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蛾和蝴蝶。虽然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，但由于缺乏易于处理的实验室系统进行常规遗传分析，实验人员受到了限制。在这个通过基因组工具进行发现的项目中，研究人员团队开发了印度谷蛾作为发现鳞翅目基因组的实验室系统。这一物种是高度肥沃的，哈代，经济的维护，允许嵌入遗传工具的蛾系的发展。特别是，研究人员优化了用于标记感兴趣的细胞和组织的尖端基因组编辑技术。这些努力将开辟新的途径，研究生理学，免疫学，生殖，发育遗传学和进化的昆虫命令的巨大多样性，包括许多物种的经济和生态重要性。为了最大限度地扩大全球影响，该项目包括培训年轻科学家，并组建一个基因组编辑协作社区，与世界各地的粮食储存单位和家庭一样，对害虫进行研究。为了解决鳞翅目常规功能基因组学缺乏模型系统的问题，这个通过基因组工具进行发现的项目将开发和传播组织-使用粉蛾Plodia interpunctella的该谱系的特异性测定和基因组编辑标准。除了是世界各地储存食物的重要害虫之外，由于独特的繁殖和饲养特征（例如，容易和低成本的维护、可注射的卵子、对近亲繁殖的抗性、成功的CRISPR靶向）、可用的基因组序列以及促进荧光标记物筛选的半透明阶段和组织。特别地，CRISPR同源性定向修复（HDR）的变化使用显性拯救测定法进行优化，其中筛选数千个注射的个体用于隐性突变的靶向修复。优化测定被设计为产生用于短等位基因置换和长DNA敲入的转化方法的体内性能的有意义的比较，并催化跨其他昆虫新兴系统的进一步创新。除了在基础研究中的应用外，这里开发的工具有望加强作物害虫和森林落叶剂的综合管理，并为重要食物链组成部分和传粉者的生物学提供信息。最后，Plodia本身显示出世界范围的分布，并对全球粮食安全构成威胁。因此，维持费用低和电子数据交换所产生的资源将在国际范围内促进这一系统的工作。作为该项目的一部分，研究人员团队在科学会议上组织研讨会，并培训有兴趣在实验室与Plodia合作的科学家社区。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NSF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.