Operation CRISPR: Deploying precision guided tools to target unique species in a complex microbiome
Rarely in nature do bacterial strains exist in isolation; they form complex microbial communities that interact with various organisms. In these communities, there are few ways to target specific strains effectively. The way people control bacteria is through antibiotics, which for the most part act indiscriminately. But often, problems stem from a single species that has invaded the microbial community; normal balanced microbiomes are not only harmless, but are often crucial and positively contribute to an environment. Because most therapies disrupt this balance, there is an obvious need for specific targeting of species in microbial communities.

Here we take advantage of conjugation, a natural horizontal gene transfer mechanism to target specific strains of bacteria and affect gene expression, which will have a potential for future applications that require targeted cell death.

Our project incorporates bacterial conjugation and CRISPRi (Clustered Regularly Interspaced Short Palindromic Repeats inhibition), a tool repurposed from a natural adaptive immunity system in bacteria, comprised of a catalytically dead Cas9 (dCas9) protein that complexes with guide RNAs (gRNA) complementary to the target bacteria’s DNA sequence. The dCas9 complexes with the gRNA and binds to DNA complimentary to the gRNA and prevents transcription, therefore repressing gene expression.
The combination of conjugation and CRISPRi allows us to create a system capable of both transferring genetic instructions from one cell to another and targeting unique species in a microbial community through a specific gene.