MAGIC tools for genome-wide mosaic analysis with existing Drosophila resources

项目摘要/摘要 该项目旨在开发一个完整的工具箱，为镶嵌分析的新技术，是兼容 几乎所有现有的黑腹果蝇资源，不需要进一步的遗传修改。 镶嵌分析是研究人类疾病分子和细胞机制的有力手段。通过 在其他杂合子动物中产生纯合细胞，镶嵌技术允许组织特异性 对多效性基因的分析，并在生物学的许多重要发现中发挥了关键作用。当前 果蝇的镶嵌技术主要依赖于FLP/FRT位点特异性重组系统，需要 一对同源染色体，每条染色体在着丝粒附近都含有一个FRT序列。然而，大多数 果蝇现有的遗传资源，如缺乏文库、转座子干扰的突变体库、 和来自野生自然种群的菌株不具有适当的FRT位点，从而阻止了它们的有效 在马赛克分析中的应用。不依赖于特定部位重组的新镶嵌技术 需要系统来释放这些现有资源的全部潜力。利用gRNA诱导的马赛克分析 交叉(魔术)是一种新的镶嵌技术，不需要特定位置的重组，并且 与未修饰的染色体相容。尽管魔法的有效性已经在 生殖系、神经系统和上皮组织，魔术试剂目前仅适用于单一 染色体臂。这个项目将首先建立一个优化的魔术工具包，可以用于马赛克 对整个果蝇组织的整个基因组进行分析。魔术屏幕也将被引导到 确定与神经元和上皮细胞的形态缺陷相关的缺陷系。 具体地说，提出了四个目标：(1)为所有果蝇建立一个优化的、完整的魔法工具包 染色体臂；(2)神奇地在不同组织的前体细胞中产生表达Cas9的菌株 应用；(3)开发抗CRISPR工具，用于安全和多功能的果蝇魔法应用；以及(4) 用魔术筛选与神经元和上皮形态发生有关的基因缺陷系。这个 在这个项目中开发的果蝇品系和结构将捐赠给布鲁明顿果蝇 STOCK中心和质粒库，便于分发给研究社区。这个 筛查结果将公之于众，以便进一步确定和表征责任人 感兴趣的实验室的基因。已经成立了一个咨询委员会，为该项目提供反馈意见。 将征求社区对要生成的候选前兆Cas9线路的意见。建议数 神奇的工具将允许在系统的基因功能分析中利用现有的遗传资源，基因组- 广泛的基因筛查，以及对自然变异的组织特异性分析。实现该项目的目标将 为果蝇社区提供重要的工具和信息，可以大大提高 了解人类疾病的现有果蝇资源。

Project Summary/Abstract This project aims to develop a complete toolbox for a new technique of mosaic analysis that is compatible with nearly all existing Drosophila melanogaster resources, without the need for further genetic modifications. Mosaic analysis is a powerful approach for studying molecular and cellular mechanisms of human disease. By generating homozygous cells in otherwise heterozygous animals, mosaic techniques allow tissue-specific analysis of pleiotropic genes and have played key roles in many important discoveries in biology. Current mosaic techniques in Drosophila primarily rely on the Flp/FRT site-specific recombination system and require a pair of homologous chromosomes that each contains an FRT sequence near the centromere. However, most existing genetic resources in Drosophila, such as deficiency libraries, transposon-disrupted mutant collections, and strains derived from wild natural populations, do not harbor appropriate FRT sites, preventing their efficient application in mosaic analysis. New mosaic techniques that do not depend on site-specific recombination systems are needed to unleash the full potential of these existing resources. Mosaic analysis by gRNA-induced crossing-over (MAGIC) is a new mosaic technique that does not require site-specific recombination and is compatible with unmodified chromosomes. Although the effectiveness of MAGIC has been demonstrated in the germline, the nervous system, and epithelial tissues, MAGIC reagents are presently only available for a single chromosome arm. This project will first establish an optimized MAGIC toolkit that can be used for mosaic analysis over the entire genome throughout Drosophila tissues. MAGIC screens will also be conducted to identify deficiency lines that are associated with morphological defects in neurons and epithelial cells. Specifically, four aims are proposed: (1) establish an optimized and complete MAGIC toolkit for all Drosophila chromosome arms; (2) generate strains expressing Cas9 in precursor cells of diverse tissues for MAGIC applications; (3) develop anti-CRISPR tools for safe and versatile MAGIC applications in Drosophila; and (4) screen deficiency lines by MAGIC for genes involved in neuronal and epithelial morphogenesis. The Drosophila strains and constructs developed in this project will be donated to the Bloomington Drosophila Stock Center and plasmid depositories, respectively, for easy distribution to the research community. The screen results will be made publicly available for further identification and characterization of responsible genes by interested labs. An advisory committee has been established to provide feedback to this project. Community inputs will be solicited regarding candidate precursor Cas9 lines to generate. The proposed MAGIC tools will allow exploitation of existing genetic resources in systematic gene-function analysis, genome- wide genetic screens, and tissue-specific analysis of natural variants. Attaining the goals of this project will provide the Drosophila community important tools and information that can greatly enhance the value of existing Drosophila resources for understanding human disease.