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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.
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The present work characterizes the porcine sex-determining region on the Y chromosome (SRY) gene and demonstrates its pivotal role in sex determination. We provide evidence that genetically male pigs with a knockout of the SRY gene undergo sex reversal of the external and internal genitalia. This discovery of SRY as the main switch for...
Citations
... A key member of this family, the DM domain-encoding DMRT1 gene, is part of the mammalian sexdetermination cascade, and it initiates sex determination in other vertebrate lineages (15,16). Similarly, the high-mobility group (HMG)-box domain, a small domain that binds the minor grove of DNA, is encoded by various genes involved in sex determination: the SRY gene that initiates male sex determination in therian mammals, various SOX genes involved in vertebrate sex determination, and mating-type (MAT) genes, which are crucial for mating type specification in fungi (17)(18)(19)(20)(21). Furthermore, the HMG-box domain is considerably widespread in other eukaryotic proteins, particularly in nonhistone components of chromatin and transcription factors, which often contain multiple copies that bind DNA nonspecifically (21). ...
... A complete sex reversion is highly unlikely in the U/V system of brown algae, given the absence of a U chromosome in males and the requirement of the U-specific region for the full expression of the female program in these organisms (30). This distinct feature sets it apart from XX/XY diploid systems, in which deletion of the master male-determining gene leads to phenotypic feminization (20). This is also unlike other haploid systems such as the Volvox system, in which deletion of VcMID (the RWP-RK domain transcription factor determinant of sperm and egg development) results in the production of functional eggs or self-fertile hermaphrodites (52). ...
In many eukaryotes, genetic sex determination is not governed by XX/XY or ZW/ZZ systems but by a specialized region on the poorly studied U (female) or V (male) sex chromosomes. Previous studies have hinted at the existence of a dominant male-sex factor on the V chromosome in brown algae, a group of multicellular eukaryotes distantly related to animals and plants. The nature of this factor has remained elusive. Here, we demonstrate that an HMG-box gene acts as the male-determining factor in brown algae, mirroring the role HMG-box genes play in sex determination in animals. Over a billion-year evolutionary timeline, these lineages have independently co-opted the HMG box for male determination, representing a paradigm for evolution’s ability to recurrently use the same genetic “toolkit” to accomplish similar tasks.
... The study of genetic diversity by the SRY gene has been conducted in several commodities in cattle. Regional SRY is a small gene of 600 base pairs (bp) and encodes a protein with 78 amino acids through the HMG (high mobility group) box region (Law et al., 2017;Kurtz et al., 2021). Y-chromosomal DNA markers are expected to have another geographical distribution and paternally inherited markers would reveal male introgression via breeding management with natural matting and artificial insemination as well (Amin, 2021). ...
This study aims to determine the kinship and genetic diversity between Limousin and Madrasin cattle based on the sex determining region-Y (SRY) gene. SRY gene is used in the analysis of genetic diversity for sex determination based on the paternal pathway (Y chromosome). The DNA samples used for this study were 10 frozen straw of Limousin cattle and 10 samples of Madrasin cattle. The method used in this research is a descriptive analysis by duplex polymerase chain reaction and analyzing the results of the SRY gene sequencing of limousine cattle and Madrasin cattle. In the present study, we showed that the SRY gene of Madrasin cattle and Limousine cattle could be amplified with SRY F and SRY R primers with a PCR product length of 318 bp. Based on these results, Madura, Limousin, and Madrasin cattle have similarities based on the SRY gene (paternal pathway)
... One potential application of CRISPR/Cas9 in semen sexing is through the modification of genes that are associated with sex determination. For example, researchers have used CRISPR/Cas9 to modify the SRY gene (Kurtz et al., 2021), which is responsible for male sex determination, in mouse embryos. This could potentially be applied to livestock animals to create sperm cells that have a higher likelihood of producing offspring of a desired sex. ...
