EDGE: Establishment of genome-editing and transgenic tools in Anolis lizards

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Genetic modification technologies have been applied to investigate gene function in a broad range of different vertebrate species. These genetic studies have produced fundamental insights into animal development and other critical biological processes. However, there are currently no effective approaches for genetically modifying any of the more than 10,000 described species of reptile. As a consequence, studies of gene function have almost completely excluded this diverse and highly successful group of animals. This project advances the brown anole lizard, Anolis sagrei, as a system for comparative studies of reptile evolution, development, and gene function. Anolis lizards have served as important models for studies of evolution, behavior, and ecology for many decades, and the brown anole is ideal for genetic studies due its small size, ease of husbandry, continuous egg production, and low cost. The project involves establishment of a new lizard model system with genome-editing capabilities to facilitate basic research studies of gene function and gene regulation in this understudied group of vertebrates. Achievement of these goals will enable the identification of genetic mechanisms that are deeply conserved across different vertebrates, as well as enable genetic studies of reptile evolution that are not currently feasible. The project also involves hosting of two Anolis workshops to train a cadre of researchers in how to create genetically modified lizards, as well as opportunities for hands-on research experiences for undergraduate and high-school students, with special emphasis on the engagement of students from underrepresented groups.This project is focused on enabling two technologies for use in the brown anole lizard: CRISPR/Cas9 genome-editing and transposon-based transgenesis. The project focuses on development of methods to surgically access maturing oocytes within adult A. sagrei females to allow the microinjection of unfertilized oocytes with genome modification reagents. Oocyte microinjection of Cas9 RNPs or transposon mRNA with appropriate transgene cassettes are performed to produce genetically modified brown anole lizards by targeted genome-editing or transgenesis, respectively. In addition, immortalized lines of A. sagrei fibroblasts are established to create a lizard cell culture system for detailed investigations of cellular mechanisms using CRISPR/Cas9. Protocols developed during this project are made freely available in the form of an Anolis Genome Manipulation Handbook. The development of genome-editing and transgenesis tools in A. sagrei will establish the first reptilian model system where functional genetic experimental approaches are available. This project is co-funded by the Evolutionary Processes program in the Division of Environmental Biology and the Behavioral Systems Cluster in the Division of Integrative Organismal Systems of the Directorate for Biological Sciences.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.