Innovative Delivery System Combining CRISPR-Cas12f for Combatting Antimicrobial Resistance in Gram-Negative Bacteria

作者：Tengfei Long, Shiying Zhou, Zilei Huang, Gong Li, Qin Zhong, Xiaojing Zhang, Yuanyuan Li, Caiping Chen, Lijuan Xia, Ran Wei, Lei Wan, Ang Gao, Hao Ren, Xiaoping Liao, Ya-Hong Liu, Liang Chen, Jian Sun · 发表于：ACS Synthetic Biology · 年份：2024 · DOI：10.1021/acssynbio.4c00112 · 被引用次数：14 · 研究领域：CRISPR and Genetic Engineering、Bacteriophages and microbial interactions、Bacterial biofilms and quorum sensing

Antimicrobial resistance poses a significant global challenge, demanding innovative approaches, such as the CRISPR-Cas-mediated resistance plasmid or gene-curing system, to effectively combat this urgent crisis. To enable successful curing of antimicrobial genes or plasmids through CRISPR-Cas technology, the development of an efficient broad-host-range delivery system is paramount. In this study, we have successfully designed and constructed a novel functional gene delivery plasmid, pQ-mini, utilizing the backbone of a broad-host-range Inc.Q plasmid. Moreover, we have integrated the CRISPR-Cas12f system into the pQ-mini plasmid to enable gene-curing in broad-host of bacteria. Our findings demonstrate that pQ-mini facilitates the highly efficient transfer of genetic elements to diverse bacteria, particularly in various species in the order of Enterobacterales, exhibiting a broader host range and superior conjugation efficiency compared to the commonly used pMB1-like plasmid. Notably, pQ-mini effectively delivers the CRISPR-Cas12f system to antimicrobial-resistant strains, resulting in remarkable curing efficiencies for plasmid-borne mcr-1 or bla KPC genes that are comparable to those achieved by the previously reported pCasCure system. In conclusion, our study successfully establishes and optimizes pQ-mini as a broad-host-range functional gene delivery vector. Furthermore, in combination with the CRISPR-Cas system, pQ-mini demonstrates its potential for broad-host delivery, hi...