CRISPR-Cas9 effectors facilitate generation of single-sex litters and sex-specific phenotypes

Background

Basic research and agriculture often only need single-sex offspring, e.g. for use in basic research experiments to investigate the development of sex organs. In agriculture, for instance, the dairy industry only needs female dairy cows, while the male calves are slaughtered. The meat industry, on the other hand, prefers male cattle because they produce more meat. In the case of pigs, it is mainly the female offspring that are used because the meat of male pigs has a strong, undesirable taste. In this instance, the male offspring are either castrated or slaughtered before reaching sexual maturity. Another example is egg production: only the female chicks are needed as laying hens, and therefore the males are killed shortly after hatching.

Genetic modification can be used to eradicate the embryos of one sex so that only the offspring of the desired sex survive. This has already been demonstrated in fruit flies, mosquitoes and zebrafish. In the present study, such experiments are performed very efficiently in mammals. In an earlier study, mice were modified using CRISPR/Cas9 gene scissors to produce more females than males. However, the small number of male offspring had severe developmental defects and died at birth (Yosef et al., 2019).

Results of the study

In the current study, the scientists developed a system to produce either male-only or female-only offspring by applying CRISPR/Cas9 genetic scissors in mice. It can also be used to induce sex-specific changes in the offspring.

The target gene of the genetic scissors in the experiments is the topoisomerase 1 (Top1) gene, which is necessary for the duplication of DNA during the cell cycle. If this gene is switched off by the genetic scissors during embryonic development, the embryos die early. The special feature of this system is the distribution of the required components of the genetic scissors: the guide RNA (gRNA) that directs the genetic scissors to the Top1 target gene is inherited from one parent, and the gene encoding the Cas9 genetic scissors is inherited from the other. Using different combinations of the genetic components, it was thus possible to produce exclusively female or male offspring in the mice.

The system described in the study can be modified in such a way that other target genes in only one sex can be specifically altered with the genetic scissors. In basic research, for example, this has the advantage that a gene knockout in the offspring does not occur in both sexes, but only in the sex under investigation.

Relevance of the results

The results have many relevant aspects: for instance, the experiments appear to make sense from certain perspectives, as they can theoretically reduce the number of laboratory animals used in basic research. These results could therefore be used to implement the 3R principle, the aim of which is either to avoid animal experiments completely (replacement), or limit the number of animals (reduction) and suffering (refinement) to an absolute minimum.

However, a closer look reveals complex ethical issues and possibly also risks for humans, animals and the environment. In principle, alternatives should be discussed and the system of current food production called into question. In general, however, there are also concerns about the risks associated with such genetically modified animals. There have been several reports of unintended genomic changes in animals, which are induced by the use of the genetic scissors or the cloning procedures required for this purpose. Thus, unintended changes in other parts of the genome can be caused or additional DNA fragments incorporated. In Europe, applications of the genetic scissors in animals are covered by the Genetic Engineering Act.

The system described here does not represent a gene drive in itself. The individual genetic components and their transmission to offspring would need further adaptation for this to happen. Theoretically, however, this would be feasible. The authors themselves stated in the discussion of the study that their experiments are not intended to establish a gene drive, and that this would not be possible with the genetic components used.