Matthieu Galtier, Antonina Krawczyk, Fabien J. Fuche, Loïc H. Charpenay, Igor Stzepourginski, Simone Pignotti, Marion Arraou, Rémi Terrasse, Andreas K. Brödel, Chloé Poquet, Gautier Prevot, Dalila Spadoni, Benjamin Buhot, Kristin Muench, Jan Havránek, Pablo Cárdenas Ramírez, Marie Rouquette, Antoine Decrulle, Olivier Kerbarh, Erica Lieberman, Camila Bramorski, Aurélie Grienenberger, Edith M. Hessel, Giuseppina Salzano, Daniel J. Garry, Aymeric Leveau, Xavier Duportet, David Bikard, Jesús Fernández-Rodríguez
Escherichia coli is not only a ubiquitous gut commensal but also an opportunistic pathogen responsible for severe intestinal and extraintestinal infections. Shiga toxin–producing E. coli (STEC) poses a notable public health threat, particularly in children, where infections can lead to bloody diarrhea and progress to hemolytic uremic syndrome, a life-threatening condition with long-term complications. Antibiotics are contraindicated in STEC infections because of their potential to induce prophages carrying Shiga toxin ( stx ) genes, triggering toxin production. Here, we developed a CRISPR-based antimicrobial strategy using a Cas12 nuclease to selectively eliminate O157 STEC clinical isolates, cleaving more than 99% of stx variants, and prevent toxin release. To enable targeted delivery, we engineered a bacteriophage-derived capsid to specifically transfer a nonreplicative DNA payload to E. coli O157, preventing its dissemination. Our therapeutic candidate, EB003, reduced bacterial burden in a murine STEC colonization model. Moreover, EB003 mitigated clinical symptoms, abrogated Stx-mediated toxicity, and accelerated epithelial repair at therapeutically relevant doses in an infant rabbit disease model. These findings demonstrate the potential of CRISPR-based antimicrobials for treating STEC infections and support further clinical development of EB003 as a precision therapeutic against antibiotic-refractory bacterial pathogens.