Single-sex litters of mice comprising only either female or male pups have been produced by means of so-called CRISPR-Cas9 gene editing technology.
The technique, developed by experts at the Francis Crick Institute and the University of Kent, works by inactivating embryos of one sex shortly after fertilisation.
This could help improve the welfare of animals in laboratory or agricultural environments where only male and female animals are required.
It is common for animals of the unrequired sex to be culled — a practice which could be drastically reduced by controlling the sex of the animals prior to birth.
This technique needs to be genetically modified by both parents in order for it to work. However, it is not suitable for forcing the sexing of designer babies.
Single-sex litters of mice — comprising only either female or male pups — have been produced by means of so-called CRISPR-Cas9 gene editing technology. These are the single-sex litters of mice created by crossing different species. They have black hair due to genetically modified cells. Their white coat comes from their non-modified genome.
The technique, developed by geneticist Charlotte Douglas of the Francis Crick Institute and colleagues, works by taking advantage of the two parts of the CRISPR-Cas9 gene editing tool.
The first is Cas9 — an enzyme which cuts DNA — while the second is the ‘guide RNA’ which carries Cas9 to the right location on the target genome, allowing for genes to be inserted, removed or replaced at the desired point.
In their proof-of-principle study, the researchers placed one of these two elements on either the X or Y chromosome of a soon-to-be father mouse — meaning that it will only be inherited by either his female or male embryos, respectively.
The other gene editing element was contributed by the mother mouse’s X chromosomes — meaning that it was inherited by all of the embryos.
“This works because we divide the genome editing process between a male or female. It is only when these two halves are merged in an embryo via breeding that it activates,” said Dr Douglas.
CRISPR/Cas9 elements were targeted at Top1 gene by the team. This is critical for DNA replication, repair and maintenance.
Dr Douglas said that an embryo with one half of the sexes cannot be developed beyond early cells stages.
Specifically, the embryos of the targeted sex — those given the detrimental mutation — fail to multiply beyond splitting into around 16 to 32 cells.
‘We’ve also shown this process works successfully in different combinations — introducing either the Cas9 or the guide RNA elements on to the mother’s or father’s chromosomes,’ the researcher added.
According to the team, this approach is 100% successful, and there are no adverse effects on the embryos that survive. However, contrary to popular belief, births with smaller litters do not occur by it.
Instead, the team found that the genetically altered litters ranged from 61–72 per cent of the size of control litters which were produced without any editing.
According to Dr Douglas and colleagues, this is likely because mice are among those animals that produce more eggs than are required during each ovarian cycle — allowing some to be lost during early develop without cutting into the litter size.
This boost does not mean that fewer breeding animals will be needed to reproduce the same amount of desired sex if gene editing is used.
It works by using two components of CRISPR-Cas9’s gene editing tool. The first is Cas9 — an enzyme which cuts DNA — while the second is the ‘guide RNA’ which carries Cas9 to the right location on the target genome, allowing for genes to be inserted, removed or replaced at the desired point. Depicted: CRISPR-Cas9 in operation
In the event the surviving offspring are required to reproduce, they only harbour one part of the CRISPR-Cas9 elements within their genome — meaning the sex selection would not naturally be passed down to the next generation.
Researchers noted that the ability to activate the element by breeding animals of different sexes with the second gene editing tool could be possible.
In this way, the techniques is distinct from ‘gene-drive’ approaches to genetic engineering — such as, for example, are proposed for eliminating malaria-carrying mosquitoes, — which seek to spread a given mutation through a population.
Researchers explained that the Top1 gene is found in most mammals and should be used to create tools for other animals.
‘This work could have immediate and valuable impact in scientific laboratories,’ explained paper author and geneticist James Turner of the Francis Crick Institute.
He said, “We have shown that it’s safe and effective when used with mice (a common mammal used for scientific and medical research.”
The technique could be used to improve animal welfare in both laboratory and agricultural settings where, for various reasons, only female or male animals are needed. It is common for animals of the unrequired sex to be culled — a practice which could be drastically reduced by controlling the sex of the animals prior to birth. Pictured: the poultry industry sort chicks by sex on conveyer belts — with the unwanted males typically macerated shortly after hatching
Peter Ellis, a paper author and expert in molecular genetics, stated that the implications of his work could have a significant impact on animal welfare. However, they should also be considered ethically and legally.
“In particular there must be extensive public debate and discussion before any agricultural potential uses can occur.
The scientific aspect of gene editing is still a lot to learn over the next few years. It is necessary to continue research, to create the specific gene editing toolkits needed for each species of animal, then to verify their safety and effectiveness.
Harry Leitch — a stem cell biologist at the MRC London Institute of Medical Sciences, who was not involved in the present study — agreed, saying ‘If applied to livestock species this could reduce the culling of animals and therefore make a significant impact on animal welfare.
“Although such technologies would be used in this setting, it would need extensive consultations and changes to the UK’s existing laws.
He cautioned that this system required genetic modification by both parents and is therefore not suitable for human reproduction.
Nature Communications published all findings.
WHAT IS CRISPR CAS9?
Crispr Cas9, a program that allows precise editing of DNA (found in bacteria), is available.
It stands for ‘Clustered Regularily Inter-Spaced Paindromic Repeats’.
It involves using a DNA cutter enzyme along with a tiny tag to tell the enzyme which part of it is being cut.
CRISPR/Cas9 uses tags to identify where the mutation is located and an enzyme to remove small parts of DNA.
Scientists can edit this tag to make specific cuts and target enzymes to certain regions of DNA.
It can be used to “silence” genes, effectively switching off their functions.
The cellular machinery removes small amounts of DNA when it repairs DNA breaks.
This allows researchers to precisely shut down certain genes within the genome.
The approach has been used previously to edit the HBB gene responsible for a condition called β-thalassaemia.