Scientists at the Francis Crick Institute, in collaboration with Kent University, used gene editing technology to create litters with only female and male mice at 100% efficiency.
The study, published today in Nature Communications, shows how the technology can be used to improve animal welfare in scientific research and even agriculture.
In scientific research and agriculture, males or females are often required. For example, laboratory studies of male or female reproduction require only animals of the sex studied. In agriculture, only females are required for laying eggs and cows. This means that it is common practice to cull animals that do not require sex after birth.
The investigators' new approach uses a two-part genetic system to inactivate embryos shortly after fertilization, allowing only the sex required for development. This gene-based approach to controlling offspring sex could greatly reduce culling in both industries.
Embryo selection is based on the fact that CRISPR-Cas9 has two elements — the Cas9 enzyme that cleaves DNA, allowing scientists to change specific regions, as well as guide RNA that brings Cas9 to the correct location on the genome. The team placed an element of the system on the father's X or Y chromosome, which means that it was inherited by female or male embryos, respectively. The other element is contributed by the mother and inherited by all embryos.
They target the Top1 gene, which is essential for DNA replication and repair. When a sperm and egg form an embryo, each embryo contains half of CRISPR-Cas9, gene editing is triggered in the embryo, and it is unable to develop beyond an early stage of approximately 16 to 32 cells.
Using this method, researchers can control the sex of a litter with 100% effect. In order to produce male litters, the researchers edited the father's X chromosome, meaning that only females inherited harmful mutations. For female litters, they edited the Y chromosome.
To their surprise, this method did not result in a 50% reduction in the number of offspring produced, but between 61%-72% of control litters. Researchers believe that this is because animals such as mice produce more eggs in each ovarian cycle than needed, so that some of them are lost during early development without reducing litter size. This means that in a situation where one sex is required, fewer breeding animals will be needed in order to produce the same number of the desired offspring sex.
Since the Top1 gene is well conserved in mammals, these results may also apply to other animals.
Charlotte Douglas, first author and former PhD student and postdoctoral scientist at the Crick, said: "This approach is effective because we divide the genome editing process into two halves, between men and women, and it is activated only by reproduction when the two halves meet in embryos. Two halves of the embryo fail to develop to very early cellular stages.
"We also show the successful operation of this process in different combinations—the introduction of Cas9 or guide RNA elements into the mother or father's chromosome."
Since surviving offspring contain only half of CRISPR-Cas9 elements in their genome, this acts as a control to prevent the transmission of sex selection to offspring unless they selectively reproduce half with heterosexual individuals containing another. This is different from genetic engineering by a "gene-driven" approach, which attempts to widely spread genetic mutations in the human population.
Gene editing also had no deleterious effect on surviving offspring.
James Turner, author and group leader of the Sex Chromosome Biology Laboratory at Crick, said: "This work may have a direct and valuable impact on scientific laboratories because we have demonstrated its safety and efficacy in mice, a common mammal used in medical and scientific research. Although many studies require both sexes, some research areas require only one. 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, said: "The impact of this work may have far-reaching implications in improving animal welfare, but should be considered at the ethical and regulatory levels.”
"In particular, extensive public dialogue and debate, as well as changes in legislation, are required before any potential use in agriculture. In science, much remains to be done, which takes years. Further research needs to first develop specific gene editing toolkits for different species and then check whether they are safe and effective."