Dalila Spadoni

Abstract Microbiome research is now demonstrating a growing number of bacterial strains and genes that affect our health 1 . Although CRISPR-derived tools have shown great success in editing disease-driving genes in human cells 2 , we currently lack the tools to achieve comparable success for bacterial targets in situ. Here we engineer a phage-derived particle to deliver a base editor and modify Escherichia coli colonizing the mouse gut. Editing of a β-lactamase gene in a model E. coli strain resulted in a median editing efficiency of 93% of the target bacterial population with a single dose. Edited bacteria were stably maintained in the mouse gut for at least 42 days following treatment. This was achieved using a non-replicative DNA vector, preventing maintenance and dissemination of the payload. We then leveraged this approach to edit several genes of therapeutic relevance in E. coli and Klebsiella pneumoniae strains in vitro and demonstrate in situ editing of a gene involved in the production of curli in a pathogenic E. coli strain. Our work demonstrates the feasibility of modifying bacteria directly in the gut, offering a new avenue to investigate the function of bacterial genes and opening the door to the design of new microbiome-targeted therapies.

Abstract Microbiome research is revealing a growing number of bacterial genes that impact our health. While CRISPR-derived tools have shown great success in editing disease-driving genes in human cells, we currently lack the tools to achieve comparable success for bacterial targets. Here we engineer a phage-derived particle to deliver a base editor and modify E. coli colonizing the mouse gut. This was achieved using a non-replicative DNA payload, preventing maintenance and dissemination of the payload, while allowing for an editing efficiency of up to 99.7% of the target bacterial population. The editing of a β-lactamase gene resulted in the stable maintenance of edited bacteria in the mouse gut at least 42 days after treatment. By enabling the in situ modification of bacteria directly in the gut, our approach offers a novel avenue to investigate the function of bacterial genes and provides an opportunity to develop novel microbiome-targeted therapies.

Abstract Escherichia coli is a ubiquitous gut commensal but also an opportunistic pathogen responsible for severe intestinal and extra-intestinal infections. Shiga toxin-producing E. coli (STEC) pose a significant public health threat, particularly in children, where infections can lead to bloody diarrhea and progress to hemolytic uremic syndrome (HUS), a life-threatening condition with long-term complications. Antibiotics are contraindicated in STEC infections due to their potential to induce prophages carrying Shiga toxin ( stx) genes, triggering toxin production. Here, we present a CRISPR-based antimicrobial strategy that selectively targets and eliminates O157 STEC clinical isolates while preventing toxin release. We designed a Cas12 nuclease to cleave >99% of all stx variants found in O157 strains, leading to bacterial killing and suppression of toxin production. To enable targeted delivery, we engineered a bacteriophage-derived capsid to specifically transfer a non-replicative DNA payload to E. coli O157, preventing its dissemination. In a mouse STEC colonization model, our therapeutic candidate, EB003, reduced bacterial burden by a factor of 3×10 3 . In an infant rabbit disease model, EB003 mitigated clinical symptoms, abrogated stx-mediated toxicity, and accelerated epithelial repair at therapeutically relevant doses. 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.

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.