Targeted elimination of Vancomycin resistance gene vanA by CRISPR-Cas9 system.
Shuan Tao, Chunwei Hu, Yewei Fang, He Zhang, Yao Xu, Lin Zheng, Luyan Chen, Wei Liang
Author Information
Shuan Tao: Department of Clinical Laboratory, The First Affiliated Hospital of Ningbo University, No 59. Liuting Road, Haishu District, Ningbo, 315010, China.
Chunwei Hu: The Biobank of The First Affiliated Hospital of Ningbo University, Ningbo, China.
Yewei Fang: Department of Clinical Laboratory, The First Affiliated Hospital of Ningbo University, No 59. Liuting Road, Haishu District, Ningbo, 315010, China.
He Zhang: Bengbu Medical College, Bengbu, China.
Yao Xu: School of Medicine, Ningbo University, Ningbo, China.
Lin Zheng: Department of Clinical Laboratory, The First Affiliated Hospital of Ningbo University, No 59. Liuting Road, Haishu District, Ningbo, 315010, China.
Luyan Chen: Department of Blood Transfusion, The First Affiliated Hospital of Ningbo University, Ningbo, China.
Wei Liang: Department of Clinical Laboratory, The First Affiliated Hospital of Ningbo University, No 59. Liuting Road, Haishu District, Ningbo, 315010, China. hslwys@163.com.
OBJECTIVE: The purpose of this study is to reduce the spread of the vanA gene by curing the vanA-harboring plasmid of vancomycin-resistant using the CRISPR-Cas9 system. METHODS: Two specific spacer sequence (sgRNAs) specific was designed to target the vanA gene and cloned into plasmid CRISPR-Cas9. The role of the CRISPR-Cas system in the plasmid elimination of drug-resistance genes was verified by chemically transformation and conjugation delivery methods. Moreover, the elimination efficiency in strains was evaluated by plate counting, PCR, and quantitative real-time PCR (qPCR). Susceptibility testing was performed by broth microdilution assay and by Etest strips (bioMérieux, France) to detect changes in bacterial drug resistance phenotype after drug resistance plasmid clearance. RESULTS: In the study, we constructed a specific prokaryotic CRISPR-Cas9 system plasmid targeting cleavage of the vanA gene. PCR and qPCR results indicated that recombinant pCas9-sgRNA plasmid can efficiently clear vanA-harboring plasmids. There was no significant correlation between sgRNA lengths and curing efficiency. In addition, the drug susceptibility test results showed that the bacterial resistance to vancomycin was significantly reduced after the vanA-containing drug-resistant plasmid was specifically cleaved by the CRISPR-Cas system. The CRISPR-Cas9 system can block the horizontal transfer of the conjugated plasmid pUC19-vanA. CONCLUSION: In conclusion, our study demonstrated that CRISPR-Cas9 achieved plasmid clearance and reduced antimicrobial resistance. The CRISPR-Cas9 system could block the horizontal transfer of plasmid carrying vanA. This strategy provided a great potential to counteract the ever-worsening spread of the vanA gene among bacterial pathogens and laid the foundation for subsequent research using the CRISPR-Cas9 system as adjuvant antibiotic therapy.
