Researchers get viable mice by editing DNA from two sperm

For many species, producing an embryo is a bit of a contest between males and females. Males want as many offspring as possible and want the females to devote as many resources as possible to each of them. Females do better by keeping their options open and distributing resources in a way to maximize the number of offspring they can produce over the course of their lives.

In mammals, this plays out through the chemical modification of DNA, a process called imprinting. Males imprint their DNA by adding methyl modifications to it in a way that alters the activity of genes in order to promote the growth of embryos. Females do similar things chemically but focus on shutting down genes that promote embryonic growth. In a handful of key regions of the genome, having only the modifications specific to one sex is lethal, as the embryo can’t grow to match its stage of development.

One consequence of this is that you normally can’t produce embryos using only the DNA from eggs or from sperm. But over the last few years, researchers have gradually worked around the need for imprinted sites to have one copy from each parent. Now, in a very sophisticated demonstration, researchers have used targeted editing of methylation to produce mice from the DNA of two sperm.

Imprinting and same-sex parents

There’s a long history of studying imprinting in mice. Long before the genome was sequenced, people had identified specific parts of the chromosomes that, if deleted, were lethal—but only if inherited from one of the two sexes. They correctly inferred that this meant that the genes in the region are normally inactivated in the germ cells of one of the sexes. If they’re deleted in the other sex, then the combination that results in the offspring—missing on one chromosome, inactivated in the other—is lethal.

Over time, seven critical imprinted regions were identified, scattered throughout the genome. And, roughly 20 years ago, a team managed to find the right deletion to enable a female mouse to give birth to offspring that received a set of chromosomes from each of two unfertilized eggs. The researchers drew parallels to animals that can reproduce through parthenogenesis, where the female gives birth using unfertilized eggs. But the mouse example obviously took a big assist via the manipulation of egg cells in culture before being implanted in a mouse.

By 2016, researchers were specifically editing in deletions of imprinted genes in order to allow the creation of embryos by fusing stem cell lines that only had a single set of chromosomes. This was far more focused than the original experiment, as the deletions were smaller and affected only a few genes. By 2018, they had expanded the repertoire by figuring out how to get the genomes of two sperm together in an unfertilized egg with its own genome eliminated.

The products of two male parents, however, died the day after birth. This is either due to improperly compensating for imprinting or simply because the deletions had additional impacts on the embryo’s health. It took until earlier this year, when a very specific combination of 20 different gene edits and deletions enabled mice generated using the chromosomes from two sperm cells to survive to adulthood.

The problem with all of these efforts is that the deletions may have health impacts on the animals and may still cause problems if inherited from the opposite sex. So, while it’s an interesting way to confirm our understanding of the role of imprinting in reproduction, it’s not necessarily the route to using this as a reliable reproductive tool. Which finally brings us to the present research.

Roll your own imprinting

Left out of the above is the nature of the imprinting itself: How does a chunk of chromosome and all the genes on it get marked as coming from a male or female? The secret is to chemically modify that region of the DNA in a way that doesn’t alter base pairing, but does allow it to be recognized as distinct by proteins. The most common way of doing this is to link a single carbon atom (a methyl group) to the base cytosine. This tends to shut nearby genes down, and it can be inherited through cell division, since there are enzymes that recognize when one of the two DNA strands is unmodified and adds a methyl to it.

Methylation turns out to explain imprinting. The key regions for imprinting are methylated differently in males and females, which influences nearby gene activity and can be maintained throughout all of embryonic development.

So, to make up for the imprinting problems caused when both sets of chromosomes come from the same sex, what you need to do is a targeted reprogramming of methylation. And that’s what the researchers behind the new paper have done.

First, they needed to tell the two sets of chromosomes apart. To do that, they used two distantly related strains of mice, one standard lab strain that originated in Europe and a second that was caught in the wild in Thailand less than a century ago. These two strains have been separated for long enough that they have a lot of small differences in DNA sequences scattered throughout the genome. So, it was possible to use these to target one or the other of the genomes.

This was done using parts of the DNA editing systems that have been developed, the most famous of which is CRISPR/CAS. These systems have a protein that pairs with an RNA sequence to find a matching sequence in DNA. In this case, those RNAs could be made so that they target imprinting regions in just one of the two mouse strains. The protein/RNA combinations could also be linked to enzymes that modify DNA, either adding methyls or removing them.

To bring all this together, the researchers started with an egg and deleted the genome from it. They then injected the heads of sperm, one from the lab strain, one from the recently wild mouse. This left them with an egg with two sets of chromosomes, although a quarter of them would have two Y chromosomes and thus be inviable (unlike the Y, the X has essential genes). Arbitrarily, they chose one set of chromosomes to be female and targeted methylation and de-methylation enzymes to it in order to reprogram the pattern of methylation on it. Once that was done, they could allow the egg to start dividing and implant it into female mice.

Rare success

The researchers spent time ensuring that the enzymes they had were modifying the methylation as expected and that development started as usual. Their general finding is that the enzymes did change the methylation state for about 500 bases on either side of the targeted site and did so pretty consistently. But there are seven different imprinting sites that need to be modified, each of which controls multiple nearby genes. So, while the modifications were consistent, they weren’t always thorough enough to result in the expected changes to all of the nearby genes.

This limited efficiency showed up in the rate of survival. Starting with over 250 reprogrammed embryos that carried DNA from two males, they ended up with 16 pregnancies, but only four that died at birth, and three live ones; based on other experiments, most of the rest died during the second half of embryonic development. Of the three live ones, one was nearly 40 percent larger than the typical pup, suggesting problems regulating growth—it died the day after birth.

All three live births were male, although the numbers are small enough that it’s impossible to tell if that’s significant or not.

The researchers suggest several potential reasons for the low efficiency. One is simply that, while the probability of properly reprogramming at least one of the sites is high, reprogramming all seven is considerably more challenging. There’s also the risk of off-target effects, where the modification takes place in locations with similar sequences to the ones targeted. They also concede that there could be other key imprinted regions that we simply haven’t identified yet.

We would need to sort that out if we want to use this approach as a tool, which might be potentially useful as a way to breed mice that carry mutations that affect female viability or fertility. But this work has already been useful even in its inefficient state, because it serves as a pretty definitive validation of our ideas about the function of imprinting in embryonic development, as well as the critical role methylation plays in this process. If we weren’t largely right about both of those, the efficiency of this approach wouldn’t be low—it would be zero.

PNAS, 2025. DOI: 10.1073/pnas.2425307122  (About DOIs).