Rationale: The emergence of bacterial pathogens resistant to most antibiotics?even those of last resort?is an urgent threat to human health, and underscores the need for new antibiotics with novel cellular targets. Antibiotics target essential cellular processes. Understanding how different essential processes interact in the cell, uncovering new essential pathways, and mapping how environmental perturbations affect essential processes will allow us to identify synergistic antibiotic therapies and define new cellular targets for antibiotics. Essential processes have been difficult to study in cells because disrupting them is lethal. However, the advent of CRISPRi (clustered regularly-interspaced short palindromic repeats interference)?a transformative new technology for performing gene knockdowns, opens to door for studying essential processes. CRISPRi consists of only two components?a non-cutting variant of the Cas9 endonuclease (dCas9) and a single guide RNA (sgRNA), which form a complex on target DNA, repressing gene expression. CRISPRi is simple to implement, scalable, and organism independent. Using CRISPRi adapted for Bacillus subtilis, I comprehensively identified interactions between antibiotics and essential genes that exposed previously unknown antibiotic-gene synergies, and identified the direct target of an uncharacterized antibiotic. Objective: During my K22, I propose to develop a suite of CRISPRi-based platforms to: transfer CRISPRi to pathogenic bacteria; perform genetic interaction (double knockdown) analysis to find new essential pathways, and identify genes involved in signal transduction to characterize and exploit new vulnerabilities in bacterial pathogens.
Antibiotic-resistant bacterial pathogens are directly responsible for the deaths of tens of thousands of people in the United States each year, and the problem is growing worse over time because we have not been able to find new weaknesses in pathogens that we can target with antibiotics. I am developing new strategies to identify such weaknesses by probing genes that are central to bacterial life on a massive scale. The knowledge I gain from my studies will be directly relevant for controlling bacterial pathogens and determining how antibiotics work.
