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 In unpublished preliminary results, the Ott/Doudna Labs also show that recombinant Cas13a in combination with HIV-1-specific guide RNAs (crRNAs) enables sensitive detection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检测取决于基于PCR的扩增之前的逆转录，这引入了 不必要的变量，不能轻易在实验室外进行。中心假设 应用是新发现的CRISPR/CAS13A蛋白的RNA结合特性适合 在不采用RT或扩增步骤的情况下，对HIV-1 RNA的敏感在家检测。这个假设是 根据Doudna实验室最近发现Cas13a结合和切割目标的伪造 单链RNA以序列特异性方式（CIS裂解），随后发挥一般RNase 可以探索基于荧光的靶RNA的活性（反裂解）。在 未发表的初步结果，OTT/Doudna实验室还表明，重组CAS13A与 HIV-1特异性指南RNA（CRRNA）可实现HIV-1 RNA的敏感检测。中心假设将是 以两种高度里程碑式驱动的方法进行测试：在创新阶段（R61），两个目标将定义 可靠HIV-1检测的最佳CAS13A同源物/CRRNA组合并优化了读出 用于家庭用途的技术。目标1：优化指导RNA（CRRNA）和CAS13A蛋白选择。申请人 将设计识别靶标HIV-1基因组的HIV特异性CRRNA和 系统地测试来自不同细菌的CAS13A同源物，用于HIV特异性顺式和反式裂解。在最后 在R61期间，为了进步到R33阶段，团队将确定≥3个优化的CRRNA HIV-1基因组中的间隔序列，选定的≥1个CAS13A同源物具有较高的HIV特异性顺式和反式 - 背景低的ssRNA清洁率。目标2：增强读出技术并优化自我 测试。在初步结果中，Fletcher Lab在PCR扩增后测量了大肠杆菌DNA检测 与基于iPhone的技术互化染料的荧光。申请人将定义最佳 CAS13A与人RNase蛋白的反式裂解序列，并优化荧光团和 用基于手机的反向镜头测量的检测寡核苷酸的淬火器部分 显微镜（CellScope）。在R61时期结束时，为了进展到R33阶段，≥1检测 序列将被鉴定出来，≥1荧光/淬火器组合将被优化 CellScope检测。 R33相由一个目的组成，严格将Cas13a-cellscope结果与 急性和长期感染的个体中的常规病毒负荷分析，并将该方法适应家庭 使用。 AIM 3：将优化的CAS13A测定参数应用于使用临床的自我测试设备 样品。该团队将在血浆，血清和全血的背景下优化HIV-1 RNA检测， 在室温下增强测试系统的稳定性，并分析冷冻保存和新鲜的患者样品 由范围队列的Deeks博士提供。预计在这笔赠款期间完成的工作将 开发了从根本上开发新技术，以便早期和经常监测家里的HIV-1感染。