Utilizing CRISPR/Cas9 Gene Editing Techniques for Knockout of Genes in Escherichia coli Bacteria

Abstract

This research explored the potential of CRISPR/Cas9 to knockout genes in Escherichia coli that encode for virulence factors such as efflux pumps and biofilms. Specifically, this study focused on the efficacy of CRISPR/Cas9 plasmid BPK764 at editing red fluorescent protein (RFP) plasmids and endogenous E. coli biofilm gene LuxS. This study required the design of single guide RNA (sgRNA) oligos which target genes of interest. These were then ligated into the plasmid via linearization of BPK764, gel extraction, and annealing. These plasmids were grown on Luria-Bertani (LB) agar and sequenced to confirm insertion. The edited plasmids were then electroporated into T7 Express E. coli cells and grown on LB plates to select colonies. These colonies were picked, induced, and grown on LB plates with selective antibiotics to determine if the Cas9 was able to edit the target gene. The experimental plates had editing efficiencies of 98%, 88.6%, and 100%. The control plates all had editing efficiencies of 0.0%. Sequencing data from LuxS suggested editing by BPK764, however further testing is necessary. The data collected from this study suggests that CRISPR/Cas9 plasmids, specifically BPK764, can be utilized to knockout genes in E. coli. Further research is required to determine if BPK764 can completely knockout endogenous E. coli genes.

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