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

基因修饰技术已被应用于广泛的不同脊椎动物物种的基因功能研究。这些遗传学研究对动物发育和其他关键的生物过程产生了基本的见解。然而，目前还没有有效的方法来对所描述的1万多种爬行动物中的任何一种进行基因改造。因此，对基因功能的研究几乎完全排除了这种多样性和高度成功的动物群体。该项目将棕色变色蜥蜴作为爬行动物进化、发育和基因功能比较研究的系统。几十年来，Anolis蜥蜴一直是进化、行为和生态学研究的重要模型，而棕色Anolis蜥蜴因其体积小、易于饲养、连续产蛋和成本低而成为遗传研究的理想动物。该项目包括建立一个新的具有基因组编辑能力的蜥蜴模型系统，以促进对这一未被研究的脊椎动物群体的基因功能和基因调控的基础研究。这些目标的实现将使识别在不同脊椎动物中高度保守的遗传机制成为可能，并使目前尚不可行的爬行动物进化的基因研究成为可能。该项目还包括主办两个Anolis研讨会，培训一批研究人员如何创造转基因蜥蜴，并为本科生和高中生提供实践研究经验的机会，特别强调来自代表性不足群体的学生的参与。该项目重点是使两种技术用于棕色变态蜥蜴：CRISPR/Cas9基因组编辑和基于转座子的转基因。该项目的重点是开发以手术方式获取成年萨格雷雌蜂体内成熟卵母细胞的方法，以允许用基因组修饰试剂显微注射未受精的卵母细胞。通过向卵母细胞显微注射Cas9 RNPs或转座子mRNA和适当的转基因小盒，分别通过靶向基因组编辑和转基因获得转基因棕色变态蜥蜴。此外，建立了蜥蜴成纤维细胞永生化系，以建立蜥蜴细胞培养系统，用于使用CRISPR/Cas9详细研究细胞机制。在该项目期间开发的协议以Anolis基因组操作手册的形式免费提供。SAGREI基因组编辑和转基因工具的开发将建立第一个可利用功能遗传学实验方法的爬行动物模型系统。该项目由环境生物学部门的进化过程计划和生物科学局综合组织系统部门的行为系统集群共同资助。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.