107 GENETIC INACTIVATION OF THE Sry GENE IN ARGALI WILD AND ROMNEY DOMESTIC SHEEP WITH CRISPR/Cas SYSTEMS FOR PRODUCING SEX-REVERSED FEMALE ANIMALS

Abstract

The Tibetan Argali (Ovis ammon hodgsoni), a wild sheep of the subfamily Caprinae (Bovidae), primarily found in the Tibetan Plateau, is categorized as near threatened on the International Union for Conservation of Nature (IUCN) Red List. For the conservation of this species, we have achieved the cloning of a full-term live Argali lamb (died shortly after birth) from a male Argali cell line that has been cryopreserved for more than 20 years. While working towards the goal of cloning a live Argali, we also recognised the need to establish a reproductive herd of animals by producing both fertile males and females. However, as in the case of Argali, it is difficult, and in some cases impossible, to obtain male and female cell lines from an endangered mammalian species. Therefore, for demonstrating a proof of concept of using an assisted reproduction technology (ART) for the conservation of endangered species or reviving extinct species, we used the Argali as an experimental model to produce both fertile males and females through ART. The Sry gene plays a central role in mammalian sex determination: mutations in the Sry gene result in the development of XY females; in mice, Sry knockout (KO) results in fertile XY females. Therefore, we created sex-reversed Argali cell lines through the KO of the Sry gene. In parallel, for potentially comparing the fertility of sex-reversed XY females between the Argali and domestic sheep [Romney (Ovis aries)], we also knocked out the Sry gene in the domestic sheep. Specifically, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system to introduce mutations to the high-mobility group (HMG) box of the Sry gene. Skin fibroblasts from either adult Argali or Romney sheep were cultured in DMEM (HyClone) supplemented with 10% fetal bovine serum (FBS). Cells were harvested at 100% confluence with 0.25% trypsin-EDTA and transfected with the CRISPR/Cas DNA constructs by using the Amaxa Nucleofector system. For each experiment, 10⁶ cells were transfected with 5 μg of CRISPR/Cas DNA constructs. After 72 hours post-transfection, cells were harvested, with half being used for genomic DNA isolation (for examining KO efficiency) and the other half for limited dilution for obtaining single-cell-derived colonies. With the Surveyor assay and a restriction enzyme digestion assay (successfully knocked-out cells lose the Dde I site in the HMG box of the Sry gene, rendering resistance to Dde I digestion), we demonstrated that the Sry KO efficiency was about 10% both in Argali and Romney sheep. By screening single-cell-derived colonies from the transfected Romney sheep cells, an Sry-KO cell line harboring a 2-nucleotide deletion in the HMG box was successfully established. Multiple vials of cells from this Sry-KO cell line have been cryopreserved and will be used for animal cloning via somatic cell nuclear transfer (SCNT) in the sheep-breeding season this fall.