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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.

项目总结