One letter DNA change switches sex development in mice

Researchers at Bar-Ilan University have discovered that changing just one letter in DNA can completely alter sex development in mice. In the new study, published in Nature Communications, a single-letter insertion in a non-coding regulatory region caused XX mice, which would normally develop as females, to develop instead as males with testis and male genitalia.

The finding is especially striking because the mutation was not made in a gene itself, but in a distant stretch of DNA that helps control a key developmental gene. The study highlights the major role of the non-coding genome -- the 98 percent of DNA that does not make proteins but helps regulate when and how genes are turned on and off.

"This is a remarkable finding because such a tiny change -- just one DNA letter out of ~2.8 billion -- was enough to produce a dramatic developmental outcome," said Dr. Nitzan Gonen, from the Goodman Faculty of Life Sciences and Institute of Nanotechnology and Advanced Materials at Bar-Ilan University. "It shows that non-coding DNA can have a profound effect on development and disease."

The mutation was introduced into a regulatory element known as Enh13, which controls the activity of Sox9, a gene essential for testis development. For ovaries to develop normally, Sox9 must be kept turned off. The researchers found that Enh13 acts as a kind of molecular battle site/switch: in males, factors that promote testis development bind to it and activate Sox9 whereas in females, factors that promote ovary development bind to it and repress Sox9.

When the researchers introduced the mutation using CRISPR genome editing, that female repression failed. As a result, Sox9 was activated in XX mice, and testis developed, leading to complete internal and external male development.

The team created several mouse models with very small mutations in Enh13, including a one-base-pair insertion and a three-base-pair deletion. Both mutations caused XX mice to develop testis. The researchers then used cell-line reporter assays to understand how the mutation disrupted the normal regulatory mechanism.

The study builds on earlier work by the same group, published in 2024, which showed that other small mutations in the same regulatory element could have the opposite effect, causing XY mice to develop as females. Together, the findings suggest that Enh13 has a dual role: it acts as an enhancer in male development, but must also be repressed in female development.

Beyond its significance for basic biology, the study may have important implications for people with Differences of Sex Development (DSD), a group of conditions that affect about 1 in 4,000 births worldwide. More than half of DSD cases still lack a genetic diagnosis, even after sequencing the protein-coding parts of the genome.

"Our findings show that it is not enough to look only at genes," said Elisheva Abberbock, the PhD student leading the research. "Important disease-causing mutations may also lie in the non-coding genome, in DNA regions that control gene activity rather than encode proteins."

The researchers believe Enh13 may be just the beginning, and that many more regulatory regions in non-coding DNA may be involved in sex determination and other developmental disorders. They are now working to identify these regions systematically and test their function.

The study was led by Elisheva Abberbock together with other researchers from Bar-Ilan University. Collaborators included Dr. Ariel Afek of the Weizmann Institute and Dr. Francis Poulat of the University of Montpellier. The research was funded by the Israel Science Foundation and an ERC Starting Grant.