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APPLICATION OF CRISPR TO REMOVE MULTIPLE BETA-LACTAMASE GENE VARIANTS**

Abstract

In modern healthcare settings, antibiotic resistant bacteria are increasingly becoming an issue. Bacteria are becoming resistant at a faster rate than we can develop drugs for, which has lead to alternatives being sought out. A solution could be to remove resistance using gene editing technology like CRISPR. This study focused on β-lactamases specifically, which confer resistance to some of the most common drugs. These enzymes work by hydrolyzing the β-lactam ring in penicillin and cephalosporin class antibiotics, and are divided into four classes based on protein sequence. It can be assumed that the active site of β-lactamases within each class are similar, supported by the fact that 3 of the 4 classes all use a serine amino acid as the core component of the active site. By applying CRISPR-Cas9, this paper examines the potential to remove multiple gene variants using a single guide RNA. The plasmids pHflu2 and pETcon-NK were used, which contain the genes for β-lactamases TEM-116 and TEM-1 and chloramphenicol and kanamycin resistance, respectively. TEM-116 and TEM-1 are variants of the TEM gene, which is a class A β-lactamase. These plasmids were inserted via electroporation into the Escherichia coli strain LC-EO3, which contains a Cas9 gene, to generate two cultures of LC-EO3 each containing one of the β-lactamase plasmids. A plasmid containing a guide RNA that targets TEM-116 was synthesized by Sigma Aldrich and called pAmpR (SRID). Plasmid pAmpR (SRID) also contains an ampicillin resistance cassette. The LC-EO3 strains containing pHflu2 and pETcon-NK will be transformed by electroporation with pAmpR (SRID). These strains will then be replica plated on Luria agar containing ampicillin, chloramphenicol or kanamycin. It is expected that the Cas9, guided by one sgRNA will cause double strand breaks in pHflu2 and potentially pETcon-NK, causing the bacteria to become sensitive to the antibiotics chloramphenicol and kanamycin.

Acknowledgements

YHC Undergraduate research initiative

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