(CN) – Scientists have created the first gene-edited reptiles: albino lizards.
Researchers from the University of Georgia used CRISPR-Cas9 to make the index finger-sized lizards providing a technique for gene editing outside of major animal models. The researchers also show that the lizards can successfully pass gene-edited alleles for albinism to their offspring.
They published the details of their work Tuesday in the journal Cell Reports.
"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," author and associate professor at the University of Georgia, Doug Menke said in a statement.
"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."
CRISPR is an acronym for Clustered Regularly Interspaced Short Palindromic Repeats, which are the trademark of a bacterial defense system that forms the basis for genome editing technology. The system that can be programmed to target specific stretches of genetic code and to edit DNA at precise locations. Researchers can then permanently modify genes in living cells and organisms and, in the future, may make it possible to correct mutations at precise locations in the human genome in order to treat genetic causes of disease.
Gene editing typically occurs by injecting CRISPR-Cas9 gene-editing reagents into freshly fertilized eggs or single-cell zygotes. Menke explained this technique cannot be used in reptiles because lizard eggs are fertilized internally and the time of fertilization cannot be predicted.
However, Menke and his research team noticed that the transparent membrane over the lizard’s ovary allowed them to see all of the developing eggs, including which eggs were going to be ovulated and fertilized next. They decided to inject the CRISPR reagents into the unfertilized eggs within the ovaries and see if the CRISPR would still work.
"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 said. "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."
According to researchers the results indicate the CRISPR components remain active for several days, or even weeks, within the unfertilized eggs. After screening the offspring, the researchers found about 6% to 9% of the oocytes, depending on their size, produced offspring with gene-editing events.
"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 said. "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."
Menke said his team had two reasons for making the lizards albino, as opposed to editing other traits. First, when the tyrosinase albinism gene is knocked out, it results in a loss of pigmentation without being lethal to the animal. Second, since humans with albinism often have vision problems, the researchers hope to use the lizards as a model to study how the loss of this 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 said.
Studying gene functions in reptiles offers new opportunities for exploring aspects of development that are best studied in non-established animal models, Menke added. And ultimately, this gene-editing technique could be translated 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 said. "Each species undoubtedly has things to tell us, if we take the time to develop the methods to perform gene editing."
The National Science Foundation, a Society for Developmental Biology Emerging Models, National Institutes of Health and the ARCS Foundation helped fund the study.
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