In Situ Targeted Mutagenesis of Gut Bacteria

Microbiome research is revealing a growing number of bacterial genes that impact our health. While CRISPR-based tools have shown success in editing human cell genes, their application to bacterial targets within their natural habitat remains a challenge. Our research introduces a pioneering solution by engineering phage-derived particles for in situ base editing of E. coli inhabiting the murine gut. The engineered particles deliver a base editor encoded in a non-replicative DNA payload, circumventing payload maintenance and dissemination, which allows for both efficient editing and controlled impact on the gut ecosystem. This novel approach achieves remarkable base editing efficiencies, reaching up to 99.7% of the target bacterial population. We first show the stable modification and inactivation of a β-lactamase gene in a model E. coli strain within the murine gut, persisting for at least 42 days post-treatment. We then leverage this approach to target a pathogenic E. coli strain and introduce a mutation in a gene involved in the production of curli, a bacterial appendage implicated in neurodegenerative diseases. Our strategy unlocks the potential for exploration of bacterial gene function within their native environment and presents an opportunity for the development of next-generation microbiome-targeted therapies.