(711d) Perturbing Bacterial Adaptive Resistance Using Crispr-Cas9 Mediated Gene Targeting: Emerging Applications in Synthetic Biology

The rate at which new antimicrobial resistances are emerging at the clinical level has surpassed our ability to develop novel therapeutics to combat them. 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 these adaptive pathways by controllably up-regulating and down-regulating the expression of genes known to be involved in bacterial tolerance. We employed emerging synthetic biology techniques to investigate gene regulatory networks involved in controlling adaptive resistance. Using deactivated CRISPR-Associated Protein 9 (dCas9), we designed a library of synthetic genetic devices to activate and inhibit native gene expression of stress-response networks. Here we show that in the presence of these synthetic constructs, significant control over bacterial fitness can be achieved during adaptation to sub-minimal inhibitory concentrations of a range of toxins, including disinfectants and antibiotics. The range of adaptive phenotypes observed allowed us to identify individual genes important in the evolution of bacterial tolerance towards specific stress conditions. Moreover, we employed this approach to design constructs targeting multiple genes simultaneously, and observed predominant negative epistasis with severe loss in fitness. 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. The techniques presented in this work offer a novel approach for both impeding the intrinsic adaptive pathways leading to antibiotic resistance and re-sensitizing antibiotic resistant pathogens to traditional therapies employed at the clinical setting.