EDGE CT: Precise genome editing in a lepidopteran insect tailored for stable transformation

人类已知的十分之一的物种是鳞翅目的飞蛾和蝴蝶。尽管这种生物多样性为了解这些昆虫的遗传学提供了新的机会，但实验者由于缺乏可操作的常规遗传分析实验室系统而受到限制。在这个通过基因组工具实现发现的项目中，研究小组开发了印度餐蛾作为鳞翅目基因组发现的实验室系统。这个物种是高度肥沃，耐寒，经济维持，允许发展蛾系嵌入遗传工具。特别是，研究人员优化了标记感兴趣的细胞和组织的尖端基因组编辑技术。这些努力将为昆虫的生理学、免疫学、生殖、发育遗传学和进化等方面的研究开辟新的途径，这些昆虫具有巨大的多样性，包括许多具有经济和生态重要性的物种。为了最大限度地发挥全球影响，该项目包括培训年轻科学家，以及组建一个基因组编辑协作社区，以对付世界各地粮食储存单位和家庭都有的害虫。为了解决鳞翅目常规功能基因组学模型系统的缺乏，这个通过基因组工具实现发现的项目将开发和传播该谱系的组织特异性检测和基因组编辑标准。除了作为世界各地储存食物的重要害虫外，由于其独特的繁殖和饲养特征（例如，易于维护和低成本，可注射的卵，对近亲繁殖的抗性，成功的CRISPR靶向），可用的基因组序列以及便于筛选荧光标记的半透明阶段和组织，这种生物被量身定制为基因组编辑。特别是，CRISPR同源性定向修复（HDR）的变异使用显性拯救试验进行优化，其中数千个注射个体被筛选用于隐性突变的靶向修复。优化实验旨在对短等位基因替换和长DNA敲入蛋白转化方法的体内性能进行有意义的比较，并催化其他昆虫新兴系统的进一步创新。除了用于基础研究之外，这里开发的工具有望加强作物害虫和森林落叶动物的综合管理，并为重要的食物链组成部分和传粉媒介提供生物学信息。最后，Plodia本身在世界范围内分布，对全球粮食安全构成威胁。因此，低成本的维护和来自边缘的资源将在国际范围内刺激这一系统的工作。作为该项目的一部分，研究小组在科学会议上组织专题讨论会，并培训一群有兴趣在实验室中使用Plodia的科学家。该项目由生物科学理事会综合有机体系统行为系统集群资助。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

One out of ten species known to humans are moths and butterflies of the Lepidoptera order. Although this biodiversity provides new opportunities to understand the genetics of those insects, experimenters have been limited by the lack of a tractable laboratory system for routine genetic analysis. In this Enabling Discovery through Genomic Tools project, the team of investigators develop the Indian-meal moth as a laboratory system for discovery in lepidopteran genomes. This species is highly fertile, hardy, economical to maintain, allowing the development of moth lines embedded with genetic tools. In particular, the investigators optimize cutting-edge genome editing techniques for marking cells and tissues of interest. These efforts will open new avenues of research about the physiology, immunology, reproduction, developmental genetics, and evolution of an insect order of enormous diversity that includes numerous species of economic and ecological importance. To maximize worldwide impact, this project includes the training of young scientists, and the assemblage of a collaborative community of genome editors working with a pest that infests grain storage units and households alike all over the world.To address the lack of model system for routine functional genomics in Lepidoptera, this Enabling Discovery through Genomic Tools project will develop and disseminate tissue-specific assays and genome editing standards fro this lineage using the mealmoth Plodia interpunctella. In addition of being an important pest of stored food across the world, this organism is tailored for genome editing due to a unique suit of reproductive and rearing features (e.g., ease and low-cost of maintenance, injectable eggs, resistance to inbreeding, successful CRISPR targeting), an available genome sequence, and translucent stages and tissues that facilitate the screening of fluorescent markers. In particular, variations of CRISPR Homology-Directed Repair (HDR) is optimized using a dominant rescue assay in which thousands of injected individuals are screened for the targeted repair of a recessive mutation. Optimization assays are designed to yield meaningful comparisons of the in vivo performances of transformation methods for short allele replacements and long DNA knock-ins, and to catalyze further innovation across other insect emerging systems. Beyond their use in fundamental research, the tools developed here are expected to potentiate the integrated management of crop pests and forest defoliators, and to inform the biology of important food chain components and pollinators. Finally, Plodia itself shows a worldwide distribution and presents a threat to global food security. The low-cost of maintenance and EDGE-derived resources will thus stimulate work in this system at an international scale. As part of this project, the team of investigators organizes symposia at scientific meetings and trains a community of scientists interested in working with Plodia in their laboratories. This project is funded by the Behavioral Systems Cluster of Integrative Organismal Systems in 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.