In collaboration with the University of Kent, scientists at the Francis Crick Institute have created female-only and male-only mice litter using gene-editing technology. They were able to create single-sex mice litters with 100% efficiency.
Animals are essential genetic tools in scientific research and global resources in agriculture. In both areas, a single-sex is often required in surplus. This means it is common practice for animals of the unrequired sex to be culled after birth.
This new method uses the two-part genetic system to inactivate embryos shortly after fertilization. This inactivation allows the development of the desired sex only. As scientists noted, such a gene-editing method could drastically reduce culling in both industries.
The embryo selection is based on the fact that there are two elements of CRISPR-Cas9 – the Cas9 enzyme that cuts the DNA. This enabled scientists to modify certain regions and the guide RNA which carries the Cas9 to the right location on the genome.
Scientists placed one element of the system on the father’s X or Y chromosome, meaning that it will only be inherited by female or male embryos respectively. The other element is contributed by the mother and is inherited by all embryos.
The Top1 gene is essential to DNA replication and repair. Scientists mainly targeted this gene. 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 around 16 to 32 cells.
Scientists produced a male-only litter by editing the father’s X chromosome. It means, only females inherited the deleterious mutation. On the other hand, they edited the Y chromosome to create a female-only litter.
This new method did not cause a 50% decrease in the number of offspring produced, rather the litter sizes were between 61% – 72% of the control litters. This is due to animals such as mice producing more eggs than required during each ovarian cycle. This allows for a proportion of them to be lost during early development without reducing litter size. 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.
Charlotte Douglas, first author and former Ph.D. student and postdoctoral scientist at the Crick, says: “This method works as we split the genome editing process in half, between a male and female, and it is only when the two halves meet in an embryo through breeding, that it is activated. Embryos with both halves cannot develop beyond very early cell stages.”
“We’ve also shown this process works successfully in different combinations – introducing either the Cas9 or the guide RNA elements onto the mother’s or father’s chromosomes.”
As the offspring endure half of the CRISPR-Cas9 elements inside their genome, this acts as a control preventing the sex selection from being passed down to further generations, except if they are specifically bred with an individual of the other gender containing the other half. This is different from genetic engineering through ‘gene-driven methods, which seek to spread a genetic mutation widely amongst a population.
James Turner, author and group leader of the Sex Chromosome Biology Laboratory at the Crick says: “This work could have an immediate and valuable impact in scientific laboratories, as we’ve shown how it is safe and effective in mice, a common mammal used in medical and scientific research. While a lot of research needs both sexes, there are areas of study where only one is needed. For example, when studying the reproductive system, sex-specific diseases, or certain hormones.”
Peter Ellis, author and senior lecturer in molecular genetics and reproduction at the University of Kent, says: “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. On the scientific side, there is also much work to be done over a number of years. Further research is needed, first to develop the particular gene-editing toolkits for different species, and then to check they are safe and effective.”
- Douglas, C., Maciulyte, V., Zohren, J. et al. CRISPR-Cas9 effectors facilitate generation of single-sex litters and sex-specific phenotypes. Nat Commun 12, 6926 (2021). DOI: 10.1038/s41467-021-27227-2