Engineering a CRISPR Interference System To Repress a Class 1 Integron in Escherichia coli.

Microbial multidrug resistance (MDR) poses a huge threat to human health. Bacterial acquisition of MDR relies primarily on class 1 integron-involved horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs). To date, no strategies other than the use of antibiotics can efficiently cope with MDR. Here, we report that an engineered CRISPR interference (CRISPRi) system can markedly reduce MDR by blocking a class 1 integron in Using CRISPRi to block plasmid R388 class 1 integron, recombinants showed halted growth upon exposure to relevant antibiotics. A microplate alamarBlue assay showed that both subgenomic RNAs (sgRNAs) R3 and R6 led to 8- and 32-fold decreases in half-maximal inhibitory concentrations (IC) for trimethoprim and sulfamethoxazole, respectively. Reverse transcription and quantitative PCR (RT-qPCR) revealed that the strain employing sgRNA R6 exhibited 97% and 84% decreases in the transcriptional levels of the cassette and , two typical ARGs, respectively. RT-qPCR analysis also demonstrated that the strain recruiting sgRNA R3 showed a 96% decrease in the transcriptional level of , and a conjugation assay revealed a 1,000-fold decrease in HGT rates of ARGs. Overall, the sgRNA R3 targeting the 31 bp downstream of the Pc promoter on the nontemplate strand outperformed other sgRNAs in reducing integron activity. Furthermore, this CRISPRi system is reversible, genetically stable, and titratable by varying the concentration of the inducer. To our knowledge, this is the first report on exploiting a CRISPRi system to reduce the class 1 integron in This study provides valuable insights for future development of CRISPRi-based antimicrobial agents and cellular therapy to suppress MDR.