MAGIC tools for genome-wide mosaic analysis with existing Drosophila resources (Equipment Supplement 2023)

利用现有果蝇资源进行全基因组嵌合分析的 MAGIC 工具（设备补充资料 2023）

• 批准号: 10808546
• 负责人: Chun Han
• 金额: $ 22.11万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-15 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10808546
• 关键词: Advisory Committees; Animals; Biology; CRISPR/Cas technology; Cells; Cellular biology; Centromere; Chromosome Arm; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Communities; Confocal Microscopy; Dedications; Defect; Developmental Biology; Disease; Drosophila genus; Drosophila melanogaster; Effectiveness; Epithelial Cells; Epithelium; Equipment; Essential Genes; Feedback; Genes; Genetic; Genetic Crossing Over; Genetic Recombination; Genetic Screening; Genomics; Goals; Guide RNA; Heterozygote; Image; Label; Libraries; Methods; Modification; Molecular; Morphogenesis; Morphology; Nerve Degeneration; Nervous System; Neurons; Output; Phenotype; Plasmids; Play; Population; Reagent; Research; Resolution; Resources; Screening Result; Series; Site; System; Techniques; Testing; Time; Tissues; Toxic effect; Transgenes; Variant; arm; cell type; confocal imaging; design; disease mechanisms study; experimental study; flexibility; gene function; genetic manipulation; genetic resource; genome-wide; human disease; imaging system; innovation; interest; mosaic; mosaic analysis; mutant; neurodevelopment; parent project; precursor cell; prevent; reagent testing; repository; screening; somatosensory; tool; whole genome

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.

项目摘要/摘要 该项目旨在开发一个完整的工具箱，以构成新的马赛克分析技术 几乎所有现有的果蝇Melanogaster资源，无需进行进一步的遗传修饰。 马赛克分析是研究人类疾病的分子和细胞机制的强大方法。经过 在其他杂合动物中产生纯合细胞，马赛克技术允许组织特异性 对多效基因的分析，并在生物学的许多重要发现中起着关键作用。当前的 果蝇中的马赛克技术主要依赖于FLP/FRT特定的重组系统，需要一个 每种同源染色体都包含丝粒附近的FRT序列。但是，大多数 果蝇中的现有遗传资源，例如缺陷库，转座破裂的突变体收集， 以及源自野生天然种群的菌株，不带有适当的FRT站点，以防止其效率 在马赛克分析中应用。不依赖特定地点重组的新的马赛克技术 需要系统来释放这些现有资源的全部潜力。通过GRNA诱导的镶嵌分析 Crossing-Over（魔术）是一种新的马赛克技术，不需要特定地点的重组，并且是 与未修饰的染色体兼容。尽管魔术的有效性已在 种系，神经系统和上皮组织，魔术试剂目前仅适用于单个 染色体臂。该项目将首先建立一个可用于马赛克的优化魔术工具包 对整个果蝇组织的整个基因组进行分析。魔术屏幕也将进行 确定与神经元和上皮细胞中形态缺陷相关的缺陷线。 具体而言，提出了四个目标：（1）为所有果蝇建立一个优化而完整的魔术工具包 染色体臂； （2）在不同组织的前体细胞中产生表达cas9的菌株以进行魔术 申请； （3）为果蝇中的安全和多功能魔术应用开发抗危机工具； （4） 魔术的筛查缺乏线，用于与神经元和上皮形态发生有关的基因。这 该项目中开发的果蝇菌株和结构将捐赠给布卢明顿果蝇 股票中心和质粒存放物分别为研究社区易于分发。这 屏幕结果将公开用于进一步的识别和表征 感兴趣的实验室的基因。已经成立了一个咨询委员会，以向该项目提供反馈。 将征求有关候选前体CAS9线的征求社区意见。提议 魔术工具将允许在系统的基因功能分析，基因组中剥削现有的遗传资源 - 广泛的遗传筛选和自然变体的组织特异性分析。实现该项目的目标将 提供果蝇社区的重要工具和信息，可以极大地提高 现有的果蝇资源以理解人类疾病。

项目成果

The Drosophila chemokine-like Orion bridges phosphatidylserine and Draper in phagocytosis of neurons.

• DOI: 10.1073/pnas.2303392120
• 发表时间: 2023-06-13
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Ji, Hui;Wang, Bei;Shen, Yifan;Labib, David;Lei, Joyce;Chen, Xinchen;Sapar, Maria;Boulanger, Ana;Dura, Jean-Maurice;Han, Chun
• 通讯作者: Han, Chun