The 2SP Site Mutation in the Bovine Natural Resistance-Associated Macrophage 1 Promoter Exhibits Antituberculosis Potential.

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.