Rationally designed pooled CRISPRi-seq uncovers an inhibitor of bacterial peptidyl-tRNA hydrolase

• Published in: Cell reports (Cambridge) 2024-11, Vol.43 (11), p.114967, Article 114967
• Main Authors: Rahman, A.S.M. Zisanur, Syroegin, Egor A., Novomisky Nechcoff, Julieta, Devarajan, Archit, Polikanov, Yury S., Cardona, Silvia T.
• Format: Article
• Language: English
• Subjects: Anti-Bacterial Agents - pharmacology; antibiotic mechanisms of action; arrayed library; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Burkholderia cenocepacia - drug effects; Burkholderia cenocepacia - genetics; Carboxylic Ester Hydrolases - genetics; Carboxylic Ester Hydrolases - metabolism; Clustered Regularly Interspaced Short Palindromic Repeats - genetics; CP: Microbiology; CRISPR-Cas Systems - genetics; CRISPRi; CRISPRi-seq; Enzyme Inhibitors - pharmacology; essential genes; Gene Library; Genes, Essential; Mutation; peptidyl-tRNA hydrolase; pooled library; Pth; ribosome-associated quality control
• ISSN: 2211-1247; 2211-1247
• DOI: 10.1016/j.celrep.2024.114967

Bacterial mutant libraries with downregulated antibiotic targets 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 constructed an arrayed essential gene mutant library (EGML) in the antibiotic-resistant bacterium Burkholderia cenocepacia using CRISPR interference (CRISPRi). By modeling depletion levels and adjusting knockdown mutant inocula, we rationally designed and optimized a CRISPR interference-mediated pooled library of essential genes (CIMPLE) approaching coverage of the bacterial essential genome with mutant sensitization. We exposed CIMPLE to an uncharacterized bacterial growth inhibitor structurally different from antibiotics and discovered that it inhibits the essential peptidyl-tRNA hydrolase. Overall, CIMPLE leverages the advantages of arrayed and pooled CRISPRi libraries to uncover unexplored targets for antibiotic action. [Display omitted] •CIMPLE is an arrayed CRISPRi-mediated pooled library of bacterial essential genes•Prediction of strain depletion allowed CIMPLE to approach essential genome coverage•CRISPRi-seq with CIMPLE identifies chemical-genetic interactions of antimicrobials•A bacterial growth inhibitor of a new class targets the peptidyl-tRNA hydrolase Bacterial knockdown libraries allow chemical-genetic profiling of antimicrobials, useful in antibiotic drug discovery. Rahman A.S.M.Z. et al. created CIMPLE, a rationally designed pooled CRISPRi library with essential genome coverage. CRISPRi-seq with CIMPLE showed that the target of PHAR(A), a growth inhibitor of a new class, is the bacterial peptidyl-tRNA hydrolase.