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 Escherichia 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: CFU/mL, with a maximum RPS of 73.13% and stable 65.79% in validation. Immunized tilapia showed elevated immune indices (161.40% higher platelets) and numerical increases in globulin, normal liver/kidney function, and improved oxidative stress resistance with no tissue damage. The vaccine did not alter intestinal microbial richness but modulated community structure, enriching beneficial taxa such as Alphaproteobacteria, suggesting a potential interaction between vaccination and gut microbiota that may contribute to enhanced host defense. CONCLUSIONS: In conclusion, this study successfully developed an 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.