has abundant signaling systems that exquisitely control its antibiotic resistance in response to different environmental cues. Understanding the regulation of antibiotic resistance will provide important implications for precise antimicrobial interventions. However, efficient genetic tools for functional gene characterizations are sometimes not available, particularly, in clinically isolated strains. Here, we established a type I-F CRISPRi (CSYi) system for programmable gene silencing. By incorporating anti-CRISPR proteins, this system was even applicable to bacterial hosts encoding a native type I-F CRISPR-Cas system. With the newly developed gene-silencing system, we revealed that the response regulator CzcR from the zinc (Zn)-responsive two-component system CzcS/CzcR is a repressor of efflux pumps MexAB-OprM and MexGHI-OpmD, which inhibits the expression of both operons by directly interacting with their promoters. Repression of MexAB-OprM consequently increases the susceptibility of to multiple antibiotics such as levofloxacin and amikacin. Together, this study provided a simple approach to study gene functions, which enabled us to unveil the novel role of CzcR in modulating efflux pump genes and multidrug resistance in . IMPORTANCE is a ubiquitous opportunistic pathogen frequently causing chronic infections. In addition to being an important model organism for antibiotic-resistant research, this species is also important for understanding and exploiting CRISPR-Cas systems. In this study, we established a gene-silencing system based on the most abundant type I-F CRISPR-Cas system in this species, which can be readily employed to achieve targeted gene repression in multiple bacterial species. Using this gene-silencing system, the physiological role of Zn and its responsive regulator CzcR in modulating multidrug resistance was unveiled with great convenience. This study not only displayed a new framework to expand the abundant CRISPR-Cas and anti-CRISPR systems for functional gene characterizations but also provided new insights into the regulation of multidrug resistance in and important clues for precise anti-pseudomonal therapies.