Wei Yongke, Yuan Mengke, Zhang Yong, Gao Yuanpeng
College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China.
Key Laboratory of Animal Biotechnology, Northwest A&F University, Yangling 712100, China.
Int J Mol Sci. 2025 Apr 29;26(9):4229. doi: 10.3390/ijms26094229.
Gene-edited cattle overexpressing natural resistance-associated macrophage 1 (NRAMP1) have demonstrated enhanced resistance to tuberculosis (TB). However, introducing synthetic sequences and selection markers may pose potential risks. The endogenous editing of target gene promoters could effectively mitigate these risks. To date, no available mutation sites in the bovine promoter have been identified to enhance host resistance to TB. In this study, we identified a unique mutation editing site, designated as 2SP, within the bovine promoter, using bioinformatics analysis and dual luciferase assays. The mutation at the 2SP site specifically increased promoter activity by 2.3-fold after H37Ra infection, without modifying promoter activity in non-infected groups. By using base editing techniques, an endogenously edited THP-1 cell line with a mutation at the homologous region of the 2SP site was generated, without introducing screening markers. In H37Ra infection experiments, the edited THP-1 cells specifically upregulated NRAMP1 expression and significantly inhibited H37Ra proliferation, while maintaining baseline NRAMP1 expression levels in the absence of infection. In this research, we identified a novel mutation site and provided a fundamental reference for the development of gene-edited cattle with enhanced resistance to TB.
过表达天然抗性相关巨噬细胞蛋白1（NRAMP1）的基因编辑牛已显示出对结核病（TB）的抗性增强。然而，引入合成序列和选择标记可能会带来潜在风险。对靶基因启动子进行内源编辑可以有效降低这些风险。迄今为止，尚未在牛启动子中鉴定出可增强宿主对结核病抗性的可用突变位点。在本研究中，我们通过生物信息学分析和双荧光素酶测定，在牛启动子内鉴定出一个独特的突变编辑位点，命名为2SP。2SP位点的突变在感染H37Ra后特异性地使启动子活性提高了2.3倍，而在未感染组中未改变启动子活性。通过使用碱基编辑技术，生成了在2SP位点同源区域发生突变的内源编辑THP-1细胞系，且未引入筛选标记。在H37Ra感染实验中，编辑后的THP-1细胞特异性上调NRAMP1表达并显著抑制H37Ra增殖，同时在未感染时维持NRAMP1表达的基线水平。在本研究中，我们鉴定出一个新的突变位点，为培育对结核病抗性增强的基因编辑牛提供了基础参考。
