Surrogate production of genome edited sperm from a different subfamily by spermatogonial stem cell transplantation

The surrogate reproduction technique provides a powerful tool for production of allogenic or xenogeneic gametes derived from endangered species or those with valuable genetic traits. Production of functional donor-derived gametes through intra- or inter-specific spermatogonial stem cell transplantation (SSCT) has been achieved in many species. However, generation of functional gametes from a phylogenetically distant species such as from a different subfamily by SSCT has never been successful. Here, using two small cyprinid fishes, Chinese rare minnow (gobiocypris rarus, for brief: Gr) and zebrafish (danio rerio), which belong to different subfamilies, as donors and recipients for SSCT, we optimized the SSCT technique and successfully obtained Gr-derived sperm carrying targeted genome modifications in zebrafish. We revealed that the transplanted Gr spermatogonia supported the host gonadal development and underwent normal spermatogenesis, resulting in a reconstructed fertile testis containing Gr spermatids and zebrafish testicular somatic cells. Interestingly, the surrogate spermatozoa resembled those of host zebrafish but not donor Gr in morphology and swimming behavior. Finally, we showed that Gr-derived genome edited sperm was successfully produced in zebrafish by cross-subfamily SSCT, when the pou5f3 and chd gene knockout Gr SSCs were used as surrogate donors. This is the first report demonstrating the surrogate production of genome edited sperm from a phylogenetically distant species, and this method is feasible to be applied to future breeding of commercial fishes.