(142a) Using Synthetic Biology to Engineer Epistasis to Deter Bacterial Adaptation

Antibiotic resistance continues to be a major public health concern, with fewer new antibiotics being developed to combat increasingly resistant bacteria. Previous antibiotic resistance research has focused primarily on a “bottom up” approach – identifying specific genetic changes that confer resistance, and developing new reactionary therapies to circumvent these changes. A “top down” approach – understanding how such resistance emerges in the first place – would enable a more preventative strategy to fight resistance. To this end, work in our lab has focused on non-genetic factors such as changes at the epigenetic level that contribute to the acquisition of antibiotic resistance.

Here we present the use of deactivated CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) –Cas9 constructs to selectively inhibit or activate sets of multiple genes in Escherichia coli. Simultaneous gene expression perturbations induced in this fashion demonstrate synthetic engineering of epigenetic epistatic interactions – the combined impact of changes in two or more genes on the overall fitness of the cell. We systematically investigate all possible combinations of gene perturbations within two sets of genes. We show strong evidence that as more genes are perturbed, the cell’s ability to adapt decreases as quantified by decreased fitness during antibiotic exposure and a slowed rate of increase in the Minimum Inhibitory Concentration of said antibiotic over time. This work demonstrates a novel approach to engineer control over the emergence of antibiotic resistance through synthetic induction of negative epigenetic epistatic interactions.