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

The surrogate reproduction technique,such as inter-specific spermatogonial stem cells(SSCs)transplantation(SSCT),provides a powerful tool for production of gametes derived from endangered species or those with desirable traits.However,generation of genome-edited gametes from a different species or production of gametes from a phylogenetically distant species such as from a different subfamily,by SSCT,has not succeeded.Here,using two small cyprinid fishes from different subfamilies,Chinese rare minnow(gobiocypris rarus,for brief:Gr)and zebrafish(danio rerio),we successfully obtained Gr-derived genome-edited sperm in zebrafish by an optimized SSCT procedure.The transplanted Gr SSCs 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.When pou5f3 and chd knockout Gr SSCs were transplanted,Gr-derived genome-edited sperm was successfully produced in zebrafish.This is the first report demonstrating surrogate production of gametes from a different subfamily by SSCT,and surrogate production of genome-edited gametes from another species as well.This method is feasible to be applied to future breeding of commercial fish and livestock.