... Producers are not likely to adopt approaches in which perpetuating the trait involves gene editing each litter or insertion of foreign DNA sequences into the genome of the breeding stock. For example, knocking out the SRY gene to induce male-to-female sex reversal would require direct editing of embryos for every litter, and integration into the parental genomes of a CRISPR/Cas vector would be needed for the production of only-female litters (Kurtz et al., 2021). Likewise, editing genes to reduce the biosynthesis of boar taint precursors would be difficult to implement due to the large number of enzymes involved, and steroid biosynthetic precursors can flow to synthetic endpoints through multiple pathways (Rydhmer et al., 2006;Squires et al., 2019). ...
Introduction: Most male pigs are surgically castrated to avoid puberty-derived boar taint and aggressiveness. However, this surgical intervention represents a welfare concern in swine production. Disrupting porcine KISS1 is hypothesized to delay or abolish puberty by inducing variable hypogonadotropism and thus preventing the need for castration.
Methods: To test this hypothesis, we generated the first KISS1-edited large animal using CRISPR/Cas9-ribonucleoproteins and single-stranded donor oligonucleotides. The targeted region preceded the sequence encoding a conserved core motif of kisspeptin. Genome editors were intracytoplasmically injected into 684 swine zygotes and transferred to 19 hormonally synchronized surrogate sows. In nine litters, 49 American Yorkshire and 20 Duroc liveborn piglets were naturally farrowed.
Results: Thirty-five of these pigs bore KISS1-disruptive alleles ranging in frequency from 5% to 97% and did not phenotypically differ from their wild-type counterparts. In contrast, four KISS1-edited pigs (two boars and two gilts) with disruptive allele frequencies of 96% and 100% demonstrated full hypogonadotropism, infantile reproductive tracts, and failed to reach sexual maturity. Change in body weight during development was unaffected by editing KISS1. Founder pigs partially carrying KISS1-disruptive alleles were bred resulting in a total of 53 KISS1 +/+, 60 KISS1 +/−, and 34 KISS1 −/− F1 liveborn piglets, confirming germline transmission.
Discussion: Results demonstrate that a high proportion of KISS1 alleles in pigs must be disrupted before variation in gonadotropin secretion is observed, suggesting that even a small amount of kisspeptin ligand is sufficient to confer proper sexual development and puberty in pigs. Follow-on studies will evaluate fertility restoration in KISS1 KO breeding stock to fully realize the potential of KISS1 gene edits to eliminate the need for surgical castration.
... More recently, the complete Y chromosome has been deleted using CRISPR/Cas9 technology in the mouse [18]. Furthermore, sex-reversal has been achieved in mice [19], rabbits [20], and pigs [21] through SRY gene knock-out, which yielded phenotypic females with an XY genotype. However, the use of these strategies involves gene editing techniques that would require approval from regulatory entities. ...
Somatic cell nuclear transfer (SCNT) is an asexual reproductive technique where cloned offspring contain the same genetic material as the original donor. Although this technique preserves the sex of the original animal, the birth of sex-reversed offspring has been reported in some species. Here, we report for the first time the birth of a female foal generated by SCNT of a male nuclear donor. After a single SCNT procedure, 16 blastocysts were obtained and transferred to eight recipient mares, resulting in the birth of two clones: one male and one female. Both animals had identical genetic profiles, as observed in the analysis of 15-horse microsatellite marker panel, which confirmed they are indeed clones of the same animal. Cytogenetic analysis and fluorescent in situ hybridization using X and Y specific probes revealed a 63,X chromosome set in the female offspring, suggesting a spontaneous Y chromosome loss. The identity of the lost chromosome in the female was further confirmed through PCR by observing the presence of X-linked markers and absence of Y-linked markers. Moreover, cytogenetic and molecular profiles were analyzed in blood and skin samples to detect a possible mosaicism in the female, but results showed identical chromosomal constitutions. Although the cause of the spontaneous chromosome loss remains unknown, the possibility of equine sex reversal by SCNT holds great potential for the preservation of endangered species, development of novel breeding techniques, and sportive purposes.
