Bacteroides are the most abundant bacteria in the human gut. Precisely editing their genomes remains difficult. Current tools rely on homologous recombination, which is inefficient in most Bacteroides, or requires isolating and culturing individual species.

In a study published in Cell Systems, a team led by Prof. DAI Lei and Prof. ZHAO Wei from the Shenzhen Institute of Advanced Technology of the Chinese Academy of Sciences developed ShCAST-based transient insertion system for Bacteroides (STIB), a CRISPR-associated transposase tool which enables efficient, site-specific insertion of large DNA fragments into Bacteroides genomes.

Researchers engineered the type V-K CRISPR transposase ShCAST for optimization of the system in three key aspects: Fusing a nicking endonuclease (nAniI) to TnsB eliminated plasmid cointegration; Fusing TnsC to Cas12k boosted on-target specificity to above 97%; adopting a non-replicating vector enabled transient expression, shortening the workflow to four days.

STIB achieved high editing efficiency across multiple Bacteroides species and genomic sites, inserting cargo up to 8.4 kilobases. Introducing an inulin utilization gene cluster enabled engineered B. thetaiotaomicron to thrive on inulin and become naturally enriched without antibiotics.

Moreover, researchers constructed a 40-species synthetic human gut bacterial community, and introduced STIB constructs, each carrying a guide RNA designed to insert a chloramphenicol resistance gene into a specific genomic site of a target species. Metagenomic sequencing of the entire community revealed highly specific enrichment of each targeted species. B. thetaiotaomicron surged from 0.3% to 98%, B. ovatus from 0.9% to 86%, and B. vulgatus from 0.3% to 69%.

With its large cargo capacity, STIB can deliver diverse functional modules to the gut ecosystem. Combined with advanced delivery vehicles such as engineered bacteriophages, it provides a foundation for precision microbiome engineering and next-generation living biotherapeutics.