Generation of sterile transgenic fluorescent marine medaka by genetic manipulation

The generation of sterile transgenic ornamental fish is the most effective way to prevent ecological risks. In this study, we initially generated a transgenic fluorescent marine medaka (Oryzias melastigma) expressing mCherry under the control of the zebrafish mylz2 promoter using the Tol2 transposase system. Subsequently, we utilized CRISPR/Cas9 technology to edit the vasa gene, also known as DDX4, in these transgenic fish Tg(mylz2:mCherry). By injecting a single guide RNA (gRNA) into one-cell-stage embryos, we induced indel DNA mutations. Moreover, the co-injection of multiple gRNAs targeting various exons resulted in the deletion of large DNA fragments. Additionally, the simultaneous delivery of multiple gRNAs significantly increased the mutation rate. The homozygous vasa mutants, characterized by a single amino acid deletion (valine) in motif IV of the Vasa protein, displayed male phenotypes. These males exhibited normal courtship behavior and were capable of inducing spawning with wild-type females. However, the eggs produced from these matings were unfertilized. Histological and gene expression analyses revealed that the gonads of these mutants contained testis tubules devoid of germ cells. Taken together, our findings suggest that the sexual differentiation of somatic gonadal cells in Oryzias species is dependent on the germ cells, and the valine in motif IV is essential for the functional integrity of the Vasa protein.