Chinese indigenous pig breeds are generally more susceptible to Mycoplasma hyopneumoniae (Mhp), however, the underlying molecular mechanisms remain poorly understood. At slaughter, Ningxiang pigs (n=77) exhibited a 2.6-fold higher Mhp detection rate and a 1.5-fold higher average Goodwin (GW) score than Duroc×Landrace×Yorkshire (DLY) pigs (n=83), (P<0.01). Here, we demonstrated that genetic variation at position 2906 of the NOD-like receptor family pyrin domain containing 3 (Nlrp3) gene in Ningxiang pigs (as compared to DLY pigs) was associated with reduced phagocytic efficiency of Porcine Alveolar Macrophages (PAMs) against Mhp, primarily by  suppressing the expression of Nlrp3 and interleukin-1β (Il-1β), thereby compromising host immune defense and enhancing susceptibility to Mhp infection in Ningxiang pigs. Genetic sequencing revealed that all PAMs derived from Ningxiang pigs harbored a Guanine (G) allele at position 2906 of the Nlrp3 gene, whereas 88.64% of DLY pig-derived PAMs carried an Adenine (A) allele at this locus. Site-directed mutagenesis of Nlrp3-2906G PAMs to Nlrp3-2906A PAMs by CRISPR-Cas9 was performed to investigate the functional consequences. Transcriptomic analysis revealed that the p.Q969 (A allele) mutation significantly upregulates Nlrp3 mRNA expression. Transcriptomic analysis and ELISA validated that this improvement was mediated by upregulated expression of Nlrp3 and Il-1β. Furthermore, functional assays demonstrated that the A allele enhanced phagocytic efficiency against pathogens by an average of 1.8- to 3.8-fold (P<0.01). Mhp Real-time fluorescence quantitative PCR (qPCR) and fluorescence tracing assays demonstrated the phagocytic efficiency of PAMs derived from Ningxiang pigs against Mhp was approximately 2.65-fold lower than that in DLY pigs (P<0.01), indicating a significant impairment in Mhp clearance. Collectively, these results suggest that the Nlrp3-2906G variant enhances Mhp susceptibility in Ningxiang pigs by impairing PAM-mediated microbial clearance, revealing a novel genetic mechanism that sheds light on the specific susceptibility of pigs to Mhp.

To Cite This Article: Zhang L, Wang Z, Zhang Z, Zhao G, Zhang S, Yu J, Liu M, Huang X, Yin C, Hu S and Wang G, 2026. CRISPR-Cas9 revealed that the Nlrp3-2906G allele impairs phagocytosis of porcine alveolar macrophages to enhance Mycoplasma hyopneumoniae susceptibility in pigs. Pak Vet J, 46(4): 940-951. http://dx.doi.org/10.29261/pakvetj/2026.066