Metagenomic editing of commensal bacteria in vivo using CRISPR-associated transposases

Although metagenomic sequencing has revealed a rich microbial biodiversity in the mammalian gut, methods to genetically alter specific species in the microbiome are highly limited. Here, we introduce Metagenomic Editing (MetaEdit) as a platform technology for microbiome engineering that uses optimized CRISPR-associated transposases delivered by a broadly conjugative vector to directly modify diverse native commensal bacteria from mice and humans with new pathways at single-nucleotide genomic resolution. Using MetaEdit, we achieved in vivo genetic capture of native murine Bacteroides by integrating a metabolic payload that enables tunable growth control in the mammalian gut with dietary inulin. We further show in vivo editing of segmented filamentous bacteria, an immunomodulatory small-intestinal microbial species recalcitrant to cultivation. Collectively, this work provides a paradigm to precisely manipulate individual bacteria in native communities across gigabases of their metagenomic repertoire.