Mice With Two Dads Reach Adulthood Thanks to CRISPR

At first glance, the seven mice skittering around their cages look like other mice. But they have an unusual lineage: They were born with DNA from two dads. The mice join an elite group of critters born from same-sex parents, paving the way for testing in larger animals, such as monkeys.

Led by veteran reproductive researchers Wei Li and Qi Zhou at the Chinese Academy of Sciences, the results “blew us away,” wrote Lluís Montoliu at the National Biotechnology Center in Madrid, who was not involved in the study.

Although mice with two dads have been born before, scientists used a completely different strategy in this study, which also provided insights into a reproductive mystery. In a process called “imprinting,” some genes in embryos are switched on or off depending on whether they come from the biological mom or dad. Problems with imprinting often damage embryos, halting their growth.

In the new study, the team hunted down imprinted genes in embryos made from same-sex parents, drawing an intricate “fingerprint” of their patterns. They then zeroed in on 20 genes and tinkered with them using the gene-editing tool CRISPR. Hundreds of experiments later, the edited embryos—made from two male donors—led to the birth of seven pups that grew to adulthood.

Imprinting doesn’t just affect reproduction. Hiccups in the process can also impair biomedical technologies relying on embryonic stem cells, animal cloning, or induced pluripotent stem cells (iPSCs). Changes in imprinting are complex and hard to predict, with “no universal correction methods,” wrote the team.

“This work will help to address a number of limitations in stem cell and regenerative medicine research,” said Li in a press release.

Genetic Civil War

The cardinal rule of reproduction in mammals is still sperm meets egg. But there are now more options, beyond nature’s design, for where these reproductive cells come from. Thanks to iPSC technology, which returns skin cells to a stem cell-like state, lab-made egg and sperm cells are now possible.

Scientists have engineered functional eggs and ovaries and created mice pups born from same-sex parents. Li’s team created the first mice born from two mothers in 2018. Compared to their peers, the mice were smaller, but they lived longer and were able to become moms.

The key was unlocking a snippet of the imprinting code.

Egg and sperm each carry half of our DNA. However, when the two sources of DNA meet, they can butt heads. For example, similar sections of the genetic code from mom could encode smaller babies for easier birth, whereas those from dad may encode larger, stronger offspring for better survival once born. In other words, balancing both sides is key.

Embryos made from same-sex gametes don’t “survival naturally,” wrote Montoliu.

Evolution has a solution: Shut off some DNA so that offspring only have one active copy of a gene, either from mom or dad. This trade-off prevents a DNA “civil war” in early embryos, allowing them to grow. Li’s team hunted down three essential DNA regions involved in imprinting and used CRISPR to delete those letters in one mom’s DNA. The edit wiped out the marks, essentially transforming the cell into a pseudo-sperm that, when injected into an egg, led to healthy baby mice.

But the process didn’t work for two dads. Here, the goal was to erase imprinted marks from male donor cells and turn them into pseudo-eggs. Despite editing up to seven genes that control imprinting, only roughly two percent of the efforts led to live births. None of the pups survived until adulthood.

Double Dad

Making offspring from two males is notoriously difficult, often triggering failure far sooner than in embryos with DNA from two mothers.

Scientists have used skin cell-derived iPSCs to make egg cells from male donors. But in previous studies, when fertilized with donor sperm, the lab-made eggs led to early embryos with severe imprinting problems. After being transferred to surrogate mothers, they eventually developed defects causing termination. The results suggested that the normal imprinting that balances gene expression from both mom and dad is critical for embryos to flourish.

There are about 200 imprinted genes currently linked to embryo development. Here, the team targeted 20 for genetic editing.

In a complicated series of experiments, they first made “haploid cells.” These cells only contain half the genetic material from a male donor. Using CRISPR, the team then individually modified each imprinting site to shut down the related gene’s activity. Some edits deleted the gene altogether; others added mutations to inhibit its function. More genetic edits to “regulatory” DNA further dampened their activity.

The result was a Frankenstein cell similar to a gamete, but carrying half the genome and with parental imprints wiped out. Next, the scientists injected the edited cells along with normal sperm—the “parental donor”—into an egg with its nucleus and DNA removed. The resulting fertilized egg now had a full set of DNA, with each half coming from male parents.

The approach worked—to a point. When transplanted into surrogate mothers, a fraction of the early embryos grew into mouse pups. Seven eventually reached adulthood. The genetic tweaks also improved placental health, a prior roadblock in the study of mice with same-sex parents.

“These findings provide strong evidence that imprinting abnormalities are the main barrier to mammalian unisexual reproduction,” said study author Guan-Zheng Luo at Sun Yat-sen University.

The work adds to a previous study that created pups from two dads. Helmed by Katsuhiko Hayashi at Osaka University, a team of scientists leveraged a curious quirk of iPSC transformation at the chromosome level—a completely different method than that pursued in the current study. Those mice grew into adults and went on to have pups of their own.

When first sharing those results at a conference, the audience was left “gasping and breathless,” wrote Montoliu.

The new study’s mice had health struggles. They had a larger frame, a squished nose, and a wider head—signs often associated with parental imprinting. They were also less anxious when roaming a large, open field than would normally be expected. Each mouse’s hippocampus, a brain area related to learning, memory, and emotions, was smaller than usual. And they were infertile, with a far shorter lifespan.

Given these problems, the method is hardly ready for clinical use. Tampering with genes in human reproductive cells is currently banned in many countries.

That said, the work is “impressive in its technical complexity,” Martin Leeb at Max Perutz Labs Vienna told Chemical and Engineering News, who was not involved in the study. “I would have personally thought it probably requires even more genetic engineering to get these bi-paternal mice born.”

The team is exploring other genetic tweaks to further improve the process and learn more about imprinting. Meanwhile, they’re planning to extend the method to monkeys, whose reproduction is far more similar to ours.