Phage-delivered CRISPR-Cas9 for strain-specific depletion and genomic deletions in the gut microbiome

Abstract

<jats:title>Abstract</jats:title><jats:p>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 <jats:italic>in vivo</jats:italic> are currently lacking. Here, we show that bacteriophage can be used as a vector for delivery of plasmid DNA to bacteria colonizing the gastrointestinal tract, using filamentous phage M13 and <jats:italic>Escherichia coli</jats:italic> engrafted in the gut microbiota of mice. We employ M13 to deliver CRISPR-Cas9 for sequence-specific targeting of <jats:italic>E. coli</jats:italic> 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 <jats:italic>E. coli</jats:italic> 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 <jats:italic>in vivo</jats:italic> 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 <jats:italic>in vivo</jats:italic> microbiome engineering using phage and a proof-of-concept for the establishment of phage-based tools for a broader panel of human gut bacteria.</jats:p>

CRISPR-based antimicrobials to obstruct antibiotic-resistant and pathogenic bacteria

“…The development of CRISPR systems as selective and titratable antimicrobials requires further study to determine therapeutic efficacy. To date, only 9 studies have included in vivo infection or colonization models in their experimental design to test the efficacy of CRISPR-based antimicrobials [ 7 , 11 , 14 , 16 , 17 , 21 , 25 , 32 , 33 ]. Of those studies, only 2 have compared the efficacy of CRISPR/Cas antimicrobials to traditional antibiotics [ 11 , 21 ].…”

Discovery and mining of enzymes from the human gut microbiome

“…The clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR-Cas) system can edit the bacterial genome in a high-throughput and targetoriented manner [70][71][72]. A platform using phage-delivered CRISPR-Cas9 has been applied to deplete one strain in a sequence-specific manner from complex gut communities [73], and eventually, we envision the use of this approach to study the function of specific genes or pathways and assess the effects of depleting particular microorganisms from the gut microbiota.…”

Mining genomes to illuminate the specialized chemistry of life

“…The biggest drawback to this approach is that it can be difficult or impossible to manipulate genes in non-model organisms, but thankfully this situation is improving thanks to the broad host range of CRISPR-Cas9 technologies. The emergence of CRISPR-Cas9-based 'microbiome editing' technologies 191,192 has even made it possible to knock out genes in specific members of a complex microbiome.…”