Precision Editing of Non-Model Bacteria in Native Microbiomes Using Mobile CRISPR-Associated Transposases

Deep sequencing shed light on the vast microbial diversity in nature, changing understanding of the crucial roles that microbiomes play in environments. Knowledge of gene functions comes from manipulating the DNA of species in isolation from their natural communities, yet fewer than 1% of microbes are culturable and even fewer are genetically tractable. Moreover, in nature, microbes live in open, dynamic, complex habitats that are difficult to recapitulate in a laboratory. To address these shortcomings, we developed a novel methodology that enables in vivo precision microbiome engineering by leveraging highly mobile vectors to effectively shuttle payloads between diverse microbes, with programmable transposases to catalyze targeted DNA integration, a technology we term MAGICAST. We optimized delivery and activity of CRISPR-associated transposases in 4 non-model Bacteroides species isolated from humans, towards development of a suite of highly mobile gene editing systems. We next introduced novel payloads with high specificity (>90% on-target), using computationally designed metagenomic spacers, and temporal persistence in Bacteroides species within the gut microbiome of live mice. We used this to enable genetic tagging of target species in vivo, to facilitate their isolation from the environment and investigation into bile acid metabolism. This work defines a new paradigm for genetic studies of gut microbiome and establishes a pipeline for genetic engineering of bacteria from nature into the laboratory. Future work will focus on expanding this technology into other microbiomes (e.g., clinical, extremophilic) to enable discovery of novel functions from undomesticated bacteria with implications in biotechnological, therapeutic, and environmental fields.