Utilizing massively parallel CRISPRi assays to investigate antibiotic tolerance

CRISPR based inteference assays are highly useful screens that have been developed for use in mammalian cells but they have not found as much utility in bacteria due to the toxcitity of the cas9 the primary workhorse for CRISPR systems. We have devevloped a system that uses an inactivated form of cas12a to rapidly screen the entire Escherichia coli genome using gene knockdown in a wide range of conditions.

We demonstrate that this approach can be used to investigate antibiotic tolerance and persisters by controlling the cas protein using an inducible promoter and thereby allowing for the reversal of growth based effects.

In addition the mechanism can be easily coupled with fluorescence markers and single gene targets can be made allowing for microscopy investigation of the physiological effects of th e cells.

We further use our technique to explore antibiotic tolerance in Escherichia coli on exposure to Naladixic acid, Carbenicillin and Tetracycline and highlight several gene categories that have gone otherwise unrecognised as being important in antibiotic survival. We further use single cell microscopy using microfluidics to attempt to illuminate the difference between different targets and their recovery using our knockdown system.