Re-engineering a mobile-CRISPR/Cas9 system for antimicrobial resistance gene curing and immunization in Escherichia coli

• Published in: Journal of antimicrobial chemotherapy 2021-12, Vol.77 (1), p.74-82
• Main Authors: He, Yu-Zhang, Kuang, Xu, Long, Teng-Fei, Li, Gong, Ren, Hao, He, Bing, Yan, Jin-Ru, Liao, Xiao-Ping, Liu, Ya-Hong, Chen, Liang, Sun, Jian
• Format: Article
• Language: English
• Subjects: Anti-Bacterial Agents - pharmacology; CRISPR-Cas Systems; Drug Resistance, Bacterial - genetics; Escherichia coli - genetics; Escherichia coli Proteins - genetics; Plasmids - genetics
• ISSN: 0305-7453; 1460-2091
• DOI: 10.1093/jac/dkab368

In this study, we developed an IS26-based CRISPR/Cas9 system as a proof-of-concept study to explore the potential of a re-engineered bacterial translocatable unit (TU) for curing and immunizing against the replication genes and antimicrobial resistance genes. A series of pIS26-CRISPR/Cas9 suicide plasmids were constructed, and specific guide RNAs were designed to target the replication gene of IncX4, IncI2 and IncHI2 plasmids, and the antibiotic resistance genes mcr-1, blaKPC-2 and blaNDM-5. Through conjugation and induction, the transposition efficiency and plasmid-curing efficiency in each recipient were tested. In addition, we examined the efficiency of the IS26-CRISPR/Cas9 system of cell immunity against the acquisition of the exogenous resistant plasmids by introducing this system into antimicrobial-susceptible hosts. This study aimed to eliminate the replication genes and antimicrobial resistance genes using pIS26-CRISPR/Cas9. Three plasmids with different replicon types, including IncX4, IncI2 and IncHI2 in three isolates, two pUC19-derived plasmids, pUC19-mcr-1 and pUC19-IS26mcr-1, in two lab strains, and two plasmids bearing blaKPC-2 and blaNDM-5 in two isolates were all successfully eliminated. Moreover, the IS26-based CRISPR/Cas9 system that remained in the plasmid-cured strains could efficiently serve as an immune system against the acquisition of the exogenous resistant plasmids. The IS26-based CRISPR/Cas9 system can be used to efficiently sensitize clinical Escherichia coli isolates to antibiotics in vitro. The single-guide RNAs targeted resistance genes or replication genes of specific incompatible plasmids that harboured resistance genes, providing a novel means to naturally select bacteria that cannot uptake and disseminate such genes.