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

项目摘要 目前HIV-1RNA的检测依赖于在基于聚合酶链式反应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执行。这一点的中心假设是 应用是新发现的CRISPR/Cas13a蛋白的RNA结合特性适合于 无需使用RT或扩增步骤即可在家中灵敏地检测HIV-1RNA。这一假设是 基于Doudna实验室最近发现的Cas13a结合和切割靶标的基础上制定的 以序列特异的方式(顺式切割)单链RNA，随后发挥一般的核糖核酸酶 可用于基于荧光的目标RNA测量的活性(反式切割)。在……里面 未发表的初步结果，Ott/Doudna实验室还显示，重组Cas13a与 HIV-1特异性指南RNA(CrRNAs)能够对HIV-1 RNA进行灵敏的检测。中心假设将是 在双管齐下、高度里程碑驱动的方法中进行测试：在创新阶段(R61)，两个目标将定义 可靠检测HIV-1的最佳Cas13a同源物/crRNA组合及其读数的优化 家用技术。目的1：优化引导RNA(CrRNA)和Cas13a蛋白的筛选。申请者 将设计HIV特异的crRNA，识别目标HIV-1基因组的保守可及区域，并 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，该团队将识别出≥3优化的crRNA ≥-1基因组中的间隔区序列，选择具有高HIV特异性顺式和反式的HIV 1Cas13a同源物。 背景较低的单链RNA裂解率。目标2：增强读出技术，优化自我 测试。在初步结果中，弗莱彻实验室在聚合酶链式反应扩增检测后测量了大肠杆菌DNA 使用基于iPhone的技术嵌入染料的荧光。申请者将确定最优 Cas13a与人核糖核酸酶蛋白的反式切割序列以及优化荧光团和 手机倒置透镜检测寡核苷酸的猝灭剂部分 显微镜(细胞显微镜)。在R61周期结束时，为了前进到R33阶段，≥1检测 序列将被鉴定，≥1荧光/猝灭剂组合将被优化用于 CellScope检测。R33阶段只有一个目标，将Cas13a-CellScope的结果与 急性和慢性感染者的常规病毒载量测定及其在家庭中的应用 使用。目的3：将优化的Cas13a检测参数应用于构建临床自检测设备 样本。该团队将优化血浆、血清和全血中HIV-1RNA的检测， 提高测试系统在室温下的稳定性，并分析冷冻保存和新鲜患者样本 由来自SCOPE队列的迪克斯博士提供。预计在这笔赠款期间完成的工作将 从根本上开发新技术，以便能够在家中及早和频繁地监测艾滋病毒-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 In unpublished preliminary results, the Ott/Doudna Labs also show that recombinant Cas13a in combination with HIV-1-specific guide RNAs (crRNAs) enables sensitive detection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.