Large scale production of sterile fish for aquaculture will increase culture performance and environmental sustainability by preventing early sexual maturation during grow-out and uncontrolled reproduction from farm escapees. While varied reproductive containment solutions have been proposed, none to date has proved fully effective, or has been widely adopted by the industry.
Here, we describe strategies to generate, breed and produce sterile fish in large scale. Our solutions rely on gene edits designed to create broodstock lines that only produce monosex, sterile populations of progeny. Thus, our design combines the benefit of sterility with sexually dimorphic performance traits in grow-out. These approaches have been validated in tilapia and are transferrable to multiple species of fish. The edited broodstock can be propagated and incorporated into breeding programs. We identified and inactivated 12 genes in two evolutionarily conserved pathways, one governing sex differentiation and the other sex competency. We isolated null alleles of genes necessary for spermiogenesis and estrogen synthesis causing male sterility and masculinization, respectively. Double edited combinations for these genes produced all-male sterile populations. Likewise, we inactivated genes which caused females to develop atrophic ovaries arrested at a previtellogenic stage or under-developed ovaries lacking oocytes. We further disrupted genes causing genetic males to sex reverse into females. Double edited combinations for these genes produced all-female, sterile populations. We successfully propagated and amplified the double edited lines via germ cell transplantation from a juvenile mutant donor into several germ cell free wild-type recipient embryos. In the resulting recipient broodstock, the edits had no effect as the genes targeted are not expressed in germ cells. With this approach, we generated fertile broodstock that successfully produced large populations of monosex, sterile progeny. Finally, we tested the performance of all-male sterile tilapia in grow-out trials. Monthly average of daily body weight gain indicated that sterile tilapia grew 12% faster than their maturing siblings starting around the time of puberty.