The team’s focal point was a tiny regulatory DNA segment known as Enh13, which serves a pivotal role in modulating the expression of SOX9, a transcription factor critical for testis development. In a typical female (XX) mouse, SOX9 remains repressed, permitting ovarian development. Conversely, in males (XY), this gene becomes active, steering the embryonic gonad toward testis formation. Enh13 functions as a molecular battleground where protein factors promoting male or female pathways compete to regulate SOX9’s activity. Small perturbations in this regulatory landscape can thus have massive developmental consequences.

Employing the precision of CRISPR genome editing, the researchers introduced a one-base-pair insertion mutation within Enh13. Remarkably, this minimal genetic tweak was sufficient to disrupt the normally female-specific repression of SOX9 in XX mice. As a result, these genetically female individuals underwent complete sex reversal, developing testes and male external genitalia in place of ovaries. The data indicates that Enh13 must be carefully regulated: acting to activate SOX9 in males while ensuring its silencing in females.

Further experiments introduced a three-base-pair deletion in the same region, which also induced the sex reversal phenotype. These complementary mutations highlight how delicate and finely tuned the regulatory controls within Enh13 are, underscoring the complexity of non-coding DNA’s functional architecture. Cell-line reporter assays provided biochemical evidence showing how these mutations impair the binding of transcriptional repressors that normally prevent SOX9 expression in females.

The implications of this discovery reach well beyond basic developmental biology. Differences of Sex Development (DSD), a group of congenital conditions affecting approximately 1 in every 4,000 births worldwide, remain puzzling due to a lack of identifiable genetic causes in over half of the cases. Traditional genetic screenings focus primarily on protein-coding genes, largely neglecting non-coding regulatory regions. This study highlights the importance of exploring these vast, non-coding landscapes where subtle mutations can have outsized effects on gene expression and phenotype.

Notably, this investigation builds on previous research published earlier in 2024 by the same group, where mutations within Enh13 led to the converse outcome: XY mice developing with female characteristics due to the failure to activate SOX9. Together, the complementary studies illustrate Enh13’s bifunctional role as both enhancer and repressor depending on the sex chromosome context, revealing a fascinating molecular switch mechanism at the heart of mammalian sex determination.

Elisheva Abberbock, the doctoral student spearheading the project, emphasized that these findings challenge the prevailing gene-centric paradigm in genetic diagnostics and research. “It’s not enough to look only at the genes themselves,” she asserted. “Non-coding DNA sequences like enhancers harbor crucial regulatory elements that can be the root cause of developmental disorders and disease when mutated.”

In the broader context of genomics, this research opens a promising frontier for the systematic identification and functional characterization of regulatory sequences involved in sex determination and beyond. The team’s ongoing work aims to map additional enhancers and silencers that influence key developmental genes, with hopes of ultimately improving diagnostic and therapeutic strategies for DSD and related conditions.

The study’s innovative approach highlights the power of CRISPR-based editing combined with functional assays to deconvolute the complex gene regulatory networks encoded in non-coding DNA. It also reinforces the emerging understanding that the 98% of the human genome once dismissed as “junk” contains vital instructions that govern development, health, and disease.

Funded by the Israel Science Foundation and an ERC Starting Grant, the collaborative effort included contributions from Dr. Ariel Afek at the Weizmann Institute and Dr. Francis Poulat at the University of Montpellier. Their collective expertise underscores the interdisciplinary nature of modern genomic research, bridging molecular biology, genetics, and computational analysis to unravel the intricacies of development.

This landmark discovery not only enhances our biological knowledge of how sex is determined in mammals but also provides a compelling example of how minimal genetic alterations in regulatory DNA can produce dramatic phenotypic changes. It challenges scientists and clinicians alike to look beyond the protein-coding sequence, emphasizing a holistic genomic perspective that could revolutionize the understanding and treatment of developmental disorders.

Subject of Research: Sexual development, gene regulation, non-coding DNA, gene enhancers, sex reversal, CRISPR genome editing

Article Title: One DNA Letter Can Trigger Complete Sex Reversal, Bar-Ilan University Study Finds

News Publication Date: 9-Apr-2026

Web References:
https://www.nature.com/articles/s41467-026-71328-9

References:
Bar-Ilan University research article in Nature Communications, DOI: 10.1038/s41467-026-71328-9

Image Credits: Courtesy Bar-Ilan University

Keywords

Sex determination, non-coding genome, Enh13, SOX9, CRISPR, gene regulation, sex reversal, Differences of Sex Development (DSD), molecular biology, developmental genetics, transcriptional enhancers, genome editing

Unlike classical sex chromosome systems seen in mammals (XY) or birds (ZW), Pogona vitticeps employs a ZZ/ZW sex chromosome mechanism in which females are ZW heterogametic and males are ZZ homogametic. However, the notable twist in this system is the ability of genetic males (ZZ) to develop as phenotypic females when exposed to elevated nest temperatures during embryogenesis. This temperature-induced sex reversal presents a compelling scenario in which environmental factors modulate genetic programming at a molecular level, highlighting a complex interplay not well understood in reptilian species.

Advances in ultra-long read sequencing technologies have been pivotal in achieving the unprecedented quality of these genomic assemblies. One study, spearheaded by a consortium including BGI, the Chinese Academy of Sciences, and Zhejiang University, utilized a combination of DNBSEQ short reads and the novel CycloneSEQ nanopore sequencer—the first animal genome assembled with this technology. In parallel, an Australian team comprising researchers from the University of Canberra and collaborators from multiple esteemed institutions, employed PacBio HiFi, Oxford Nanopore ultralong reads, and Hi-C chromatin conformation capture sequencing to generate a complementary female genome assembly. Together, these resources enable unprecedented resolution of the Pogona vitticeps’ sex chromosomes.

