CRISPR-Cas9-Mediated Construction of a Streptococcus agalactiae Vaccine for Tilapia and Evaluation of Its Protective Efficacy

Background Streptococcus agalactiae (GBS) causes severe tilapia streptococcosis with heavy aquaculture losses; existing vaccines have administration or efficacy limitations. This study used CRISPR-Cas9 to construct recombinant E. coli DH5α-ORF4-GFP (targeting GBS ScpB gene ORF4 fragment), optimized tilapia immersion immunization doses/frequencies, and evaluated the vaccine’s protective efficacy, biosafety and regulatory effects via multi-dimensional assays. Results The optimal regimen was single immersion at 1.5×10⁴ CFU/mL, with a maximum RPS of 73.12% and stable 65.26% in validation. Immunized tilapia showed elevated immune indices (161.40% higher platelets) and enhanced globulin synthesis, normal liver/kidney function, and improved oxidative stress resistance with no tissue damage. The vaccine did not change intestinal microbial richness but optimized its structure, enriching beneficial taxa like Alphaproteobacteria via the microbiota-immunity axis, enhancing host’s defense capacity via the "microbiota-immunity" axis. Conclusions In conclusion, this study successfully developed a highly effective and safe genetically engineered vaccine against GBS in tilapia. The precise CRISPR-Cas9-mediated construction strategy and confirmed immune protective effect provide a novel technical approach for controlling this disease in aquaculture and offer important references for the development of related genetically engineered vaccines.