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Researchers have, for the first time, succeeded in using CRISPR-Cas9 to genetically engineer the reptilian genome. Scientists at the University of Georgia developed a method to mutate the tyrosinase gene (TYR) in Anolis sagrei, an important model system used to study reptile biology and evolution. Rasys et al. saw fit to design this study around TYR because a wide variety of vertebrate species possess this gene, and loss-of-function tyr mutants are viable. Furthermore, since tyrosinase plays a critical role in the production of the skin pigment melanin, effects of mutations to tyr, such as albinism, can be easily observed from the lizard’s phenotype.
Up till now, genetic manipulation of the Anolis genome has been a particularly challenging endeavor due to the complex nature of their reproductive development. Factors such as internal fertilization, extended storage of sperm within females, the fragility of yolk-filled oocytes and the simultaneous timelines of embryonic and egg shell development, all make delivering CRISPR-Cas9 components into oocytes difficult to time and technically challenging to execute. To get around these challenges, researchers surgically exposed the Anolis ovary and microinjected Cas9 ribonucleoprotein complex into immature, unfertilized follicles. They then sealed the surgical incision and allowed the female lizard to recover. Fertilization and egg development through to hatching were allowed to progress normally. Of the 146 oocytes that were injected from a total of 21 female lizards, 9 F0 animals bearing mutations in tyr were hatched. Excitingly, 4 of those hatched were albino and possessed loss-of-function mutations in both tyr alleles. The other 5 animals were heterozygous, carrying loss-of-function mutations in just one tyr allele, and possessed pigmentation similar to wildtype Anolis lizards.
This research work highlights a new cost effective and time-efficient way to study gene function in reptiles, and is a method that can likely be successfully adapted to a wide variety of lizard and snake species.
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