CRISPR-Cas9 Used to Create Single Sex Litters in Mice with 100% Efficiency

CRISPR-Cas9 Used to Create Single Sex Litters in Mice with 100% Efficiency

The CRISPR-Cas9 gene editing tool has been used in a proof-of-concept study to produce single-sex mice litters with 100% efficiency.

The ability to influence the sex of animals has great value in both scientific research and agriculture. Certain animal studies can only be conducted on a single-sex – studies on female reproduction for example. In agriculture, cows are required by the dairy industry as only females produce milk and only female chickens produce eggs. Since the sex ratio is 50:50, that means that around half of animals – the males – are culled at birth. That is neither efficient nor desirable.

Researchers at the Francis Crick Institute and the University of Kent turned to the CRISPR gene editing tool to test whether it is possible to safely create single-sex litters in mice to solve this problem. The researchers found a way to inactivate embryos shortly after fertilization to only allow a specific sex to develop.

The CRISPR-Cas9 gene editing tool uses the Cas9 enzyme component to cut the DNA, with the location of that cut determined by a guide RNA. The guide RNA ensures the Cas9 enzyme only makes the cut at a very specific point. The tool is used to alter the function of genes. The researchers used the tool to insert an element into either the father’s X or Y chromosome, which will see it passed on to female embryos if on the X chromosome or male embryos if added to the Y chromosome. The other chromosome is inherited from the mother and is inherited by male and female embryos. In this case, the CRISPR-Cas9 system was split across the chromosomes, with the mother contributing the guide RNA component and the father contributing the Cas9 enzyme or vice versa.

The researchers used this tool to target the Top1 gene, which is involved in DNA replication. When the Cas9 and guide RNA are successfully combined, an edit is made to the Top1 gene which disrupts DNA replication and inactivates the embryos, preventing a specific sex from developing beyond the 16 to 32 cell stage.

The researchers tested their technique by breeding 72 litters, with half targeting each sex and every pup born was of the desired sex. Since the ratio of male to female pups is 50:50 in normal litters, the researchers naturally expected litter sizes to be half the size, but in the experiments, the litter size was 61% to 72% the size of the unedited control group.

The researchers found no undesirable side effects in the sex-selected mice, which could go on to have normal litters with a 50/50 sex ratio unless they were bred with mice with the corresponding part of the CRISPR system added.

Since the Top1 gene is conserved across all mammal species, this technique could be used to breed single sex litters in any mammal, although tests would need to be conducted to determine whether this system works effectively in other species of mammal.

“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,” said Peter Ellis, author and senior lecturer in molecular genetics and reproduction at the University of Kent

You can read more about the study in the paper – CRISPR-Cas9 effectors facilitate generation of single-sex litters and sex-specific phenotypes – which was recently published in Nature Communications. DOI: 10.1038/s41467-021-27227-2