Abstract

Gut Bacteroides are abundant and critical to human health, yet most are genetically cumbersome, non-model microbes. A widely applicable editing tool for Bacteroides is essential for gut microbiome manipulation. Here, we develop STIB (ShCAST-based transient insertion system for Bacteroides ), an efficient genome-editing tool derived from CRISPR-associated transposases that enables rapid and site-specific insertions independent of homologous recombination. By fusing a nicking homing endonuclease to the transposase and an ATPase to Cas12k, we systematically optimize STIB to minimize plasmid cointegration and achieve >97% on-target insertion. STIB exhibits broad applicability across different genomic loci in diverse Bacteroides species, including non-model species. Finally, we apply STIB to achieve species- and site-specific editing of distinct Bacteroides species within a complex synthetic gut microbiota. Overall, STIB expands the toolbox for the functional investigation and engineering of the human microbiome. A record of this paper’s transparent peer review process is included in the supplemental information.