利用Cas9大规模基因敲除技术在HIV-1潜伏细胞上筛选及鉴定与HIV潜伏相关的关键宿主基因

The long-term persistence of latent viral reservoir that comprises the HIV latent infection cell has been the major obstacle towards finding a method to completely cure human immunodeficiency virus type 1 (HIV-1).Several epigenetic and non-epigenetic mechanisms have been implicated in the regulation of viral latency, but is still unclear which genes play a key role in the establishment and maintenance of HIV latency. Therefore, our study intends to ues CRISPR-Cas large-scale gene knockout technology, which is a Cas9-sgRNA lentivirus pool targeted 18080 known human genes, then we will infect HIV-1 latent cell line by lentivirus , and sort out the activation latent cell before carrying on the depth of the second generation sequencing and obtaining candidate genes related to latency. The candidate genes will be classified by according to bioinformatics analysis; For screening candidate genes, we will respectively design targeting single or multiple candidate genes lentiCRISPRv2-sgRNA to knockout candidate genes in HIV-1 latent cell line, then detect latent cells after the knockout genotype and phenotype changes, and discuss the possible involved in signaling pathway and action mechanism of latent HIV-1. We also will create a latently infected human primary T-cells and further perform functional verification mechanism of the screening candidate genes and discuss the involved mechanism. Our research will screen and identify new latent function of key genes involved in regulating the HIV-1 and help clarify the molecular mechanism of latent infections. The most important thing is that will provide latent HIV-1 new potential target for therapeutic intervention.

人类免疫缺陷病毒（HIV）难以在体内被完全清除的一个重要原因是由于HIV潜伏感染细胞的长期存在。尽管,HIV-1潜伏的分子机制已报道涉及包括表观及非表观遗传在内的多种因素,但宿主细胞哪些基因对HIV潜伏建立及维持起关键作用仍不清楚。为此,本课题在前期研究基础上,利用Cas9基因敲除技术，制备针对人18080个基因的Cas9-sgRNA慢病毒库,在HIV潜伏感染细胞系进行大规模基因敲除，获得能被激活的潜伏感染细胞; 经二代测序分析,获得潜伏相关的候选基因; 在HIV潜伏细胞系上对候选基因进行基因敲除，检测分析敲除后潜伏细胞基因型及表型变化, 探讨其参与HIV潜伏的信号通路以及机制。 在HIV潜伏感染的人原代T淋巴细胞模型上，进一步对筛选出的候选基因进行功能性验证。本研究旨在筛选并鉴定出新的参与调控HIV潜伏的关键基因，将有助于阐明HIV潜伏的分子机制,也将为HIV潜伏干预治疗提供新的靶点。

人类免疫缺陷病毒（Human immunodeficiency virus，HIV）在患者体内难以被完全清除，主要是由于HIV-1潜伏感染细胞组成的病毒潜伏库长期存在所导致的。然而，HIV-1潜伏细胞是如何形成及维持的机制，目前还未完全阐明。因此，在本研究中，我们通过全基因组CIRPSR-Cas9敲除文库（GECKO文库）及激活文库（SAM文库），使用针对人已知19050个基因的Cas9-sgRNA慢病毒文库分别感染感染HIV-1潜伏感染细胞，并分选出激活的潜伏细胞，进行二代深度测序，获得与潜伏相关的候选基因2356个。对于筛选出丰度排名前十的潜伏相关候选基因，分别设计构建针对单个候选基因的lenti CIRPSR RV2-sgRNA或者lenti CRISPR SAM-sgRNA载体，在HIV-1潜伏细胞系上敲除或者激活候选基因，检测敲除或者激活后潜伏细胞表型的变化，最后筛选并鉴定出三个基因PEBP1、FKBP3和FBXO34与HIV-1潜伏有关；并进一步分别证实了 PEBP1通过失活MAPK和IKK信号通路，抑制NF-κB入核促进HIV-1潜伏；FKBP3通过与YY1互作，招募HDAC1/2到HIV-1 LTR LSF位点促进组蛋白去乙酰化导致HIV-1潜伏；FBXO34通过与hnRNP U互作，促进其泛素化和降解，干预HIV-1潜伏。以上发现，不仅丰富了HIV-1潜伏感染的分子机制，重要的是将为HIV-1潜伏的干预治疗提供新的潜在靶点，为病毒潜伏储藏库清除的原创性治疗方案提供新的思路。