Disrupting Bacterial Adaptive Resistance Using dCas9 and dCas9-? Gene Perturbations: Emerging CRISPR Applications in Synthetic Biology

The rate at which new antimicrobial resistances evolve has surpassed our ability to develop novel therapeutics to combat them. This has engendered a crisis that necessitates a greater understanding of how resistance emerges. Recent studies have suggested that, when exposed to antimicrobials, bacteria enter an “adaptive resistance” state by exploring multiple pathways sampling a dynamic gene regulatory space.

We investigated the impact of non-genetic factors in bacterial adaptive pathways by controllably up-regulating and down-regulating the expression of genes known to be involved in tolerance. To this end, we employed emerging synthetic biology techniques to investigate gene regulatory networks involved in controlling adaptive resistance. Using deactivated CRISPR-Cas9 (dCas9) and dCas9 fused with the omega subunit of RNA polymerase (dCas9-ω), we designed and characterized a library of synthetic genetic devices to activate and inhibit native gene expression of stress-response networks.

Here we demonstrate the effect these constructs have on Escherichia coli phenotypes during exposure to sub-minimal inhibitory concentrations of a range of toxins, including antibiotics and disinfectants. We show that significant control over bacterial fitness and growth phenotypes can be achieved, reducing (or improving) the cells’ ability to proliferate during stress. The range of adaptive phenotypes observed allowed us to identify individual genes important in the evolution of bacterial tolerance towards specific stress conditions. We ultimately discern genes responsible for the development of bacterial tolerance, providing direction for novel therapeutics which can impede intrinsic adaptive pathways leading to antimicrobial resistance.