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

项目总结 识别基因的功能和对疾病生物学的影响是后现代生命科学研究的首要目标。 基因组时代。功能基因组学也巩固了我们理解遗传变异的意义的能力 人类人口。然而，功能基因组学工具仍然存在重大差距，这将减缓我们的进展 应用基因组学来揭开疾病生物学的面纱。目前，高效地汇集全基因组的方法 筛选需要根据生长优势选择细胞或进行物理纯化(例如，通过FACS或 用于单细胞分析)。许多疾病过程的特点是复杂的细胞表型，包括 细胞或细胞器形态的缺陷，分子成分的亚细胞定位，或细胞的运动性。其他 感兴趣的关键表型可能涉及瞬变状态(如有丝分裂)、细胞-细胞相互作用或需要动态 活细胞中的分析(例如，心脏或神经细胞电生理活动的光学记录)。以图像为基础， 使用过度表达和RNA干扰的高含量筛选已经发现了参与 复杂的表型，包括有丝分裂、突触发生和胚胎发生。然而，这种以微板为基础的 克隆细胞群体的筛选不是定期在基因组规模上进行的，因为费用，劳动力， 以及所需的自动化专业知识。在这个项目中，我们开发了一种新的基因组扰动和筛选 将混合微扰与单细胞阵列成像分析的主要优点相结合的概念 复杂表型的读数。具体地说，我们筛选集合的基因组扰动(使用CRISPR-Cas9 单导RNA)使用显微镜读出单细胞的表型并识别受干扰的基因 水平通过原位测序和合成测序的方法。此方法具有高度可伸缩性，因为 试剂和仪器的成本很低(现在全基因组测序只需几万美元 屏幕)。在此，我们请求提供行政补充，以应用我们现有奖励中开发的技术 与东北波士顿大学Rob Davey研究小组一起筛查SARS-CoV-2感染细胞系 配备并积极处理高遏制病毒的新兴传染病实验室 病原体包括SARS-CoV-2。这项关于抗病毒宿主细胞程序的工作属于 原始赠款。我们将密切协调多种生物模型中光学联合筛查的快速执行 戴维博士正在进行的常规CRISPR基因组筛选活动。数据丰富的全基因组光学 筛查数据将确定SARS-CoV-2-宿主界面在病毒生命周期中的新方面并取得进展 我们对候选疗法的理解以及支持产生新的治疗假说 应对新冠肺炎大流行。

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.