close
Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Jan 12;118(2):e2008743118.
doi: 10.1073/pnas.2008743118.

Knockout of the HMG domain of the porcine SRY gene causes sex reversal in gene-edited pigs

Stefanie Kurtz  1 , Andrea Lucas-Hahn  1 , Brigitte Schlegelberger  2 , Gudrun Göhring  2 , Heiner Niemann  3 , Thomas C Mettenleiter  4 , Björn Petersen  5
Affiliations

Knockout of the HMG domain of the porcine SRY gene causes sex reversal in gene-edited pigs

Stefanie Kurtz et al. Proc Natl Acad Sci U S A. .

Abstract

The sex-determining region on the Y chromosome (SRY) is thought to be the central genetic element of male sex development in mammals. Pathogenic modifications within the SRY gene are associated with a male-to-female sex reversal syndrome in humans and other mammalian species, including rabbits and mice. 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. Here, we successfully generated a knockout of the porcine SRY gene by microinjection of two CRISPR-Cas ribonucleoproteins, targeting the centrally located "high mobility group" (HMG), followed by a frameshift mutation of the downstream SRY sequence. This resulted in the development of genetically male (XY) pigs with complete external and internal female genitalia, which, however, were significantly smaller than in 9-mo-old age-matched control females. Quantitative digital PCR analysis revealed a duplication of the SRY locus in Landrace pigs similar to the known palindromic duplication in Duroc breeds. Our study demonstrates the central role of the HMG domain in the SRY gene in male porcine sex determination. This proof-of-principle study could assist in solving the problem of sex preference in agriculture to improve animal welfare. Moreover, it establishes a large animal model that is more comparable to humans with regard to genetics, physiology, and anatomy, which is pivotal for longitudinal studies to unravel mammalian sex determination and relevant for the development of new interventions for human sex development disorders.

Keywords: CRISPR/Cas9; HMG domain; RNPs; porcine SRY gene; sex reversal.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
The dPCR biplex assay of WT samples revealed half of the copy number of the monoallelic KDM6A gene compared to the biallelic GGTA1 gene, as expected. A similar copy number of the monoallelic SRY gene compared to the biallelic GGTA1 gene shows a duplication of the SRY locus in Landrace pigs. Reprinted with permission from ref. .
Fig. 2.
Fig. 2.
(A) Schematic illustration of the gRNAs SRY_1 and SRY_2 (yellow underlined) targeting an ∼72-bp segment in the 5′ flanking region of the HMG domain (red box) of the SRY gene. (B) Location of two sgRNA target sites SRY_1 and SRY_3 (yellow underlined) flanking the HMG-box (red box) of the SRY gene. The primers amplifying the SRY exon are indicated with green arrows. Reprinted with permission from ref. .
Fig. 3.
Fig. 3.
A total of 12 healthy piglets were born after intracytoplasmic microinjection of two CRISPR-Cas9 RNP complexes (SRY_1 and SRY_3) into IVF-produced zygotes and surgical embryo transfer to recipients. Three genetically male piglets (714/1, 715/2, and 715/7) had a complete set of female external genitalia. The deletion of the SRY gene did not affect the growth rate compared to WT controls (SI Appendix, Figs. S15 and S16). Reprinted with permission from ref. .
Fig. 4.
Fig. 4.
PCR-based detection of the edited SRY gene in piglets (714/1 and 715/1 to 715/11) generated via microinjection of CRISPR-Cas9 RNP complexes (SRY_1 and SRY_3). Three piglets (715/2, 715/7, and 714/1, indicated with white asterisk) showed deletions of ∼300 bp within the SRY gene compared to a male WT control (WT 578 F7). The male WT control showed an expected band of ∼500 bp. A female WT (WT 578 F4) served as negative control. Reprinted with permission from ref. .
Fig. 5.
Fig. 5.
Sanger sequencing of the purified PCR product of the SRY-KO piglets (715/2, 715/7, and 714/1) showed genetic modifications within the SRY locus. Piglet 715/7 displayed a deletion of 292 bp and piglet 715/2 of 266 bp. Piglet 714/1 showed two different modifications with a deletion of 298 bp and an indel formation of −298 bp and +1 bp. Reprinted with permission from ref. .
Fig. 6.
Fig. 6.
Karyotyping of cells from the SRY-KO piglet 715/2 confirmed the male genotype of this piglet. The karyotypes of piglet 715/7 and 714/1 are shown in SI Appendix, Fig. S7.
Fig. 7.
Fig. 7.
(A) Hematoxylin and eosin staining of porcine ovarian tissue from the SRY-KO piglet 715/2 (Left) and female WT control (Right) 34 d after birth. No structural differences were shown. (B) Histological analysis of the ovarian tissue of the SRY-KO pig (Left) compared to the female WT control from same litter (microinjection [MI] WT control, Right) at the age of 9 mo. A higher amount of loose connective tissue (indicated with black arrows) in the 9-mo-old SRY-KO pig revealed fat deposits within the ovarian tissue. The ovarian tissue from SRY-KO pigs showed no follicular development compared to MI WT controls (black asterisk) at 9 mo of age. (Scale bars, 500 μm.) Reprinted with permission from ref. .
Fig. 8.
Fig. 8.
Uteri and ovaries of the 9-mo-old SRY-KO, XY pig (714/1) and the age-matched WT, XX piglet (control from same litter). (A) Substantial size differences of external female genitalia were apparent in the 9-mo-old SRY-KO pig compared to the female WT control. (B) The ovaries of the 9-mo-old SRY-KO, XY pig were significantly smaller than the ovaries of the WT, XX pig and showed no follicle development.
Fig. 9.
Fig. 9.
Immunohistological staining of FOXL2-positive cells (red) in ovaries of two 9-mo-old SRY-KO pigs (upper images: SRY-KO pig 1,255, lower images: SRY-KO pig 1,262). Cell clusters of FOXL2-positive cells (indicated with white arrows) were detected in the cortical region of the porcine ovaries. SiR-Hoechst–stained nuclei (blue) are shown. The merged images revealed positive FOXL2 staining in the nuclei of the cells. The experiments were repeated three times with similar results. (Scale bars, 20 μm.) Reprinted with permission from ref. .
See this image and copyright information in PMC

References

    1. Waters P. D., Wallis M. C., Marshall Graves J. A., Mammalian sex–Origin and evolution of the Y chromosome and SRY. Semin. Cell Dev. Biol. 18, 389–400 (2007). - PubMed
    1. She Z.-Y., Yang W.-X., Molecular mechanisms involved in mammalian primary sex determination. J. Mol. Endocrinol. 53, R21–R37 (2014). - PubMed
    1. Parma P., Radi O., Molecular mechanisms of sexual development. Sex Dev. 6, 7–17 (2012). - PubMed
    1. Gubbay J., et al. , A gene mapping to the sex-determining region of the mouse Y chromosome is a member of a novel family of embryonically expressed genes. Nature 346, 245–250 (1990). - PubMed
    1. Daneau I., Ethier J. F., Lussier J. G., Silversides D. W., Porcine SRY gene locus and genital ridge expression. Biol. Reprod. 55, 47–53 (1996). - PubMed

Publication types

MeSH terms

LinkOut - more resources