... Before weaning, fibroblasts were obtained by ear biopsy from transgenic Cas9 positive piglets. Fibroblasts were isolated as previously described [34]. When fibroblasts reached confluency, cells were either frozen or further processed. ...
Due to its close resemblance, the domesticated pig has proven to be a diverse animal model for biomedical research and genome editing tools have contributed to developing porcine models for several human diseases. By employing the CRISPR-Cas9 system, porcine embryos or somatic cells can be genetically modified to generate the desired genotype. However, somatic cell nuclear transfer (SCNT) of modified somatic cells and embryo manipulation are challenging, especially if the desired genotype is detrimental to the embryo. Direct in vivo edits may facilitate the production of genetically engineered pigs by integrating Cas9 into the porcine genome. Cas9 expressing cells were generated by either random integration or transposon-based integration of Cas9 and used as donor cells in SCNT. In total, 15 animals were generated that carried a transposon-based Cas9 integration and two pigs a randomly integrated Cas9. Cas9 expression was confirmed in muscle, tonsil, spleen, kidney, lymph nodes, oral mucosa, and liver in two boars. Overall, Cas9 expression was higher for transposon-based integration, except in tonsils and liver. To verify Cas9 activity, fibroblasts were subjected to in vitro genome editing. Isolated fibroblasts were transfected with guide RNAs (gRNA) targeting different genes (GGTA1, B4GALNT2, B2M) relevant to xenotransplantation. Next generation sequencing revealed that the editing efficiencies varied (2–60%) between the different target genes. These results show that the integrated Cas9 remained functional, and that Cas9 expressing pigs may be used to induce desired genomic modifications to model human diseases or further evaluate in vivo gene therapy approaches.
... RNA was isolated and cDNA was synthesized following a protocol previously established in our laboratory [20]. The coding sequences (CDSs) of the LsSox2 and LsSox9 genes were cloned by RT-PCR. ...
... Two pairs of primers were designed for PCR amplification and qRT-PCR of LsSox2 and LsSox9 based on the results of L. sieboldii transcriptome data previously generated in our laboratory. PCR, sequencing, and sequence assembly were performed following a protocol previously established in our laboratory [20], and sequences were submitted to NCBI under Accession numbers OM243913 and OM243914. ...
The Sox family plays essential roles as transcription factors in vertebrates; however, little is known about the Sox family in Lutraria sieboldii. L. sieboldii are pleasant to eat with a short growth cycle and have become one of the best bottom-seeded enrichment species in Guang Xi. In this study, Sox2 (named LsSox2) and Sox9 (named LsSox9) from L. sieboldii were cloned, and their expression patterns were analyzed. The length of the LsSox2 gene coding sequence was 1011 bp, encoding 336 amino acids, and LsSox9 was 1449 bp, encoding 482 amino acids. LsSox2 had its highest expression levels in the ovary, which were 356 times those in testis, whereas LsSox9 presented higher expression in testis, which was 6 times more highly expressed than in the ovary. LsSox2 exhibited the highest expression during the morula stage, which was 20 times that of the D-shaped larvae or zygote. LsSox9 exhibited two expression peaks, one at the four-cell stage and the other at the trochophore stage, while the lowest expression was in the zygote. LsSox9 was 73 times more highly expressed in the four-cell stage than in the zygote stage. During gonadal development, LsSox2 presented the highest expression in the mature ovary, which was 756 times more highly expressed than in mature testis. LsSox9 presented higher expression in testis at the emission stage which was 6 times more highly expressed than in the ovary. These results indicate that LsSox2 and LsSox9 may play important roles in embryonic and gonadal development.
