Abstract
SummaryAntibacterial agents target the products of essential genes but rarely achieve complete target inhibition. Thus, the all-or-none definition of essentiality afforded by traditional genetic approaches fails to discern the most attractive bacterial targets: those whose incomplete inhibition results in major fitness costs. In contrast, gene “vulnerability” is a continuous, quantifiable trait that relates the magnitude of gene inhibition to the effect on bacterial fitness. We developed a CRISPR interference-based functional genomics method to systematically titrate gene expression in Mycobacterium tuberculosis (Mtb) and monitor fitness outcomes. We identified highly vulnerable genes in various processes, including novel targets unexplored for drug discovery. Equally important, we identified invulnerable essential genes, potentially explaining failed drug discovery efforts. Comparison of vulnerability between the reference and a hypervirulent Mtb isolate revealed incomplete conservation of vulnerability and that differential vulnerability can predict differential antibacterial susceptibility. Our results quantitatively redefine essential bacterial processes and identify high-value targets for drug development.
Highlights
Essential bacterial genes orchestrate core biological processes and represent the targets of most antibacterial drugs
We used the ability of Sth1dCas9 to recognize non-canonical protospacer adjacent motifs (PAMs) that lead to a gradient of target knockdown (Rock et al, 2017)
This CRISPR interference (CRISPRi) library is biased toward single guide RNAs (sgRNAs) targeting predicted in vitro essential genes (DeJesus et al, 2017) because knockdown of these genes is predicted to reduce bacterial fitness and enable vulnerability quantification
Summary
Essential bacterial genes orchestrate core biological processes and represent the targets of most antibacterial drugs. There is growing appreciation that partial inhibition of some essential genes results in strong fitness costs, whereas other essential genes can tolerate substantial inhibition with little effect on bacterial fitness (Hawkins et al, 2020; Jost et al, 2020; Keren et al, 2016; Wei et al, 2011). This expression-fitness relationship is defined as gene vulnerability (Barry et al, 2009; Wei et al, 2011). Despite the growing appreciation of variable expression-fitness relationships, quantification of gene vulnerability remains intractable with traditional genetic approaches and has yet to be defined systematically for any pathogen
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