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

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Microbial multidrug resistance (MDR) is a significant global health threat.
• Horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs) via class 1 integrons is a primary mechanism driving MDR.
• Current strategies to combat MDR are limited, with antibiotic use being the primary intervention.

Purpose of the Study:

• To investigate the efficacy of an engineered CRISPR interference (CRISPRi) system in reducing MDR in Escherichia coli.
• To determine the impact of CRISPRi-mediated class 1 integron inhibition on the expression of ARGs and HGT rates.
• To evaluate the potential of CRISPRi as a novel therapeutic strategy against MDR.

Main Methods:

• An engineered CRISPRi system was utilized to target and block the class 1 integron in Escherichia coli.
• Microplate alamarBlue assays were performed to determine changes in antibiotic susceptibility (IC50 values).
• Reverse transcription and quantitative PCR (RT-qPCR) were employed to measure the transcriptional levels of ARGs and integrase genes.
• Conjugation assays were conducted to quantify the rates of ARG HGT.

Main Results:

• CRISPRi targeting of the class 1 integron significantly decreased antibiotic resistance, with 8- and 32-fold reductions in IC50 for trimethoprim and sulfamethoxazole, respectively.
• Transcriptional levels of key ARGs (dfrB2 and sul1) were reduced by up to 97% and 84%, respectively.
• CRISPRi effectively reduced the expression of the integrase gene (intI1) by 96% and decreased ARG HGT rates by 1,000-fold.
• sgRNA R3, targeting downstream of the Pc promoter, demonstrated superior inhibition of integron activity.

Conclusions:

• Engineered CRISPRi systems can effectively suppress class 1 integron activity in Escherichia coli, leading to reduced MDR.
• This CRISPRi-based approach offers a reversible, genetically stable, and titratable method to combat ARGs and HGT.
• This study presents the first report of using CRISPRi to reduce class 1 integrons in E. coli, paving the way for novel CRISPRi-based antimicrobial therapies.