Highly efficient CRISPR‐mediated base editing for the gut Bacteroides spp. with pnCasBS‐CBE

Abstract Bacteroidales are the most abundant order of bacteria in the healthy human gut and have the potential as a therapeutic agent. We constructed a pnCasBS‐CBE system for base editing in the Bacteroides thetaiotaomicron to expand their genetic toolkit, which is able to efficiently convert a C:G to a T:A in the genome. As a functional proof‐of‐concept, we used the pnCasBS‐CBE system to successfully introduce nonsynonymous mutation and stop codons to the genes involved in carbohydrate metabolism. The system also allowed for multiplexed gene editing with a single plasmid, enabling efficient editing of up to four genes in a single experiment. Furthermore, the pnCasBS‐CBE editing system was validated and successfully applied in four other non‐model gut Bacteroides species for genome editing. An unbiased genome‐wide SNPs analysis indicated that the pnCasBS‐CBE system showed high fidelity and applicability. Thus, this study provides a powerful clustered regularly interspaced short palindromic repeats (CRISPR)‐mediated genome editing toolbox for functional genomic analysis in Bacteroidales.