... This experiment also revealed that all of the sequences necessary for Sry expression are present within this 14.5-kb region. Human patients exhibiting XY gonadal dysgenesis and loss-of-function studies in mice, pig, and rabbit have demonstrated that SRY/Sry disruption causes sex reversal, such that chromosomal males (XY-type) are anatomically female Jäger et al. 1990;Sinclair et al. 1990;Lovell-Badge and Robertson 1990;Wang et al. 2013;Kato et al. 2013;Song et al. 2017;Kurtz et al. 2021). These experiments suggested that SRY functions as the master regulator of male sex determination in all eutherian mammals (Waters et al. 2007). ...
... In a knockout study in pigs, a frameshift mutation in one copy of the SRY gene did not induce male-to-female sex reversal. These results suggest that expression from one SRY copy is sufficient for the development of male genitalia, or that only one of the two SRY copies is expressed, and the frameshift mutation was introduced into the unexpressed SRY copy (Kurtz et al. 2021). ...
To deceive predators, palatable species often resemble colour patterns and morphology of unpalatable species, which is called Batesian mimicry. In two closely related swallowtail butterflies, Papilio polytes and P. memnon, only females mimic model poisonous butterflies, and females have mimetic and non-mimetic types. Responsible loci (H for P. polytes and A for P. memnon) for mimicry were identified in the homologous chromosomal region as supergene containing doublesex (dsx) and a few genes. The supergene sequences between H and h (A and a) alleles are highly diversified due to recombination suppression, which is caused by chromosomal inversion in P. polytes, but no inversion in P. memnon. In both species, higher expression of mimetic dsx (dsx-H and dsx-A) in female pupal wings induced mimetic colouration, and the expression of genes inside the supergene was similar to that of mimetic dsx. In contrast, colouration patterns and supergene structure were considerably different between the two species. Furthermore, the chemical features of pale-yellow in hindwings, which are important both for sexual and mimicry strategies in P. polytes, seemed different in P. memnon. Here, we summarise the similarities and differences in mimetic traits between two butterflies and discuss how two mimicry supergenes have evolved.KeywordsFemale-limited Batesian mimicrySupergeneIn vivo electroporation-mediated RNAiCost–benefit balance of mimicryPapilio butterflies
doublesex
... Most human male-to-female sex reversal syndrome cases are believed to be caused by a mutation located in the HMG domain of SRY [64,65]. Recent gene-editing research demonstrated that knockout of the HMG domain of the porcine SRY gene could result in male-to-female sex reversal [66]; however, additional tests are necessary to confirm the fertility of these transgenic animals. Interestingly, a study found that XX mice carrying a chimeric SRY/SOX construct (replacing the HMG domain of SRY with the HMG domain of SOX3 or SOX9) also exhibited sexual reversal [67], suggesting that SOX3 and SOX9 HMG domains can functionally replace SRY HMG domain. ...
Sex determination is crucial for the transmission of genetic information through generations. In mammal, this process is primarily regulated by an antagonistic network of sex-related genes beginning in embryonic development and continuing throughout life. Nonetheless, abnormal expression of these sex-related genes will lead to reproductive organ and germline abnormalities, resulting in disorders of sex development (DSD) and infertility. On the other hand, it is possible to predetermine the sex of animal offspring by artificially regulating sex-related gene expression, a recent research hotspot. In this paper, we reviewed recent research that has improved our understanding of the mechanisms underlying the development of the gonad and primordial germ cells (PGCs), progenitors of the germline, to provide new directions for the treatment of DSD and infertility, both of which involve manipulating the sex ratio of livestock offspring.
... The development of high-precision genome editing tools, such as targeted nucleases, has accelerated advances in fundamental human medicine, animal science, animal breeding, as well as disease diagnosis (Doudna and Charpentier, 2014;Kurtz et al., 2021;Rieblinger et al., 2021;Xie et al., 2021). In particular, the genome editing system known as CRISPR technology has grown rapidly since it was first reported (Jinek et al., 2012) and has become one of the most popular technologies. ...
Advances in CRISPR-Based Functional Genomics and Nucleic Acid Detection in Pigs
