Rationally Designed Pooled CRISPRi-seq Uncovers a Novel Inhibitor of Bacterial Peptidyl-tRNA Hydrolase

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. Summary Bacterial mutant libraries in which antibiotic targets are downregulated are useful tools to functionally characterize novel antimicrobials. These libraries are used for chemogenetic profiling as target-compound interactions can be inferred by differential fitness of mutants during pooled growth. Mutants that are functionally related to the antimicrobial mode of action are usually depleted from the pool upon exposure to the drug. Although powerful, this method can fail when the unequal proliferation of mutant strains before exposure causes mutants to drop out of the library pool. To address this issue, we constructed an arrayed essential gene mutant library (EGML) in the antibiotic-resistant bacterium Burkholderia cenocepacia using CRISPR interference (CRISPRi) and analyzed the growth parameters of individual mutant strains. We then modelled depletion levels during pooled growth and used the model to rationally design an optimized CRISPR interference-mediated pooled library of essential genes (CIMPLE). By adjusting the initial inoculum of the knockdown mutants, we achieved coverage of the bacterial essential genome with mutant sensitization. We exposed CIMPLE to a recently discovered antimicrobial of a novel class and discovered it inhibits the peptidyl-tRNA hydrolase, an important component of ribosome recycling during translation. In summary, we demonstrate the utility of CIMPLE and CRISPRi-seq to uncover the mechanism of action of novel antimicrobial compounds. ### Competing Interest Statement The authors have declared no competing interest.