Construction of fish without intermuscular bones based on cytosine base editing system

To verify the editing effect of new cytosine base editor (CBE) in fish, this study took the runx2b that regulates the formation of intermuscular bones (IBs) in fish as the target gene, and explored its editing efficiency, phenotype and related gene expression on the constructed mutation zebrafish line. The results showed that the mutation efficiency in the F₀ generation was 60%. Skeletal staining demonstrated a 55% reduction in IBs in F₀ generation mutants compared to wild-type controls. In the F₁ generation produced by self-crossing of the F₀ generation, only heterozygotes and homozygotes were observed, with homozygotes exhibiting completely IBs absence and stable inheritance across generations. Western Blot analysis confirmed the absence of runx2b protein in mutant, while RT-qPCR showed significantly downregulated expression of osteoblast differentiation and mineralization-related genes (entpd5a, sp7, and alpl) in mutants. The results indicate that CBE not only achieves high editing efficiency in fish, but also enables IBs free phenotypes, shortens homozygotes generation time, and expands the utility of base editing in aquatic species. This study provides a foundation for applying GE in breeding economic fish.