The knockout of the HMG-Box Domain of the porcine SRY-gene by CRISPR/Cas RNP microinjection causes sex reversal in gene-edited pigs
The sex-determining region on Y chromosome (SRY) is presumed as a main genetic switch of male sex development. Mutations within the SRY gene are associated with a male-to-female sex reversal syndrome in humans and other mammalian species such as mice and rabbits. However, the underlying mechanism causing this syndrome is not definitely known. To understand the genetic pathway of SRY, the investigation and analysis of morphological changes are essential. For the first time, we successfully generated a knockout of the SRY gene in pigs using microinjection of two Clustered regularly interspaced short palindromic repeats (CRISPR) – associated protein - 9 nuclease (Cas9) ribonucleoprotein (RNP) complexes targeting the centrally located “high mobility group”-box domain (HMG) of the SRY gene. Mutation within the porcine HMG-box domain resulted in the development of complete external and internal female genitalia in genetically male piglets. Uteri and oviducts displayed equal morphology compared to female wildtype controls, only the ovaries showed smaller size in sex reversal piglets. In contrast to the HMG-box domain deletion, a deletion within the 5’ flanking region of the HMG domain was not associated with sex reversal. In summary, this study demonstrates for the first time the main role of the HMG-box domain of the SRY gene triggering the male sex determination in pigs.