Harnessing the RNA-Binding Properties of Cas13a for HIV-1 Self-Testing

利用 Cas13a 的 RNA 结合特性进行 HIV-1 自检

• 批准号: 10456229
• 负责人: Melanie Maria Ott
• 金额: $ 88.94万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10456229
• 关键词: AIDS/HIV problem; Acquired Immunodeficiency Syndrome; Acute; Address; Bacteria; Binding; Biological; Biological Assay; Biology; Blood; CRISPR/Cas technology; Car Phone; Cellular Phone; Chronic; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cryopreservation; DNA; Dengue; Detection; Development; Devices; Diagnostic; Diagnostic tests; Dyes; Early Diagnosis; Economically Deprived Population; Epidemic; Escherichia coli; Failure; Fluorescence; Funding; Genome; Goals; Gold; Grant; Guide RNA; HIV; HIV Genome; HIV-1; Home; Homologous Gene; Human; Human immunodeficiency virus test; Incidence; Individual; Infection; Interruption; Knowledge; Laboratories; Life; Liquid substance; Measurement; Measures; Methods; Microscopy; Mission; Monitor; Oligonucleotides; Patients; Phase; Plasma; Procedures; Property; Proteins; Public Health; RNA; RNA Binding; RNA purification; Recombinants; Reporter; Reproducibility; Research; Research Support; Resource-limited setting; Reverse Transcription; Ribonucleases; Sampling; Scientist; Serum; Site; Structure; System; Technology; Temperature; Testing; United States National Institutes of Health; Viral Load result; Whole Blood; Work; ZIKA; acute infection; assay development; base; chronic infection; cohort; design; detection method; detection platform; fluorophore; healthspan; home test; innovation; isothermal amplification; lens; microbial; multidisciplinary; new technology; novel; preference; reagent testing; self testing; stability testing; viral RNA; viral detection

PROJECT ABSTRACT Current HIV-1 RNA detection relies on reverse transcription before PCR-based amplification, which introduces unwanted variables and cannot be easily performed outside a laboratory. The central hypothesis of this application is that the RNA-binding properties of newly discovered CRISPR/Cas13a proteins are suitable for sensitive at-home detection of HIV-1 RNAs without employing RT or amplification steps. This hypothesis was formulated on the basis of the recent discovery by the Doudna Lab that Cas13a binds and cleaves target single-stranded RNAs in a sequence-specific manner (cis cleavage) and subsequently exerts general RNase activity (trans cleavage) that can be exploited for fluorescence-based measurement of the target RNA. In unpublished preliminary results, the Ott/Doudna Labs also show that recombinant Cas13a in combination with HIV-1-specific guide RNAs (crRNAs) enables sensitive detection of HIV-1 RNAs. The central hypothesis will be tested in a two-pronged, highly milestone-driven approach: in the innovation phase (R61), two aims will define the optimal Cas13a homologue/crRNA combination for reliable HIV-1 detection and optimize the read-out technology for home use. Aim 1: To optimize guide RNA (crRNA) and Cas13a protein selection. The applicants will design HIV-specific crRNAs recognizing conserved accessible regions of the target HIV-1 genome and systematically test Cas13a homologs from different bacteria for HIV-specific cis and trans cleavage. At the end of the R61 period in order to progress to the R33 phase, the team will have identified ≥3 optimized crRNA spacer sequences in the HIV-1 genome, selected ≥1 Cas13a homologs with high HIV-specific cis- and trans- ssRNA cleavage rates with low background. Aim 2: To enhance read-out technology and optimize for self- testing. In preliminary results, the Fletcher Lab measured E. coli DNA after PCR amplification detecting fluorescence from an intercalating dye with iPhone-based technology. The applicants will define the optimal sequence for trans-cleavage by Cas13a versus human RNase proteins as well as optimize fluorophore and quencher moieties on the detection oligonucleotide for measurements with mobile phone-based reverse lens microscopy (CellScope). At the end of the R61 period in order to progress to the R33 phase, ≥1 detection sequence will have been identified and ≥1 fluorescence/quencher combination will have been optimized for CellScope detection. The R33 phase consists of one aim, rigorously comparing Cas13a-CellScope results with conventional viral load assays in acutely and chronically infected individuals and adapting the method to home use. Aim 3: To apply optimized Cas13a assay parameters towards building a self-testing device using clinical samples. The team will optimize HIV-1 RNA detection in the context of plasma, serum and whole blood, enhance stability of the test system at room temperature and analyze cryopreserved and fresh patient samples provided by Dr. Deeks from the SCOPE cohort. It is anticipated that the work completed during this grant will develop fundamentally new technology to enable early and frequent monitoring of HIV-1 infection at home.

项目摘要 当前的 HIV-1 RNA 检测依赖于基于 PCR 的扩增之前的逆转录，这引入了 不需要的变量并且不能在实验室外轻松地进行。这个假设的中心假设 应用是新发现的 CRISPR/Cas13a 蛋白的 RNA 结合特性适用于 无需使用 RT 或扩增步骤即可在家中灵敏地检测 HIV-1 RNA。这个假设是 根据 Doudna 实验室最近发现的 Cas13a 结合和切割靶标制定的 以序列特异性方式（顺式切割）形成单链 RNA，随后发挥通用 RNase 作用 活性（反式切割），可用于基于荧光的目标 RNA 测量。在 Ott/Doudna 实验室未发表的初步结果还表明，重组 Cas13a 与 HIV-1 特异性引导 RNA (crRNA) 能够灵敏地检测 HIV-1 RNA。中心假设将是 以双管齐下、高度里程碑驱动的方法进行测试：在创新阶段（R61），将定义两个目标 用于可靠 HIV-1 检测并优化读数的最佳 Cas13a 同源物/crRNA 组合 家用技术。目标 1：优化向导 RNA (crRNA) 和 Cas13a 蛋白选择。申请人 将设计 HIV 特异性 crRNA，识别目标 HIV-1 基因组的保守可及区域，并且 系统地测试来自不同细菌的 Cas13a 同源物的 HIV 特异性顺式和反式切割。在最后 为了进入 R33 阶段，团队将鉴定出 ≥3 个优化的 crRNA HIV-1基因组中的间隔序列，选择≥1个具有高HIV特异性顺式和反式的Cas13a同源物 低背景的 ssRNA 切割率。目标 2：增强读出技术并优化自我 测试。初步结果中，弗莱彻实验室在PCR扩增检测后测量了大肠杆菌DNA 使用基于 iPhone 的技术从嵌入染料发出荧光。申请人将确定最佳方案 Cas13a 与人 RNase 蛋白的反式切割序列以及优化荧光团和 检测寡核苷酸上的猝灭剂部分，用于使用基于手机的反向镜头进行测量 显微镜（CellScope）。在 R61 阶段结束时，为了进入 R33 阶段，≥1 次检测 序列将被识别，并且 ≥1 荧光/猝灭剂组合将被优化 CellScope 检测。 R33 阶段包含一个目标，将 Cas13a-CellScope 结果与 对急性和慢性感染个体进行常规病毒载量测定并使该方法适应家庭 使用。目标 3：应用优化的 Cas13a 检测参数构建临床自检设备 样品。该团队将优化血浆、血清和全血中的 HIV-1 RNA 检测， 增强测试系统在室温下的稳定性并分析冷冻保存的新鲜患者样本 由 SCOPE 队列中的 Deeks 博士提供。预计本次赠款期间完成的工作将 开发根本性的新技术，以便能够尽早、频繁地在家中监测 HIV-1 感染。