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

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.