Targeted genomic editing of human gut Bacteroides species based on CRISPR-associated transposases
Abstract
Bacteroides are a group of highly abundant bacteria in the gut and play critical roles in human health and diseases, while most of them are non-model microbes and genetically cumbersome. The development of a widely applicable editing tool for Bacteroides is much needed for the manipulation of human gut microbiome. In this study, we develop STIB (ShCAST-based Transient Insertion system for Bacteroides), a novel genome editing tool derived from CRISPR-associated transposases that enable 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 to achieve >97% on-target insertion. We demonstrate that STIB exhibits broad applicability across different genomic loci in distinct Bacteroides, including some non-model species. Finally, we apply STIB to achieve species- and site-specific editing of multiple Bacteroides species in a complex synthetic gut microbiota. Overall, our study provides a novel genome editing tool for Bacteroides species and expands the toolbox for functional investigation and engineering of the human microbiome.
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Declaration of Competing Interests
The authors declare no competing interests to disclose.
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