PT  - JOURNAL ARTICLE
AU  - Stefanie Kurtz
AU  - Andrea Lucas-Hahn
AU  - Brigitte Schlegelberger
AU  - Gudrun Göhring
AU  - Heiner Niemann
AU  - Thomas C. Mettenleiter
AU  - Björn Petersen
TI  - The knockout of the HMG domain of the porcine SRY gene causes sex reversal in gene-edited pigs
AID  - 10.1101/617589
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 617589
4099  - http://biorxiv.org/content/early/2020/04/30/617589.short
4100  - http://biorxiv.org/content/early/2020/04/30/617589.full
AB  - The sex-determining region on the Y chromosome (SRY) is thought to be the central genetic element 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 mechanisms are largely unknown. To understand the biological function of the SRY gene, a site-directed mutational analysis is required to investigate associated phenotypic changes at the molecular, cellular and morphological level. In our study, we successfully generated a knockout of the porcine SRY gene by 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” (HMG) domain of the SRY gene. Mutations within this region resulted in the development of complete external and internal female genitalia in genetically male pigs. The internal female genitalia including uteri, ovaries, and oviducts, revealed substantial size differences in 9-months old SRY-knockout pigs compared to age-matched female wild type controls. In contrast, a deletion within the 5’ flanking region of the HMG domain was not associated with sex reversal. Results of this study demonstrates for the first time the central role of the HMG domain of the SRY gene in male sex determination in pigs. Moreover, quantitative analysis by digital PCR revealed evidence for a duplication of the porcine SRY locus. Our results pave the way towards the generation of boars exclusively producing phenotypically female offspring to avoid surgical castration without anesthesia in piglets. Moreover, the study establishes a large animal model that is much more similar to humans in regard of physiology and anatomy and pivotal for longitudinal studies.Competing Interest StatementThe authors have declared no competing interest.