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

利用 CRISPR-Cas13a 的 RNA 切割特性应用于 HIV 检测和潜伏期

• 批准号: 10004494
• 负责人: Parinaz Fozouni
• 金额: $ 5.05万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10004494
• 关键词: Apoptosis; Apoptotic; Bacteria; Binding; Biological; Biological Assay; CD4 Positive T Lymphocytes; CRISPR/Cas technology; Cell Cycle Arrest; Cell Line; Cell Survival; Cells; Cleaved cell; Clinic; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA Polymerase II; DNA-Directed RNA Polymerase; Dengue; Detection; Development; Diagnostic; Fluorescence; Genetic Transcription; Genome; Gold; HIV; HIV Long Terminal Repeat; HIV-1; Histone Acetylation; Homologous Gene; In Vitro; Individual; Integration Host Factors; Laboratories; Measurement; Methods; Molecular; Monitor; Patients; Play; Population; Predisposition; Property; Proteins; Proviruses; RNA; RNA Binding; RNA Viruses; Reporter; Reproducibility; Reverse Transcription; Ribonucleases; Role; Sampling; Site; Stress; Structure; T-Cell Activation; Testing; Transactivation; Transcript; ZIKA; antiretroviral therapy; assay development; base; curative treatments; cytokine release syndrome; design; experimental study; fluorophore; improved; in vitro Assay; in vivo; innovation; interest; knock-down; latent infection; new technology; novel; preference; premature; prevent; promoter; recruit; response; small hairpin RNA; tool; viral RNA; viral detection

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.

项目概要/摘要 尽管人类免疫缺陷病毒的治疗已取得很大进展，但持续存在的 带有整合的 HIV 原病毒的 CD4+ T 细胞群（潜在的储存库）仍然是艾滋病病毒感染的主要障碍。 治愈。直接检测和靶向病毒 RNA 的改进工具可以极大地支持理解、量化、 并消灭潜在的储存库。最近，人们发现 CRISPR-Cas13a（以前称为 C2c2）可以 以序列特异性方式结合单链靶 RNA，并发挥一般 RNase 活性 被靶RNA激活。这种非特异性或附带裂解可用于基于荧光的 检测特定 RNA，之前已用于检测寨卡病毒和登革热 RNA 病毒。 然而，这些检测策略需要逆转录、扩增和 T7 转录步骤 这可能会降低测定的可重复性并引入偏差。目前 HIV-1 的黄金标准 RNA 检测、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、灵敏地检测 HIV RNA 的新方法。 此外，由于操作 RNA 的方法有限，直接在体内研究 HIV RNA 一直具有挑战性 细胞内。一种对理解 HIV 潜伏机制特别感兴趣的体内 RNA 很短， 流产的 TAR 转录本。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 相互作用调节 HIV 转录的因子之前已被使用 shRNA，很少有研究在 HIV 潜伏和感染的背景下针对新生 TAR RNA 本身。这 CRISPR-Cas13a作为分子工具的发展可以让我们在体外直接检测HIV RNA并靶向 体内特定的HIV RNA。我们假设 CRISPR-Cas13a 组件（Cas13a 同源物、crRNA 设计和荧光报告 RNA）可以直接检测和定量 HIV RNA，并且 CRISPR-Cas13a 可用于体内切割短 TAR RNA 转录物，这可能有助于 HIV潜伏期和细胞凋亡。我们的目标是开发 CRISPR-Cas13 作为研究和检测的多功能工具 艾滋病毒。总之，拟议的实验将利用一种新颖的 CRISPR 技术来直接研究 HIV-1 RNA 检测并将阐明基于 RNA 的潜伏机制，这有助于确定潜在的 HIV 治疗方法 目标。