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

项目总结/摘要 该项目旨在为镶嵌分析的新技术开发一个完整的工具箱， 几乎所有现有的果蝇资源，而不需要进一步的遗传修饰。 镶嵌分析是研究人类疾病的分子和细胞机制的有力手段。通过 在杂合动物中产生纯合细胞，镶嵌技术允许组织特异性 分析多效性基因，并在生物学的许多重要发现中发挥了关键作用。电流 果蝇中的嵌合体技术主要依赖于Flp/FRT位点特异性重组系统， 一对同源染色体，每个染色体在着丝粒附近含有一个FRT序列。但大多数 果蝇中现有的遗传资源，如缺陷文库，转座子破坏的突变体收集， 和来源于野生自然种群的菌株，不具有适当的FRT位点，阻止了它们的有效表达。 应用于镶嵌分析。不依赖于位点特异性重组的新镶嵌技术 需要建立各种制度，以充分发挥这些现有资源的潜力。通过gRNA诱导的嵌合体分析 交换（MAGIC）是一种新的镶嵌技术，不需要位点特异性重组， 与未修饰的染色体相容。虽然MAGIC的有效性已经在 生殖细胞、神经系统和上皮组织，MAGIC试剂目前仅可用于单个 本项目将首先建立一个优化的MAGIC工具包，可用于镶嵌 整个果蝇组织的基因组分析。魔术屏幕也将进行， 鉴定与神经元和上皮细胞中的形态缺陷相关的缺陷线。 具体而言，本文提出了四个目标：（1）建立一个优化的、完整的适用于所有果蝇的MAGIC工具包 （2）产生在用于MAGIC的不同组织的前体细胞中表达Cas9的菌株 （3）开发抗CRISPR工具，用于果蝇中安全和多功能的MAGIC应用;以及（4） 通过MAGIC筛选参与神经元和上皮形态发生的基因的缺陷系。的 在这个项目中开发的果蝇品系和构建体将捐赠给布卢明顿果蝇研究所 库存中心和质粒寄存处，分别为方便分发给研究界。的 筛选结果将公开提供，以进一步确定和表征负责的 感兴趣的实验室。已设立一个咨询委员会，为该项目提供反馈。 将征求社区关于候选前体Cas9细胞系的意见。拟议 MAGIC工具将允许利用现有的遗传资源进行系统的基因功能分析，基因组分析， 广泛的遗传筛选和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.