic Azad University, Islamshahr Branch
    4. Faculty of Basic Sciences, Islamic Azad University, Karaj Branch


  • Introduction: Colorectal cancer (CRC) remains a leading cause of cancer-related mortality worldwide, with a complex etiology involving genetic predisposition and gut microbiome dysbiosis. Accumulating evidence identifies Fusobacterium nucleatum (Fn) as a clinically meaningful driver of CRC: recent multi-cohort sequencing studies detect Fn in 35–45% of tumors, with enrichment approaching 50% in stage II–III cases. Meta-analyses encompassing over 4,000 patients consistently link Fn positivity to higher recurrence risk and reduced response to fluoropyrimidine-based chemotherapy. Furthermore, the Fna C2 lineage—the most oncogenic subclade—appears in 29.2% of CRC specimens versus only 4.8% of healthy controls (P < 5.6 × 10⁻¹⁵). Similarly, colibactin, a genotoxin produced by pks⁺ Escherichia coli, contributes to the rising incidence of early-onset CRC. Despite these established oncogenic roles, no approved therapeutic directly suppresses these microbial drivers, underscoring an urgent need for precision microbial interventions.
  • Methods: We developed a dual-platform CRISPR-based therapeutic strategy. First, we engineered Bifidobacterium strains—which naturally accumulate in tumor hypoxic zones at densities near 10⁷ CFU/g—to deliver CRISPR antimicrobials specifically targeting oncogenic Fn within the tumor microenvironment. Second, we constructed a self-transmissible conjugative plasmid system to deliver CRISPR interference (CRISPRi) into multiple pks⁺ E. coli strains, silencing transcription of colibactin biosynthetic genes. The efficacy of both platforms was evaluated in mouse models of CRC, assessing targeted microbial reduction, preservation of commensal diversity, DNA damage, and tumor burden. Mechanistic validation included quantification of FadA adhesin expression, amyloid formation, and downstream oncogenic signaling.
  • Results: The engineered Bifidobacterium platform achieved 95–99% reduction of targeted Fn populations in vivo while sparing the surrounding microbiome—a precision unattainable with systemic antibiotics. CRISPRi-mediated silencing of colibactin biosynthetic genes abolished pks⁺ E. coli genotoxicity without the resistance mutations associated with wild-type Cas9-mediated bacterial inhibition. In mice, conjugation-mediated CRISPRi significantly reduced DNA damage and pks⁺ E. coli colonization while preserving commensal diversity, and notably lowered tumorigenesis, outperforming a pharmacologic inhibitor in a mouse CRC model. Mechanistic studies revealed that CRISPRi-mediated inactivation of the Rnf complex in Fn reduced FadA-mediated amyloid formation and fusobacterial invasion of CRC cells, with significant suppression of spheroid formation in HCT116 cells.
  • Conclusion: This study establishes CRISPR-engineered microbiome as a programmable "living therapeutic" platform that selectively eliminates oncogenic bacteria and neutralizes microbial genotoxins without disrupting commensal communities. By precisely targeting high-risk Fn lineages and colibactin-producing E. coli, this strategy addresses two distinct microbial drivers of CRC pathogenesis. These findings support the translation of CRISPR-microbiome therapeutics into clinical evaluation for CRC management, with potential to reduce inflammatory drive, restore chemotherapy responsiveness, and lower recurrence rates in CRC patients harboring oncogenic microbial signatures.
  • Keywords: CRISPR interference, Fusobacterium nucleatum, colibactin, colorectal cancer,microbiome engineering,

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