High-content optical pooled genome-wide screens of SARS-CoV-2 infection

SARS-CoV-2 感染的高内涵光学汇集全基因组筛查

基本信息

批准号：
10166221
负责人：
Paul Clark Blainey
金额：
$ 35.78万
依托单位：
BROAD INSTITUTE, INC.
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-01 至 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10166221
关键词：
2019-nCoV Acute Address Administrative Supplement Affect Antiviral Agents Antiviral Response Automation Award Biological Assay Biological Models Biological Sciences Biology Boston COVID-19 COVID-19 pandemic CRISPR/Cas technology Cardiac Cell Communication Cell Line Cell Separation Cell model Cells Clone Cells Clustered Regularly Interspaced Short Palindromic Repeats Combined Modality Therapy Communities Complex Containment DNA Sequence Data Defect Detection Development Disease Drug Combinations Drug Interactions Drug Synergism Drug toxicity Electrophysiology (science)Embryonic Development Emerging Communicable Diseases Expression Profiling Flow Cytometry Generations Genes Genetic Screening Genetic Variation Genomics Grant Growth Guide RNA Human Human Cell Line Image Immune response In Situ Infection Infectious Diseases Research Integration Host Factors Investigation Knowledge Life Cycle Stages Methods Microscopy Mitosis Modeling Molecular Monkeys Morphology Neurons Nucleocapsid Proteins Optics Organelles Outcome Pathogenesis Pattern Phenotype Play Population Predisposition Process Proteins RNA Interference Reagent Research Research Personnel Role Running Screening Result Speed Technology Testing Therapeutic Universities Viral Virus Virus Replication Work base cell motility coronavirus disease cost cytotoxicity drug candidate drug development drug mechanism fitness follow-up functional genomics gene function genome wide screen genome-wide genomic tools improved in situ sequencing instrumentation interest novel novel therapeutics overexpression pathogen pathogenic virus phenotypic biomarker programs response screening single cell analysis synaptogenesis temporal measurement therapeutic candidate therapeutic development trafficking transcriptome sequencing viral RNA viral detection

项目摘要

PROJECT SUMMARY Identifying gene function and impact on disease biology are overarching aims of life science research in the post- genomic era. Functional genomics also underpins our ability to understand the meaning of genetic variation in human populations. However, crucial gaps remain in the functional genomics tool set that will slow our progress in applying genomics to unravel disease biology. Currently, efficiently pooled methods for genome-wide screening require either selection of cells based on growth advantage or physical purification (e.g. by FACS or for single-cell analysis). Many disease processes are characterized by complex cellular phenotypes including defects in cell or organelle morphology, subcellular localization of molecular components, or cell motility. Other key phenotypes of interest may involve transient states (eg mitosis), cell-cell interactions, or require dynamic assays in live cells (eg, optical recording of electrophysiological activity of cardiac or neural cells). Image-based, high-content screens using overexpression and RNA interference have uncovered novel genes involved in complex phenotypes, including mitosis, synaptogenesis, and embryogenesis. However, such microplate-based screens of clonal cell populations are not regularly conducted at the genomic scale due to the expense, labor, and automation expertise required. In this program, we developed a new genomic perturbation and screening concept that combines major advantages of pooled perturbation with imaging assays for single-cell arrayed readout of complex phenotypes. Specifically, we screen pooled genomic perturbations (with CRISPR-Cas9 single-guide RNAs) using microscopy to read out phenotypes AND to identify perturbed genes at the single-cell level via in situ sequencing with a sequencing by synthesis approach. This approach is highly scalable because reagent and instrumentation costs are modest (now a few tens of thousands of dollars for a genome-wide screen). Here we request an administrative supplement to apply the technology developed in our existing award to screens for SARS-CoV-2 infection of cell lines with Rob Davey’s group at the Boston University Northeast Emerging Infectious Disease Laboratory that is equipped and actively working with high-containment viral pathogens including SARS-CoV-2. This work on antiviral host cell programs is within the scientific scope of the original grant. We will tightly coordinate the rapid execution of optical pooled screens in multiple biological models with Dr. Davey’s ongoing conventional CRISPR genomic screening activity. The data-rich genome-wide optical screening data will identify new aspects of the SARS-CoV-2-host interface across the viral life cycle and advance our understanding of candidate therapeutics as well as support the generation of new therapeutic hypotheses to address the COVID-19 pandemic.

项目概要识别基因功能及其对疾病生物学的影响是后生命科学研究的首要目标基因组时代也支撑了我们理解遗传变异意义的能力。然而，功能基因组学工具集仍然存在重大差距，这将减缓我们的进步。应用基因组学来阐明疾病生物学。目前，有效地汇集了全基因组方法。筛选需要根据生长优势选择细胞或物理纯化（例如通过 FACS 或用于单细胞分析）。许多疾病过程都以复杂的细胞表型为特征，包括细胞或细胞器形态、分子成分的亚细胞定位或细胞运动的缺陷。感兴趣的关键表型可能涉及瞬时状态（例如有丝分裂）、细胞间相互作用，或需要动态活细胞测定（例如，基于图像的心脏或神经细胞的电生理活动的光学记录）。使用过表达和 RNA 干扰的高内涵筛选发现了参与复杂的表型，包括有丝分裂、突触发生和胚胎发生，然而，这种基于微孔板的方法。由于费用、劳动力、在此计划中，我们开发了一种新的基因组扰动和筛选方法。结合了混合扰动与单细胞阵列成像分析的主要优点的概念具体来说，我们筛选汇集的基因组扰动（使用 CRISPR-Cas9）。单向导RNA）使用显微镜读取表型并识别单细胞中受到干扰的基因通过合成测序方法进行原位测序，这种方法具有高度可扩展性，因为。试剂和仪器成本适中（现在全基因组只需几万美元）在此，我们请求行政补充，以应用我们现有奖项中开发的技术。与波士顿大学东北部的 Rob Davey 团队一起筛查细胞系的 SARS-CoV-2 感染配备并积极研究高遏制病毒的新兴传染病实验室这项关于抗病毒宿主细胞计划的工作属于该研究的科学范围。我们将紧密协调光学联合筛选在多个生物模型中的快速执行。 Davey 博士正在进行的传统 CRISPR 基因组筛选活动，数据丰富的全基因组光学。筛选数据将确定整个病毒生命周期中 SARS-CoV-2-宿主界面的新方面并推进我们对治疗候选者的理解以及支持新治疗假设的产生应对 COVID-19 大流行。