Rahman, A. S. M. Z.; Syroegin, E. A.; Novomisky Nechcoff, J.; Timmerman, L.; Devarajan, A.; Polikanov, Y. S.; Cardona, S. T.

Pooled knockdown libraries of essential genes are useful tools for elucidating the mechanisms of action of antibacterial compounds, a pivotal step in antibiotic discovery. However, achieving genomic coverage of antibacterial targets poses a challenge due to the uneven proliferation of knockdown mutants during pooled growth, leading to the unintended loss of important targets. To overcome this issue, we introduce CIMPLE (CRISPRi-mediated pooled library of essential genes), a rationally designed pooled knockdown library built in a model antibiotic-resistant bacteria, Burkholderia cenocepacia. By analysing growth parameters of clonal knockdown populations of an arrayed CRISPRi library, we predicted strain depletion levels during pooled growth and adjusted mutant relative abundance, achieving genomic coverage of antibacterial targets during antibiotic exposure. We demonstrate the utility of CIMPLE for chemogenetic profiling of known antibacterials and a previously discovered bacterial growth inhibitor of a new class. CRISPRi-Seq with CIMPLE, followed by biochemical validation, revealed that the novel compound targets the ribosome-associated quality control apparatus (RQC) by inhibiting the peptidyl-tRNA hydrolase (Pth). Overall, CIMPLE leverages the advantages of arrayed and pooled CRISPRi libraries to uncover unexplored targets for antibiotic action.