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: 10423661
• 负责人: Melanie Maria Ott
• 金额: $ 88.45万
• 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 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10423661
• 关键词: Harnessing; RNA; Binding; Properties; Cas13a

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 In unpublished preliminary results, the Ott/Doudna Labs also show that recombinant Cas13a in combination with HIV-1-specific guide RNAs (crRNAs) enables sensitive detection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易地进行该中心假设。 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。 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。 Ott/Doudna 实验室未发表的初步结果还表明，重组 Cas13a 与 HIV-1 特异性指导 RNA (crRNA) 能够灵敏地检测 HIV-1 RNA 的中心假设是。 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 目标 1：优化引导 RNA (crRNA) 和 Cas13a 蛋白选择。 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 最后稳定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。 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 目标 2：增强读出技术并优化自我检测。 初步结果是，弗莱彻实验室在PCR扩增检测后测量了大肠杆菌DNA。 申请人将使用基于 iPhone 的技术来确定嵌入染料的荧光。 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 在 R61 期结束时进行显微镜检查 (CellScope)，以便进入 R33 期，≥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 目标 3：应用优化的 Cas13a 测定参数来构建临床自检设备。 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 团队的 Deeks 博士提供，预计在这笔资助期间完成的工作将 从根本上开发新技术，以便能够对家庭中的 HIV-1 感染进行早期和频繁的监测。