Laboratory mice are crucial to scientific testing and research. In some instances, however, one is more crucial than the one, namely when studying the reproductive system, sex-specific diseases or certain hormones.
In the lab, as well as in farming and agriculture, it’s common practice for animals of the unrequired sex to be culled after birth. Now, scientists have devised a better way work with non-crucial animals—make sure they are not born in the first place.
Using the CRISPR-Cas9 gene editing technique, scientists at the Francis Crick Institute in collaboration with University of Kent have succeed in creating female-only and male-only mice litters with 100% efficiency. And while safety and ethicality are always top of mind when it comes to CRISPR-Cas9, the researchers confirmed there are no harmful effects of the gene edit in the surviving offspring and—most importantly—the “sex-selection” is not passed down to further generations.
The new method hinges on the fact that there are two elements of CRISPR-Cas9—the Cas9 enzyme that cuts the DNA, and the guide RNA that carries the Cas9 to the right location on the genome. In this study, published in Nature Communications, the scientists placed one element of the gene editing system on the father’s X or Y chromosome, meaning it was only to be inherited by female or male embryos, respectively. The other element was contributed by the mother, and was inherited by all embryos.
When an embryo formed from a sperm and egg, each containing one half of CRISPR-Cas9, the gene-editing was triggered in the embryo and it was not able to develop beyond a very early stage of 16 to 32 cells. To produce a male-only litter, the researchers edited the father’s X chromosome, meaning only females inherited the deleterious mutation, while for a female-only litter, the team edited the Y chromosome.
To induce embryo loss prior to implantation, the scientists chose to target the DNA replication and repair gene Topoisomerase 1 (Top1). This worked extremely well, allowing the team to control the sex of the litter 100% of the time using the CRISPR-based method.
While the researchers were initially worried about negatively affecting the size of the litter when controlling sex, they were pleasantly surprised to find the method did not lead to a 50% decrease in offspring. Rather, litter sizes were between 61 to 72% of the control litters.
“This is because animals such as mice produce more eggs than required during each ovarian cycle, allowing for a proportion of them to be lost during early development without reducing litter size,” the researchers explain in their paper. “This means that in situations where one sex is needed, fewer breeding animals will be required in order to produce the same number of the desired sex of offspring.”
While the work may have an immediate impact in the lab, the researchers acknowledged there is more to be done, especially if the method makes its way to agriculture—which is a possibility considering the Topoisomerase 1 gene is well conserved across mammals.
“The implications of this work are potentially far-reaching when it comes to improving animal welfare, but should be considered at ethical and regulatory levels. In particular, before any potential use in agriculture, there would need to be extensive public conversation and debate, as well as changes to legislation,” said Peter Ellis, co-author and senior lecturer in molecular genetics and reproduction at University of Kent. “On the scientific side, there is also much work to be done. Further research is needed, first to develop the particular gene editing toolkits for different species, and then to check they are safe and effective.”
Photo: The black coat of the mouse refers to the genetically modified cells, the white is the non-modified cells. The researchers breed these animals to produce offspring which are 100% genetically modified, containing one half of CRISPR-Cas9. Credit: The Francis Crick Institute