Massive assay for single viruses in single cells

单细胞中单一病毒的大规模检测

• 批准号: RGPIN-2020-06377
• 负责人: Filion, Guillaume
• 金额: $ 2.62万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744894
• 关键词: Massive; assay; single; viruses; cells

The genome of every eukaryotic cell is coated in a molecular meshwork called chromatin. As the first layer between genes and their environment, chromatin plays the roles of information repository and coordinator of gene expression. Importantly, genomes also contain genetic parasites such as transposons and integrated retroviruses. Through its ability to regulate gene expression, chromatin also acts as a safeguard of the genome against genetic aggressions. The molecular mechanisms by which chromatin defends the genome are still poorly understood. It has long been observed that transposons and retroviruses tend to be silenced by the host cell but it is still unknown how they are recognized and identified as threats. The aim of my research programme is to understand this particular mechanism of genetic immunity. More specifically, this project is designed to study how genetic aggressors are identified and silenced by the host cell. To study this important process, we must first develop a new genetic technology and bespoke software packages that will allow us to simultaneously track the fate of viruses in millions of individual cells. We are developing a novel high-throughput technology called SHIVA (Secondary Hyper-Indexing of Virus Activity) precisely for that purpose. With a complete record of parasite activity in every cell, we can identify patterns of expression that in turn inform us about the mechanism at work. With this approach, we can test the existence of recognition mechanisms to identify genetic parasites. We propose to establish a proof of principle using HIV as a model virus because of the large amount of available tools in this system. Importantly, the SHIVA technology can be used with any natural or artificial insertion sequences, including viruses, transposons and transgenes. Finally, we can combine SHIVA with genetic tools such as CRISPR screens to identify the genes that can recognize genetic parasites and contribute to the natural genetic immunity of eukaryotic cells. In summary, this research program paves the way to understand how eukaryotic genomes identify and neutralize integrated foreign genetic elements.

每一个真核细胞的基因组都包裹在一个叫做染色质的分子网中。染色质作为基因与其环境之间的第一层，起着基因表达的信息库和协调者的作用。重要的是，基因组还包含遗传寄生虫，如转座子和整合逆转录病毒。通过其调节基因表达的能力，染色质还可以作为基因组抵御遗传侵害的保障。染色质保护基因组的分子机制仍然知之甚少。长期以来，人们一直观察到转座子和逆转录病毒倾向于被宿主细胞沉默，但人们仍然不清楚它们是如何被识别和识别为威胁的。我的研究计划的目的是了解遗传免疫的这种特殊机制。更具体地说，这个项目的目的是研究遗传侵略者如何被宿主细胞识别和沉默。为了研究这一重要过程，我们必须首先开发一种新的基因技术和定制的软件包，使我们能够同时追踪数百万个单个细胞中病毒的命运。我们正在开发一种新的高通量技术，称为SHIVA（二级病毒活动超索引），正是为了这个目的。有了每个细胞中寄生虫活动的完整记录，我们就能识别出表达模式，进而了解起作用的机制。通过这种方法，我们可以测试识别机制的存在，以识别遗传寄生虫。我们建议用HIV作为模型病毒建立一个原理证明，因为这个系统中有大量可用的工具。重要的是，SHIVA技术可用于任何自然或人工插入序列，包括病毒、转座子和转基因。最后，我们可以将SHIVA与CRISPR筛选等遗传工具结合起来，鉴定出能够识别遗传寄生虫并有助于真核细胞天然遗传免疫的基因。总之，本研究计划为了解真核生物基因组如何识别和中和整合的外源遗传元素铺平了道路。