CRISPR Hinderance of Adaptation of Organisms (CHAOS) Based Therapeutics

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 sgRNAs 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 manipulated gene expression epistasis using multiplexed dCas9 perturbations in an attempt to curb bacterial adaption to antibiotics. Here we present evidence that multiplexed dCas9 perturbations induce significant negative epistasis, and reduce fitness of Escherichia coli more than 50% during antibiotic exposure. Greater epistasis correlates with increasing nodes within affected protein interaction networks, with perturbations affecting essential metabolic pathways appearing to induce the strongest epistasis. Furthermore, greater negative epistasis corresponds with significantly diminished adaptation rates, thereby increasing antibiotic susceptibility over time. This suggests that multiplexed dCas9 perturbations can restrict adaptation and curb the emergence of antibiotic resistance. Such phenomenon could curtail antibiotic resistance evolution when administered as a co-therapy with last-resort antibiotics.