UTILIZATION OF CRISPR INTERFERENCE IN IDENTIFICATION OF NOVEL ANTIBIOTIC TARGET IN BURKHOLDERIA PSEUDOMALLEI

Abstract

"Melioidosis is a life-threatening disease caused by Burkholderia pseudomallei. In the absence of vaccines and the limitation of antibiotic therapy due to the inherent antibiotic resistance of this bacterium, new drugs are needed to improve treatment and prevention. Since
B. pseudomallei is classified as a select agent, genetic tools for genetic manipulations of this pathogen are limited. In the characterization of gene functions, gene deletion mutation using an allelic exchange is among the most common genetic manipulation tools available to this microorganism, but not all genes can be deleted, especially an essential gene. Essential gene is a gene encoding bacterial components essential for growth and survival which is an attractive target for the development of novel antibiotics. In this study, the CRISPR interference (CRISPRi) technology was used to identify the novel essential genes in B. pseudomallei strain 1026b. BP1026B_I0557, a gene encoding putative exported proteins with unknown functions, was selected as a candidate essential gene in this study. We confirmed that BP1026B_I0557 is essential for B. pseudomallei growth and survival by depleting its expression using CRISPRi. Unfortunately, no growth defect was observed in strains expressing sgRNA targeting BP1026B_I0557, which indicated that BP1026B_I0557 might not be essential."