Harnessing the RNA-Cleaving Properties of CRISPR-Cas13a for Applications to HIV Detection and Latency

项目摘要/摘要 尽管在治疗人类免疫缺陷病毒方面取得了很大进展，但持续存在的 整合了HIV前病毒的CD4+T细胞群体是潜在的储蓄者，仍然是 解药。改进的直接检测和靶向病毒RNA的工具可以极大地支持理解、量化 消除潜伏油气藏。最近，CRISPR-Cas13a(以前称为C2c2)，被发现 以序列特异性的方式结合单链靶RNA，并对其发挥一般的核糖核酸酶活性 被靶RNA激活。这种非特异性或侧枝裂解可用于基于荧光的 检测特定的RNA，以前曾用于检测寨卡病毒和登革热RNA病毒。 然而，这些检测策略需要反转录、扩增和T7转录步骤 这可能会降低分析的重复性并引入偏差。目前HIV-1的黄金标准 RNA检测，即RT-PCR，目前也需要逆转录步骤。迫切需要 开发在临床和实验室环境中直接和灵敏地检测艾滋病毒RNA的新方法。 此外，由于操纵RNA的方法有限，直接在体内研究HIV RNA一直是具有挑战性的 在细胞内。一种对了解HIV潜伏期机制特别感兴趣的体内RNA是短的， 流产的焦油成绩单。它们还被认为在防止受感染细胞凋亡方面发挥作用。虽然 TAR RNA与之相互作用以调节HIV转录的因子先前已被用 对于shRNA，很少有研究针对艾滋病毒潜伏期和感染背景下的新生TAR RNA本身。这个 CRISPR-Cas13a作为一种分子工具的开发使我们能够在体外直接检测HIV RNA和靶向 体内特定的HIV RNA。我们假设CRISPR-Cas13a组件(Cas13a)的严格优化 同系物、crRNA设计和荧光报告RNA)可以直接检测和量化HIV RNAs，CRISPR-Cas13a可以在体内被用来切割可能有助于 与艾滋病毒潜伏和细胞凋亡有关。我们的目标是开发CRISPR-CAS13作为研究和检测的通用工具 艾滋病病毒。总之，拟议的实验将利用一种新的CRISPR技术来直接检测HIV-1RNA 并将阐明基于RNA的潜伏期机制，这可能有助于识别潜在的艾滋病毒治愈 目标。

PROJECT SUMMARY/ABSTRACT Although much progress has been made in treating human immunodeficiency virus, the presence of a persistent population of CD4+ T cells with integrated HIV proviruses, the latent reservoir, remains the major barrier to a cure. Improved tools to directly detect and target viral RNAs could greatly support efforts to understand, quantify, and eliminate the latent reservoir. Recently, CRISPR-Cas13a (formerly referred to as C2c2), was discovered to bind single-stranded target RNAs in a sequence-specific manner and exert general RNase activity upon activation by the target RNA. This non-specific or collateral cleavage can be exploited for fluorescence-based detection of specific RNAs and has been previously used for detection of Zika and Dengue RNA viruses. However, these detection strategies required reverse transcription, amplification, and T7 transcription steps which may diminish the reproducibility of the assay and introduce biases. The current gold standard of HIV-1 RNA detection, RT-PCR, also currently requires a reverse transcription step. There is a critical need for developing new methods to directly and sensitively sense HIV RNAs both in the clinic and laboratory setting. Additionally, directly studying HIV RNAs in vivo has been challenging due to limited methods to manipulate RNAs within cells. One in vivo RNA of special interest in understanding the mechanisms of HIV latency are short, abortive TAR transcripts. They are also thought to play a role in preventing apoptosis of infected cells. Although factors that TAR RNA interacts with to modulate HIV transcription have been previously knocked-down using shRNAs, few studies have targeted nascent TAR RNA itself in the context of HIV latency and infection. The development of CRISPR-Cas13a as a molecular tool can allow us to directly detect HIV RNAs in vitro and target specific HIV RNAs in vivo. We hypothesize that rigorous optimization of CRISPR-Cas13a components (Cas13a homolog, crRNA design, and fluorescent reporter RNA) can allow for direct detection and quantification of HIV RNAs, and that CRISPR-Cas13a can be utilized in vivo to cleave short TAR RNA transcripts that may contribute to HIV latency and apoptosis. We aim to develop CRISPR-Cas13 as a versatile tool for the study and detection of HIV. Together, the proposed experiments will harness a novel CRISPR technology towards direct HIV-1 RNA detection and will elucidate RNA-based mechanisms of latency, which could help identify potential HIV cure targets.