CRISPR/Cas-Based Genome Editing for Human Gut Commensal <i>Bacteroides</i> Species

Abstract

<i>Bacteroides</i> is the most abundant genus in the human gut microbiome and has been increasingly used as model organisms for studying the function and ecology of the gut microbiome. However, genome editing tools for such commensal gut microbes are still lacking. Here we developed a versatile, highly efficient CRISPR/Cas-based genome editing tool that allows markerless gene deletion and insertion in human gut <i>Bacteroides</i> species. We constructed multiple CRISPR/Cas systems in all-in-one <i>Bacteroides</i>-<i>E. coli</i> shuttle plasmids and systematically evaluated the genome editing efficiency in <i>Bacteroides thetaiotaomicron</i>, including the mode of Cas protein expression (constitutive, inducible), different Cas proteins (FnCas12a, SpRY, SpCas9), and sgRNAs. Using the anhydrotetracycline (aTc)-inducible CRISPR/FnCas12a system, we successfully deleted large genomic fragments up to 50 kb to study the function of metabolic gene clusters. Furthermore, we demonstrated that CRISPR/FnCas12a can be broadly applied to engineer multiple human gut <i>Bacteroides</i> species, including <i>Bacteroides fragilis</i>, <i>Bacteroides ovatus</i>, <i>Bacteroides uniformis</i>, and <i>Bacteroides vulgatus</i>. We envision that CRISPR/Cas-based genome editing tools for <i>Bacteroides</i> will greatly facilitate mechanistic studies of the gut commensal and the development of engineered live biotherapeutics.