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.

项目概要/摘要 该项目旨在开发一个完整的工具箱，用于镶嵌分析新技术，该技术与 几乎所有现有的果蝇资源，无需进一步的基因改造。 镶嵌分析是研究人类疾病的分子和细胞机制的有效方法。经过 在杂合动物中产生纯合细胞，镶嵌技术允许组织特异性 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。当前的 果蝇中的嵌合技术主要依赖于 Flp/FRT 位点特异性重组系统，并且需要 一对同源染色体，每条染色体在着丝粒附近都包含一个 FRT 序列。然而，大多数 果蝇现有的遗传资源，例如缺陷文库、转座子破坏的突变体集合， 和源自野生自然种群的菌株，没有适当的 FRT 位点，从而妨碍了它们的有效利用 在马赛克分析中的应用不依赖于位点特异性重组的新镶嵌技术 需要系统来释放这些现有资源的全部潜力。 gRNA 诱导的镶嵌分析 交叉（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。尽管MAGIC的有效性已经在 种系、神经系统和上皮组织，MAGIC 试剂目前仅适用于单一 染色体臂。本项目首先会建立一个优化的MAGIC工具包，可用于马赛克 对整个果蝇组织的整个基因组进行分析。 MAGIC屏幕也将进行 识别与神经元和上皮细胞形态缺陷相关的缺陷系。 具体来说，提出了四个目标：（1）为所有果蝇建立一个优化且完整的MAGIC工具包 染色体臂； (2) 为MAGIC生成在不同组织的前体细胞中表达Cas9的菌株 应用程序； (3) 开发抗 CRISPR 工具，用于果蝇中安全且多功能的 MAGIC 应用；和（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。这 该项目中开发的果蝇菌株和构建体将捐赠给布卢明顿果蝇 分别是库存中心和质粒保藏库，以便于分发给研究界。这 筛查结果将公开，以进一步识别和定性责任人 感兴趣的实验室的基因。已经成立了一个咨询委员会来为该项目提供反馈。 将就要生成的候选前体 Cas9 系征求社区意见。拟议的 MAGIC 工具将允许在系统基因功能分析、基因组分析中利用现有遗传资源。 广泛的遗传筛选和自然变异的组织特异性分析。实现该项目的目标将 为果蝇界提供重要的工具和信息，可以极大地提高果蝇的价值 用于了解人类疾病的现有果蝇资源。

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