Using CRISPR-Cas9 to Modify gyrA to Remove Nalidixic Acid Resistance in Clinically Important Pathogens

Abstract

Diseases caused by drug resistant bacteria are one of the leading causes of death in the United States, and they are becoming a pressing public health concern due to the lack of new antibiotics and the evolution of multidrug resistance. Drug resistance is an inequitable quandary, disproportionately affecting minorities and people of lower socio-economic status. Here, we propose to use CRISPR-Cas9-based gene editing to restore antibiotic susceptibility in resistant Escherichia coli. Recent studies have used CRISPR-Cas9 gene editing to successfully target and modify resistance genes to increase antibiotic susceptibility. We chose this system to test proof-of-principle due to the implications towards treatment of nalidixic acid resistant E. coli urinary tract infections in women, which are a growing clinical problem. Potentially, the results of this study could then be applicable to other drug-resistant infections. We bioinformatically designed a CRISPR-Cas9 construct that could revert gyrA mediated nalidixic acid resistance in E. coli, resulting in cells sensitive to antibiotics. Our goal is to develop a proof-of-concept antimicrobial strategy utilizing a CRISPR-Cas9 system delivered via bacteriophage M13 to edit a point mutation in the gyrA gene of nalidixic acid-resistant E. coli, thereby restoring antibiotic sensitivity and contributing to the broader effort to combat antimicrobial resistance. We isolated a series of mutants resistant to nalidixic acid, characterized them, and determined that our target was a single D87G point mutation in gyrA. The following thesis describes the progress made towards building, transforming, and testing this construct.