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.

项目摘要确定基因功能和对疾病生物学的影响是生命科学研究的总体目的基因组时代。功能基因组学还支持我们理解遗传变异含义的能力人口。但是，关键差距仍然存在于功能基因组学工具集中，这将减缓我们的进度在阐明疾病生物学的过程中。目前，有效地汇总了全基因组的方法筛查需要根据生长优势或物理纯化选择细胞（例如FACS或用于单细胞分析）。许多疾病过程的特征是复杂的细胞表型，包括细胞或细胞器形态缺陷，分子成分的亚细胞定位或细胞运动。其他感兴趣的关键表型可能涉及瞬态状态（例如有丝分裂），细胞 - 细胞相互作用或需要动态活细胞的测定（例如，心脏或神经细胞电生理活性的光学记录）。基于图像，使用过表达和RNA干扰的高含量筛选发现了参与的新型基因复杂的表型，包括有丝分裂，突触发生和胚胎发生。但是，这种基于微板的克隆细胞种群的筛查由于费用，劳动，，不定期在基因组量表上进行和自动化专业知识。在此计划中，我们开发了一种新的基因组扰动和筛选概念将汇总扰动的主要优势与单细胞阵列的成像测定法相结合复杂表型的读数。具体而言，我们筛选了汇总基因组扰动（使用CRISPR-CAS9 使用显微镜读取表型并在单细胞处识别扰动基因的单个指南RNA）通过合成方法进行测序通过原位测序进行水平。这种方法是高度可扩展的，因为试剂和仪器成本很小（现在全基因组的数万美元屏幕）。在这里，我们请求一种行政补充来应用我们现有奖励中开发的技术与罗布·戴维（Rob Davey）的小组在波士顿大学（Boston University Northeast 新兴的传染病实验室等效，并积极地使用高控制病毒包括SARS-COV-2的病原体。这项关于抗病毒宿主细胞程序的工作在科学范围内原始赠款。我们将在多个生物模型中紧密协调光学合并屏幕的快速执行随着Davey博士持续的常规CRISPR基因组筛查活动。全基因组的光学富含数据的光学筛选数据将在整个病毒生命周期中确定SARS-COV-2-host接口的新方面并提高我们对候选疗法的理解，并支持新疗法假设的产生解决COVID-19-19大流行。