Massive assay for single viruses in single cells

每个真核细胞的基因组都被包裹在一种叫做染色质的分子网络中。染色质作为基因与环境之间的第一层，扮演着基因表达的信息库和协调者的角色。重要的是，基因组中还含有转座子和整合型逆转录病毒等遗传寄生虫。通过它调节基因表达的能力，染色质也起到了保护基因组免受遗传侵袭的作用。染色质保护基因组的分子机制仍然知之甚少。长期以来，转座子和逆转录病毒往往被宿主细胞沉默，但仍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，这个项目旨在研究宿主细胞如何识别和沉默遗传侵略者。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，使我们能够同时跟踪病毒在数百万个单独细胞中的命运。我们正在开发一种新的高通量技术，称为SHIVA(病毒活动的二级超索引)，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，我们就可以确定表达模式，进而告诉我们工作机制。使用这种方法，我们可以测试识别机制的存在，以识别遗传寄生虫。我们建议建立一个以艾滋病毒为模型病毒的原则证明，因为这个系统中有大量可用的工具。重要的是，Shiva技术可以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筛查等遗传工具结合起来，识别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。总而言之，这项研究计划为理解真核基因组如何识别和中和整合的外源基因成分铺平了道路。

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.