CRISPR Perturbation of Gene Expression Alters Bacterial Fitness under Stress and Reveals Underlying Epistatic Constraints

Abstract

The evolution of antibiotic resistance has engendered an impending global health crisis that necessitates a greater understanding of how resistance emerges. The impact of nongenetic factors and how they influence the evolution of resistance is a largely unexplored area of research. Here we present a novel application of CRISPR-Cas9 technology for investigating how gene expression governs the adaptive pathways available to bacteria during the evolution of resistance. We examine the impact of gene expression changes on bacterial adaptation by constructing a library of deactivated CRISPR-Cas9 synthetic devices to tune the expression of a set of stress-response genes in Escherichia coli. We show that artificially inducing perturbations in gene expression imparts significant synthetic control over fitness and growth during stress exposure. We present evidence that these impacts are reversible; strains with synthetically perturbed gene expression regained wild-type growth phenotypes upon stress removal, while maintaining divergent growth characteristics under stress. Furthermore, we demonstrate a prevailing trend toward negative epistatic interactions when multiple gene perturbations are combined simultaneously, thereby posing an intrinsic constraint on gene expression underlying adaptive trajectories. Together, these results emphasize how CRISPR-Cas9 can be employed to engineer gene expression changes that shape bacterial adaptation, and present a novel approach to synthetically control the evolution of antimicrobial resistance.

Multiplexed deactivated CRISPR-Cas9 gene expression perturbations deter bacterial adaptation by inducing negative epistasis

“…The first set included activation of four universal stress response genes important for adaptation 27,28 : mutS (DNA mismatch repair), soxS (SOX pathway regulator), tolC (multidrug efflux pump), and recA (SOS response activator) (Fig. 2a).…”

“…2a). These four genes were determined in our previous work to be particularly important during bacterial adaptation to a broad range of antibiotics and other stressors, and we reasoned that upregulating these genes would serve as the most likely avenue influencing adaptation 22,27,28 . Furthermore, we hypothesized that activating these genes would serve as the worst-case scenario, as we expect broad upregulation of stress response to provide multiple avenues of adaptive escape and encourage positive epistasis.…”

“…Multiplexed perturbations could thus constrain an organism from reaching an adapted state, and offers a tangible avenue towards curbing the ability of bacteria to adapt to antibiotic treatment. Previous work in our lab investigating five strains harboring multiplexed gene expression perturbations 25,26 showed their tendency to exhibit detrimental growth phenotypes 27,28 . Additionally, independently beneficial mutations of sequences related to gene expression were found to cause deleterious fitness impacts when combined jointly, further supporting the credibility of this hypothesis 29 .…”

Origin, Evolution, and Loss of Bacterial Small RNAs

“…Given that the abundance of mRNA targets as well as the consequence of mRNA regulation will be different based on the organism’s stage of growth, it is highly likely that differential expression patterns indicate diverged sRNA function. This is supported by the observation that differences in gene expression affect bacterial fitness and adaptation during exposure to stress (50). …”

In situ genotyping of a pooled strain library after characterizing complex phenotypes

“…For example, one can make pooled dCas9 libraries based on plasmids harboring both a genotype-identifying barcode and a sgRNA gene (Dixit et al, 2016;Jaitin et al, 2016;Peters et al, 2016;Garst et al, 2017;Otoupal et al, 2017) for labeling genetic loci (Chen et al, 2013) or knocking down/activating genes throughout the chromosome. Alternatively, pooled A Examples of channels and cells in the custom-made microfluidic device which are imaged in both phase contrast (top) and fluorescence microscopy (bottom).…”