Reproductive containment of fish using a gene-on/off strategy derived from CRISPR/Cas9 system

Reproductive containment delays germplasm degradation and is a promising solution for potential ecological concerns associated with genetically modified fish, while also helping protect intellectual property rights related to fish varieties. However, current methods for fish fertility control, which typically involve interfering with germ cell survival or migration using molecular techniques, face a major challenge—modified traits are not often inheritable. Here, we developed a novel strategy to control fish fertility using an induced gene-editing system derived from CRISPR/Cas9. Targeting DANA as the gene-editing site, we established two transgenic zebrafish lines, one carrying a piwi : Cas9 cassette that expresses germ-cell specific Cas9, and the other expressing DANA -targeting guide RNA. In the germ cells of the two hybrid offspring, extensive DNA breaks were expected to occur in DANA , sufficient to disrupt genome structure and induce germ cell apoptosis. These effects were confirmed by Sanger sequencing, immunofluorescence, and TUNEL assays. Germ cells in all hybrid fry were reduced, with approximately 17% showing a complete absence of germ cells. Upon maturation, gonadal development was impaired in 80% of the hybrid fish, including 20% with completely undeveloped gonads and 60% with significantly decreased egg production or fertilization rate. Collectively, these results indicate that our germ cell-specific CRISPR/Cas9 strategy may resolve the paradox between inheritability and fertility control in genetically modified fish. Our findings also provide new insights into the establishment of large-scale universal fertility control technologies for fish.