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

项目摘要 目前的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容易地进行。这个问题的核心假设是 新发现的CRISPR/Cas 13 a蛋白的RNA结合特性适用于 灵敏的HIV-1 RNA在家检测，无需RT或扩增步骤。了该假设 根据Doudna实验室最近发现Cas 13 a结合并切割靶点， 以序列特异性方式（顺式切割）切割单链RNA，随后发挥一般的RNA酶 活性（反式切割），可用于靶RNA的基于荧光的测量。在 Ott/Doudna实验室还显示，重组Cas 13 a与 HIV-1特异性引导RNA（crRNA）能够灵敏地检测HIV-1 RNA。核心假设是 在高度里程碑驱动的双管齐下的方法中进行测试：在创新阶段（R61），两个目标将定义 用于可靠的HIV-1检测的最佳Cas 13 a同源物/crRNA组合，并优化读出 家用技术。目的1：优化指导RNA（crRNA）和Cas 13 a蛋白的筛选。申请人 将设计识别靶HIV-1基因组保守可及区域的HIV特异性crRNA， 系统地测试来自不同细菌的Cas 13 a同源物的HIV特异性顺式和反式切割。年底 为了进入R33阶段，研究小组将鉴定出≥3种优化的crRNA HIV-1基因组中的间隔区序列，选择≥1个具有高HIV特异性顺式和反式 ssRNA切割速率，具有低背景。目标2：提高读出技术，优化自 试验.在初步结果中，弗莱彻实验室测量了E。PCR扩增后检测大肠杆菌DNA 荧光从嵌入染料与iPhone为基础的技术。申请人将定义最佳的 用于Cas 13 a相对于人RNase蛋白的反式切割的序列以及优化荧光团， 检测寡核苷酸上的猝灭剂部分，用于使用基于移动的手机的反向透镜进行测量 显微镜（CellScope）。在R61阶段结束时，为了进入R33阶段，≥1次检测 序列将已确定，且≥1种荧光/猝灭剂组合将已优化， CellScope检测。R33阶段包括一个目标，严格比较Cas 13 a-CellScope结果， 在急性和慢性感染个体中进行常规病毒载量测定，并使该方法适用于家庭 使用.目的3：将优化的Cas 13 a测定参数应用于构建使用临床应用的自测试装置。 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，并分析冻存和新鲜患者样本 由SCOPE队列的Deeks博士提供。预计在此期间完成的工作将 开发全新的技术，以便能够在家中早期和频繁地监测HIV-1感染。

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 In unpublished preliminary results, the Ott/Doudna Labs also show that recombinant Cas13a in combination with HIV-1-specific guide RNAs (crRNAs) enables sensitive detection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.