Li Linai, Hu Yuxiang, Wang Dan, Li Xin, Bao Shengjuan, Deng Taibing, Wang Qinglan
Institute of Respiratory Health, Frontiers Science Center for Disease-related Molecular Network, West China Hospital, Sichuan University, Chengdu, China.
School of Clinical Medicine, North Sichuan Medical College, Nanchong, China.
Front Microbiol. 2025 Jul 9;16:1608274. doi: 10.3389/fmicb.2025.1608274. eCollection 2025.
The increasing global prevalence of infections presents a significant clinical challenge due to the pathogen's intrinsic resistance to multiple antibiotics and poor treatment outcomes. Despite the necessity of genetic tools for studying its physiology, pathogenesis, and drug resistance, efficient methods for large-fragment deletions remain underdeveloped. Here, we report a CRISPR/Cas9-based dual-sgRNA system employing CRISPR1-Cas9 (Sth1Cas9), enabling efficient large-fragment knockout in with deletion efficiencies exceeding 90% at certain loci and spanning up to 16.7 kb. Furthermore, we systematically optimized the modular arrangement of genetic components in Cas9/dual-sgRNA expression plasmids and refined their construction workflow, achieving a significant reduction in cassette loss rates while enabling single-step plasmid assembly. Notably, deletion efficiency was position-dependent rather than correlated with target size, suggesting an influence of chromatin structure on editing outcomes. As the first CRISPR/Cas9-based platform capable of kilobase-scale deletions in , this system advances functional genomics studies and facilitates targeted investigations into virulence and antibiotic resistance mechanisms.
由于病原体对多种抗生素具有内在抗性且治疗效果不佳，全球感染患病率的上升带来了重大的临床挑战。尽管需要遗传工具来研究其生理学、发病机制和耐药性，但用于大片段缺失的有效方法仍未得到充分发展。在此，我们报告了一种基于CRISPR/Cas9的双sgRNA系统，该系统采用CRISPR1-Cas9（Sth1Cas9），能够在中实现高效的大片段敲除，在某些位点的缺失效率超过90%，跨度可达16.7 kb。此外，我们系统地优化了Cas9/双sgRNA表达质粒中遗传元件的模块化排列，并改进了其构建工作流程，在实现单步质粒组装的同时，显著降低了盒式丢失率。值得注意的是，缺失效率取决于位置，而非与靶标大小相关，这表明染色质结构对编辑结果有影响。作为首个能够在中进行千碱基规模缺失的基于CRISPR/Cas9的平台，该系统推动了功能基因组学研究，并有助于对毒力和抗生素耐药机制进行靶向研究。