The assemblies themselves are monumental achievements in genomic science. Both projects delivered high-contiguity genomes of approximately 1.75 gigabase pairs, with near-complete telomere to telomere coverage and minimal gaps confined predominantly to microchromosomes. Importantly, the sex chromosomes were fully assembled as single scaffolds, allowing research teams to delineate the pseudo-autosomal region (PAR) on chromosome 16 where Z and W chromosomes recombine. This level of chromosomal resolution is critical for dissecting sex-determining loci and understanding the evolutionary dynamics of sex chromosome differentiation.

Through comparative genomic analyses of the Z and W chromosomes, the studies identified a non-recombining sex determination region (SDR) enriched in candidate genes central to male sex differentiation. Foremost among these were the Anti-Müllerian hormone gene (Amh), its receptor (Amhr2), and the bone morphogenetic protein receptor 1A gene (Bmpr1a). Amh, well-known for its role in the regression of Müllerian ducts during vertebrate sexual development, exhibited a significant male-biased expression pattern across developmental stages, positioning it as the likely master sex-determining gene in the central bearded dragon.

Intriguingly, these findings diverge from classical models exemplified by mammals and birds, where dominant master sex-determining genes such as Sry or Dmrt1 drive sexual fate. To date, no analogous single sex-determining gene has been definitively identified in reptiles, making the discovery of Amh’s central role in Pogona vitticeps a landmark contribution. The dosage-sensitive presence of Amh and its receptor on the Z chromosome, existing in double copy in males (ZZ) and single copy in females (ZW), suggests a mechanism whereby gene dosage influences sexual fate, nuanced further by environmental temperature inputs.

Adding complexity, the transcription factor Nr5a1, located within the PAR, exhibited differential expression patterns implicating it in the modulation of Amh. Nr5a1 is known to bind regulatory regions of Amh, potentially orchestrating a gene regulatory network governing sex determination that integrates both chromosomal and hormonal signals. Such a multifactorial regulatory model underscores the intricate biological choreography underlying sex differentiation in this species.

The utilization of nanopore sequencing technologies, especially the CycloneSEQ platform, enabled the recovery of approximately 124 million base pairs of previously uncharacterized genomic sequences, enhancing annotation completeness. This additional genetic material contains numerous genes and regulatory elements critical for unraveling the mechanisms by which temperature influences sex determination, providing an unprecedented window into epigenetic and transcriptional modulation.

From a broader perspective, these genomic resources pave the way for accelerating research across vertebrate developmental biology and evolutionary genetics. Arthur Georges, senior author from the University of Canberra, articulated the significance of having such high-resolution squamate models available for comparative studies, bridging gaps with mammalian and avian model organisms. The insights gleaned here have profound implications for understanding gene-environment interactions, sexual development, and the evolution of sex chromosome systems.

In the Chinese-led project, Qiye Li from BGI emphasized the strategic choice of Pogona vitticeps to inaugurate their novel sequencing technology, aligning scientific innovation with cultural symbolism during China’s Year of the Dragon. The unbiased, ultra-long reads afforded by CycloneSEQ transcended traditional challenges posed by repetitive and GC-rich sequences, yielding a robust and contiguous assembly. Li also highlighted the complementary nature of the two independent datasets and the exciting prospects for exploring the evolutionary genesis of the ZW system in related species.

Noteworthy is the elevated confidence resulting from two separate teams converging independently on the same candidate master sex-determining genes. This concordance, underpinned by open data sharing, provides a solid foundation for subsequent functional studies. Nonetheless, several aspects of the regulatory network remain to be elucidated, particularly regarding the interactions among transcription factors and environmental cues guiding this unique sex determination process.

The convergence of high-throughput sequencing innovation and integrative biological inquiry embodied in these publications marks a transformative moment in vertebrate genomics. As the scientific community digests these findings, forthcoming research is poised to dissect mechanistic pathways, elucidate evolutionary trajectories, and harness this knowledge for broader biological and conservation applications. The central bearded dragon emerges not only as a fascinating evolutionary subject but also as a genomic beacon illuminating the complexities of sex determination.

A webinar featuring the lead authors, scheduled for 26th August at 10:00 am UTC, will offer attendees a unique opportunity to engage directly with the investigators and delve deeper into the nuances of this groundbreaking work.

Subject of Research: Animals

Article Title: A near telomere to telomere phased genome assembly and annotation for the Australian central bearded dragon

News Publication Date: 19-Aug-2025

References:
Guo Q, Pan Y, Dai W et al., A near-complete genome assembly of the bearded dragon Pogona vitticeps provides insights into the origin of Pogona sex chromosomes. GigaScience 2025.
Patel HR, Alreja K, Reis AML, et al., A near telomere to telomere phased genome assembly and annotation for the Australian central bearded dragon Pogona vitticeps. GigaScience 2025.

Image Credits: Duminda Dissanayake

Keywords: sex determination, Pogona vitticeps, bearded dragon, genome assembly, telomere-to-telomere sequencing, nanopore sequencing, CycloneSEQ, PacBio HiFi, ZZ/ZW sex chromosomes, Anti-Müllerian hormone, Amh, Amhr2, gene dosage, temperature-dependent sex determination