Precise and tunable genetic tools are essential for high-throughput functional genomics. To address this need in the important gram-negative pathogen, we developed and characterized a tightly regulated CRISPR-interference (CRISPRi) system that enables precise and tunable repression of essential genes. The system utilizes a rhamnose-inducible promoter to control both the-derived dCas9 and gene-specific sgRNAs, each encoded on separate plasmids for modularity and efficiency. The combination of tight regulation and high conjugation efficiency facilitated the rapid and facile construction of strains with regulated depletion of 16 essential genes spanning diverse pathways. Comparison of phenotypes across the different genetically depleted strains, including growth rate, susceptibility to antibiotics, and changes in transcriptional programs, revealed novel aspects of gene function or small-molecule mechanism of action. Finally, the rhamnose-inducible CRISPRi system supports the generation and stable maintenance of pooled mutant libraries, thereby paving the way for future genome-wide, systematic assessment of individual gene vulnerabilities, which will provide critical insights for target prioritization in antibiotic discovery efforts against this recalcitrant pathogen.IMPORTANCECRISPR-interference (CRISPRi) has become an invaluable tool for studying genetics. In particular, the ability to knockdown (KD) genes enables the study of essential genes and their role in cell survival. However, a tightly regulated gene KD system is required to gain valuable insights into these vulnerable genes by virtue of their essentiality. We report a tightly regulated CRISPRi system to study the biology of essential gene perturbations in, an important gram-negative pathogen that causes severe infections and is increasingly resistant to current antibiotics. This system enables characterization of both chemical genetic interactions between small molecules and specific gene depletions and the impact of genetic perturbations on transcriptional networks. Genetic perturbations using CRISPRi can thus further our understanding of basic biology with translation toward future antimicrobial development. Pseudomonas aeruginosa Streptococcus pasteurianus Pseudomonas aeruginosa
