Droplet-Assisted RNA targeting by single-cell sequencing to dissect the single-cell heterogeneity of RNA virus infection

通过单细胞测序进行液滴辅助 RNA 靶向分析 RNA 病毒感染的单细胞异质性

• 批准号: 9888327
• 负责人: Iwijn De Vlaminck
• 金额: $ 19.78万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-06 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9888327
• 关键词: Bar Codes; Biological; Biological Assay; Biology; Catalogs; Cause of Death; Cells; Cellular Immunity; Characteristics; DNA sequencing; Disease; Drug resistance; Event; Gastroenteritis; Gene Expression Profiling; Genes; Genetic Recombination; Genetic Variation; Genome; Genotype; HIV; Heterogeneity; IFNAR1 gene; Immune response; Individual; Infection; Influenza A virus; Integration Host Factors; Interferons; Investigation; L Cells; Lead; Mammalian Orthoreovirus; Measurement; Measures; Messenger RNA; Microfluidics; Modeling; Modernization; Molecular; Molecular Analysis; Mus; Mutation; Nucleosome Core Particle; Nucleotides; Organoids; Outcome; Phenotype; Pilot Projects; Poly(A) Tail; Population; Population Analysis; Process; RNA; RNA Sequences; RNA Virus Infections; RNA Viruses; Reovirus; Resolution; Reverse Transcription; Rotavirus; Sampling; Small Intestines; Surveys; Tail; Technology; Testing; Time; Transcript; Tropism; Variant; Viral; Viral Genome; Viral Vector; Virus; Virus Diseases; Virus Replication; Zika Virus; base; cell type; cost; design; disorder control; droplet sequencing; experimental study; flexibility; gene therapy; high throughput screening; influenzavirus; innovation; interest; mammalian genome; mathematical ability; novel; novel diagnostics; receptor; response; single cell analysis; single cell sequencing; single-cell RNA sequencing; technology development; tool; transcriptome; whole genome

Fast evolving RNA viruses, such as rotavirus, influenza virus, human immunodeficiency virus, and zika virus, are a leading cause of death worldwide and represent a major challenge for global disease control. Despite their miniscule genomes, often comprised of only a few thousand nucleotides and a handful of genes, it remains exceedingly difficult to study the infection biology of RNA viruses via modern gene sequencing technologies. RNA viruses evolve quickly. So quickly that even at the level of the individual infected cell, RNA viruses do not occur as a single viral genotype but rather as a swarm of closely related genotypes. This genetic diversity is a key determinant of the capacity of RNA viruses to escape cellular immunity, evolve drug resistance and cause emergent disease. The natural immune response of the infected cell in turn is highly heterogeneous, and dependent on cell type and state. The extreme heterogeneity of RNA virus infections is difficult to survey with current molecular technologies which are largely limited to analyzing populations of infected cells. To overcome this limitation, we have recently created a single cell RNA sequencing technology that combines multiplexed amplicon sequencing with single cell transcriptional profiling: Droplet Assisted RNA Targeting by single-cell Sequencing (DART-seq). DART-seq implements a combination of droplet-microfluidics based co- encapsulation of single cells with barcoded primer beads and ultra-deep DNA sequencing. With DART-seq it is possible to catalog the diversity of viral genome sequences within single infected cells, and at the same time record the cellular response to viral infection. The cost per-cell of DART-seq is less than one dollar, and a single DART-seq assay can yield measurements across thousands of cells in a biological sample. In a proof-of-principle study, we have used DART-seq to profile viral-host interactions and viral genome dynamics in single cells infected with mammalian orthoreovirus (REOV) strain Type 3 Dearing (T3D). Here we propose to expand upon these initial technology development experiments to interrogate the infection biology of REOVs and other segmented RNA viruses including rotavirus, a common cause of gastroenteritis, and influenza A virus. We will furthermore investigate the utility of DART-seq as a tool to perform high-throughput screens of the relationship between viral genotypes and single-cell infectivity and the relationship between host cell type or state and infection permissivity. This innovative project introduces a high-throughput single-cell analysis technology with transformative potential for investigations of the infection biology of RNA viruses. These studies are highly translational: DART-seq can lead to novel diagnostic approaches and can find application as a read-out tool for gene therapies that employ viral vectors.

快速进化的RNA病毒，如轮状病毒、流感病毒、人类免疫缺陷病毒和寨卡病毒，是全球死亡的主要原因，也是全球疾病控制的重大挑战。尽管RNA病毒的基因组很小，通常仅由几千个核苷酸和少数基因组成，但通过现代基因测序技术研究RNA病毒的感染生物学仍然极其困难。 RNA病毒进化迅速。速度如此之快，以至于即使在单个感染细胞的水平上，RNA病毒也不会以单一病毒基因型的形式出现，而是以一群密切相关的基因型的形式出现。这种遗传多样性是 RNA 病毒逃避细胞免疫、进化耐药性和引起突发疾病能力的关键决定因素。受感染细胞的自然免疫反应又具有高度异质性，并且取决于细胞类型和状态。 RNA病毒感染的极端异质性很难用当前的分子技术来调查，这些技术很大程度上仅限于分析受感染的细胞群。为了克服这一限制，我们最近创建了一种单细胞 RNA 测序技术，将多重扩增子测序与单细胞转录分析相结合：单细胞测序液滴辅助 RNA 靶向 (DART-seq)。 DART-seq 实现了基于液滴微流体的单细胞共封装与条形码引物珠和超深 DNA 测序的组合。通过 DART-seq，可以对单个感染细胞内病毒基因组序列的多样性进行编目，同时记录细胞对病毒感染的反应。每个细胞的 DART-seq 成本不到一美元，并且一次 DART-seq 检测可以对生物样本中的数千个细胞进行测量。在一项原理验证研究中，我们使用 DART-seq 来分析感染哺乳动物正呼肠孤病毒 (REOV) 3 型 Dearing (T3D) 菌株的单细胞中的病毒与宿主相互作用和病毒基因组动态。在这里，我们建议扩展这些初步技术开发实验，以探究 REOV 和其他分段 RNA 病毒（包括轮状病毒（胃肠炎的常见原因）和甲型流感病毒）的感染生物学。我们将进一步研究 DART-seq 作为工具对病毒基因型与单细胞感染性之间的关系以及宿主细胞类型或状态与感染许可率之间的关系进行高通量筛选的实用性。这个创新项目引入了一种高通量单细胞分析技术，具有研究 RNA 病毒感染生物学的变革潜力。这些研究具有高度转化性：DART-seq 可以带来新的诊断方法，并且可以作为使用病毒载体的基因治疗的读出工具。