Phage-delivered CRISPR-Cas9 for strain-specific depletion and genomic deletions in the gut microbiota
The recognition that the gut microbiome has a profound influence on human health and disease has spurred efforts to manipulate gut microbial community structure and function. Though various strategies for microbiome engineering have been proposed, methods for phage-based genetic manipulation of resident members of the gut microbiota in vivo are currently lacking. Here, we show that bacteriophage can be used as a vector for delivery of plasmid DNA to bacteria colonizing the gastrointestinal
... strointestinal tract, using filamentous phage M13 and Escherichia coli engrafted in the gut microbiota of mice. We employ M13 to deliver CRISPR-Cas9 for sequence-specific targeting of E. coli leading to depletion of one strain of a pair of fluorescently marked isogenic strains competitively colonizing the gut. We further show that when mice are colonized by a single E. coli strain, it is possible for M13-delivered CRISPR-Cas9 to induce genomic deletions that encompass the targeted gene. Our results suggest that rather than being developed for use as an antimicrobial in the gut microbiome, M13-delivered CRISPR-Cas9 may be better suited for targeted genomic deletions in vivo that harness the robust DNA repair response of bacteria. With improved methods to mitigate undesired escape mutations, we envision these strategies may be developed for targeted removal of strains or genes present in the gut microbiome that are detrimental to the host. These results provide a highly adaptable platform for in vivo microbiome engineering using phage and a proof-of-concept for the establishment of phage-based tools for a broader panel of human gut bacteria.