(619c) CHAOS: A Novel Strategy for Restricting Bacterial Evolution By Inducing Epistatic Interactions

The emergence of multidrug-resistant “superbugs” continues to pose a looming global health crisis that necessitates novel therapeutic strategies to curb their threat. The advent of CRISPR technologies has expanded our capacity to investigate such strategies. In particular, deactivated versions of the Cas9 (dCas9) enzyme enable relatively facile manipulation of gene expression in a multiplexed fashion that was once prohibitively difficult and expensive to perform.

Employing dCas9 with multiplexed gRNAs permits manipulation of specific bacterial gene expression profiles during the critical stage of early antibiotic exposure. Previous work has consistently revealed that upon exposure to antibiotics, bacterial transcriptomes adjust to new levels that impart improved fitness phenotypes. This arises from the natural variation in gene expression across the population that exists as a bet-hedging tool to enable subsets of the population to survive during sudden environmental changes. We postulated that this heterogeneity can act as a “double-edged sword” – if a subset of random expression states provides an advantage to stress, then another subset will similarly prove disadvantageous.

Whether or not a particular expression state is advantageous or deleterious depends upon the degree of epistasis between each change. Epistasis describes the nonlinear interactions between two or more simultaneous genetic changes and is widely recognized for its role in shaping evolutionary trajectories. While research into epistasis has largely focused on simultaneous mutations, similar effects have been observed from changes in gene expression.

In this study, we develop a novel strategy for deterring the evolution of antibiotic resistance, dubbed the Controlled Hindrance of Adaptation of OrganismS or “CHAOS”. This approach involves the systematic manipulation of gene expression in a multiplexed fashion to induce negative epistatic interactions. We begin by demonstrating that systematically combining dCas9 perturbations causes significant negative epistasis, cutting fitness of Escherichia coli up to 2-fold during antibiotic exposure. We then show that this induced negative epistasis restricts the rate at which antibiotic minimum inhibitory concentration (MIC) increases during multiple days of increasing exposure. Multiplexed perturbations consistently decrease MICs in a clinically relevant timeframe of two weeks. Finally, we demonstrate that alternative perturbation approaches using Peptide Nucleic Acids can be used to replicate this epistatic effect in clinically isolated multi-drug resistant bacteria, demonstrating its therapeutic potential.

Together, these results suggest that CHAOS multiplexed gene expression perturbations can restrict adaptation, and help to curb the emergence of antibiotic resistance.