CRISPR works on individual cells, so the most effective way is to edit an organism when it only has one cell. That means that it’s usually performed on freshly-fertilized eggs, or zygotes – the single-celled precursor to an embryo. And for mammals, that works just fine.
But reptiles are another matter. In lizards, fertilization happens internally in the cloaca, but it’s impossible to tell exactly when it takes hold. It’s also much more difficult to fertilize reptile eggs in vitro and transfer them.
So, the researchers on the new study tested whether it would work on unfertilized lizard eggs. They injected CRISPR reagents directly into eggs still in the animals’ ovaries, with the aim of knocking out the tyrosinase albinism gene. That way any mutant lizards would be born albino, making it very obvious if the procedure worked.
Sure enough it did, and interestingly, the process even affected alleles (gene variants) from the father, even though his genes weren’t added until after the procedure. That suggests that the CRISPR reagents stay active within the eggs for several days or even weeks.
"Because we are injecting unfertilized eggs, we thought that we would only be able to perform gene editing on the alleles inherited from the mother,” says Doug Menke, corresponding author of the study. “Paternal DNA isn't in these unfertilized oocytes. We had to wait three months for the lizards to hatch, so it's a bit like slow-motion gene editing. But it turns out that when we did this procedure, about half of the mutant lizards that we generated had gene-editing events on the maternal allele and the paternal allele."
Afterwards, the team screened the offspring and found that between six and nine percent of the eggs went on to produce gene-edited offspring. That might not sound like a great success rate, but considering the process had never worked in reptiles at all before, this is still a big leap forward.
"Relative to the very established model systems that can have efficiencies up to 80 percent or higher, six percent seems low, but no one has been able to do these sorts of manipulations in any reptile before," says Menke. "There's not a large community of developmental geneticists that are studying reptiles, so we're hoping to tap into exciting functional biology that has been unexplored.”
The study potentially opens up the CRISPR technique for use in a range of other animals that previously couldn’t be genetically edited. The team also says that this new, easier method for creating albino lizards could help them study albinism in humans. In particular, since some lizards have similar eye structures to humans, they could be a better models for why humans with albinism often have vision problems.
According to newatlas