Massive assay for single viruses in single cells

每一个真核细胞的基因组都被一种叫做染色质的分子网络包裹着。染色质作为基因与环境之间的第一层，在基因表达中起着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，如转座子和整合的逆转录病毒。通过其调节基因表达的能力，染色质也可以作为基因组抵御遗传侵袭的保障。染色质保护基因组的分子机制仍然知之甚少。长期以来，人们已经观察到转座子和逆转录病毒倾向于被宿主细胞沉默，但仍然不知道它们是如何被识别和鉴定为威胁的。我的研究计划的目的是了解这种特殊的遗传免疫机制。更具体地说，该项目旨在研究遗传攻击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遗传技术和定制的软件包，使我们能够同时跟踪数百万个细胞中病毒的命运。我们正在开发一种名为SHIVA（Secondary Hyper-Indexing of Virus Activity）的新型高通量技术，正是为了实现这一目标。有了每个细胞中寄生虫活动的完整记录，我们可以识别表达模式，进而告诉我们工作机制。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。我们建议使用HIV作为模型病毒建立一个原理证明，因为在这个系统中有大量的可用工具。重要的是，SHIVA技术可用于任何天然或人工插入序列，包括病毒、转座子和转基因。最后，我们可以将联合收割机SHIVA与CRISPR筛选等遗传工具相结合，以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。总之，这项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件铺平了道路。

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.