Read Time:

In a world first, researchers at the University of Georgia have used CRISPR-Cas9 to make gene-edited reptiles: albino lizards that are roughly the size of your index finger.



The study provides a technique for gene-editing outside of traditionally used animal models: "For quite some time we've been wrestling with how to modify reptile genomes and manipulate genes in reptiles, but we've been stuck in the mode of how gene editing is being done in the major model systems," says corresponding author Doug Menke, associate professor at the University of Georgia. "We wanted to explore anole lizards to study the evolution of gene regulation, since they've experienced a series of speciation events on Caribbean islands, much like Darwin's finches of the Galapagos."



In the study, the scientists also demonstrate that the lizards successful pass gene-edited alleles for albinism to their offspring.



Their findings are published in the journal Cell Reports. As lizards have internal fertilization processes that cannot be predicted, the traditional route of injecting CRISPR-Cas9 gene-editing reagents into freshly fertilized eggs or single-cell zygotes was not an option. Instead, the researchers opted to inject CRISPR reagents directly into the unfertilized eggs within the ovaries. The transparent membrane covering the ovary in lizards permits visibility of developing eggs in vivo, including the eggs that are due to be ovulated and fertilized next.



Slow-motion gene-editing



"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. Paternal DNA isn't in these unfertilized oocytes," Menke says. "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."



These findings suggest that the CRISPR reagents remain active for a several days or perhaps even weeks within the unfertilized eggs. Once the offspring were born, the researchers screened them and found that roughly six – nine percent of the oocytes produced offspring with gene-editing events. This was dependent on the size of the lizard.



The authors address the fact that this percentage seems particularly low: "Relative to the very established model systems that can have efficiencies up to 80% or higher, 6% seems low, but no one has been able to do these sorts of manipulations in any reptile before," Menke says. "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."



Why make the lizards albino?



Menke and team explain that there are two reasons as to why they chose to make the lizards albino:





When the tyrosinase albinism gene is knocked out, it results in a loss of pigmentation but is not lethal to the animal



Humans with albinism typically have vision problems, and so the scientists hope to use the lizards as a model to explore how the loss of the gene impacts retina development





"Humans and other primates have a feature in the eye called the fovea, which is a pit-like structure in the retina that's critical for high-acuity vision. The fovea is absent in major model systems, but is present in anole lizards, as they rely on high-acuity vision to prey on insects," Menke says.



Taking the leap to study gene function in reptiles opens new doors for exploring the aspects off development that are best studied in non-established animal models, according to Menke. There is the potential of translating the gene-editing technique for use in other animals.



"We never know where the next major insights are going to come from, and if we can't even study how genes work in a huge group of animals, then there's no way to know if we've explored everything there is to explore in the realm of gene function in animals," Menke says. "Each species undoubtedly has things to tell us, if we take the time to develop the methods to perform gene editing."



Reference: Rasys et al. 2019. CRISPR-Cas9 Gene Editing in Lizards through Microinjection of Unfertilized Oocytes. DOI: https://doi.org/10.1016/j.celrep.2019.07.089. Cell Reports.