Genome engineering of the human gut microbiome.

The human gut microbiome, a complex ecosystem, significantly influences host health, impacting crucial aspects such as metabolism and immunity. To deepen our comprehension and control of the molecular mechanisms orchestrating the intricate interplay between gut commensal bacteria and human health, the exploration of genome engineering for gut microbes is a promising frontier. Nevertheless, the complexities and diversities inherent in the gut microbiome pose substantial challenges to the development of effective genome engineering tools for human gut microbes. In this comprehensive review, we provide an overview of the current progress and challenges in genome engineering of human gut commensal bacteria, whether executed in vitro or in situ. A specific focus is directed towards the advancements and prospects in cargo DNA delivery and high-throughput techniques. Additionally, we elucidate the immense potential of genome engineering methods to deepen our understanding of the human gut microbiome and engineer the microorganisms to enhance human health.

CRISPR/Cas-Based Genome Editing for Human Gut Commensal Bacteroides Species.

Bacteroides 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 Bacteroides species. We constructed multiple CRISPR/Cas systems in all-in-one Bacteroides-E. coli shuttle plasmids and systematically evaluated the genome editing efficiency in Bacteroides thetaiotaomicron, 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 Bacteroides species, including Bacteroides fragilis, Bacteroides ovatus, Bacteroides uniformis, and Bacteroides vulgatus. We envision that CRISPR/Cas-based genome editing tools for Bacteroides will greatly facilitate mechanistic studies of the gut commensal and the development of engineered live biotherapeutics.