Utilizing massively parallel CRISPRi assays to investigate persistence during antibiotic exposure

Bacterial persisters are characterised by a subgroup of cells within a population that have significant tolerance to antibiotics. This tolerance is primarily achieved due to the reduction of cell growth and metabolic activity which allows the bacteria to wait out the stress that would otherwise kill the cell and enabling the population to regrow once the stress is removed. The process has previously been attributed, at least in part, to toxin-antitoxin systems that allow the cell to inactivate itself in a probabilistic manner, however it is still unclear as to the global mechanisms that cause this.

Using our previously developed massively parallel CRISPRi assay[1], we explore the entire Escherichia coli genome identifying genes or operons that both enhance or impair the persistence mechanism. We show that the CRISPRi can replace the function of the toxin-antitoxin systems, inhibiting the cells metabolism and improving survival under certain antibiotics. We follow several highlighted genes, both beneficial and detrimental, using single cell microscopy in a microfluidic device to quantiify the phenotypic changes during and after antibiotic exposure.

[1] https://doi.org/10.1073/pnas.1918685117