CRISPR-Cas13-based rapid HIV-1 test

艾滋病毒是世界上最致命的传染病之一。艾滋病毒传播主要是由急性 不知道自己感染状况的感染者或接受抗逆转录病毒治疗但未感染病毒的感染者 抑制目前可用的艾滋病毒快速检测，包括第四代艾滋病毒抗原/抗体组合检测， 缺乏在急性感染和病毒反弹的早期阶段检测HIV所需的灵敏度。Nucleic 酸性扩增试验（NAAT）检测HIV-1 RNA，最早可在感染后5至10天内检测到。 病毒传播，提供了上级的灵敏度，目前是在急性期检测HIV的金标准。 感染和病毒反弹。然而，NAAT是费力的，耗时的，并且依赖于昂贵的 仪器和制冷部件，阻碍了它们用于自我测试。 因此，本项目的目标是开发和验证一种货架稳定，无仪器的CRISPR- 基于Cas 13的快速检测，用于血液或唾液中的高灵敏度HIV-1检测。这种高风险高回报的 这种方法与现有的NAAT有根本的不同，现有的NAAT涉及多个样品制备步骤（RNA 提取和扩增）或需要实验室仪器或冷冻组件。我们的快速HIV-1 测试将采用具有增强的附带活性和稳定性的工程化Cas 13 a酶，并冻干 分析组件，延长保质期，同时提供可在家中执行的简化工作流程 设置（类似于COVID-19抗原快速自检）。手机应用程序会自动分析 侧流装置，并以清晰、易于解释的方式呈现测试结果。这样做的理由 申请人的初步数据支持了这项研究， 使用基于CRISPR-Cas 13的快速侧向流测定和RRM 2-L Cas 13 a酶的开发， 具有增强的附属物活性和稳定性。在R61阶段，我们将追求以下具体目标： 1)描述不同艾滋病毒感染人群唾液和血液中的HIV-1 RNA水平; 2)研究用户友好型设备操作策略; 3)开发一种货架稳定、无需仪器的CRISPR-Cas 13快速检测方法，用于高灵敏度的HIV-1检测。 如果R61阶段的里程碑得以实现，我们将进一步开发和表征该平台，并验证其 通过追求以下具体目标，在R33阶段使用临床HIV标本实现功能： 4)进一步开发和表征基于CRISPR-Cas 13的侧向流快速HIV-1检测; 5)使用临床标本验证基于CRISPR-Cas 13的HIV-1快速检测的功能。 这种方法是创新的，因为它结合了速度，简单性和负担得起的横向流为基础的 CRISPR-Cas为基础的核酸检测的高灵敏度的诊断测试，它是显着的 因为它将提高艾滋病毒自我检测的有效性，以便在ART期间进行早期诊断和患者监测 因此，我们必须采取有效的预防和治疗措施，从而最大限度地减少艾滋病毒的无意传播，并降低与艾滋病毒有关的发病率和死亡率。

HIV is one of the most deadly infectious diseases in the world. HIV transmission is largely driven by acutely infected individuals who are unaware of their status or those receiving antiretroviral therapy but not virally suppressed. Currently available rapid HIV tests, including fourth-generation HIV antigen/antibody combo tests, lack the sensitivity needed for detecting HIV during early stages of acute infection and viral rebound. Nucleic acid amplification tests (NAATs) that detect HIV-1 RNA, which can be detected in as early as 5 to 10 days after viral transmission, offer superior sensitivity and are currently the gold standard for detecting HIV during acute infection and viral rebound. However, NAATs are laborious, time-consuming and rely on expensive instrumentation and refrigerated components, hindering their use for self-testing. Therefore, the objective of this project is to develop and validate a shelf-stable, instrument-free CRISPR- Cas13-based rapid test for highly sensitive HIV-1 detection in blood or saliva. This high-risk, high-reward approach is fundamentally different from existing NAATs that involve multiple steps of sample preparation (RNA extraction and amplification) or require laboratory instrumentation or refrigerated components. Our rapid HIV-1 test will employ an engineered Cas13a enzyme with enhanced collateral activity and stability, and lyophilized assay components for enhanced shelf life while offering a simplified workflow that can be performed in home settings (similar to COVID-19 antigen rapid self-tests). A phone app will automatically analyze the readout from the lateral flow device and present the test results in a clear, simple-to-interpret manner. The rationale for this research is supported by the applicants' preliminary data demonstrating highly sensitive detection of viral RNA using a rapid, lateral flow CRISPR-Cas13-based assay and the development of an RRM2-L Cas13a enzyme with enhanced collateral activity and stability. In the R61 phase, we will pursue the following specific aims: 1) Characterize HIV-1 RNA levels in saliva and blood from a diverse population of people with HIV; 2) Investigate strategies for user-friendly device operation; 3) Develop a shelf-stable, instrument-free CRISPR-Cas13 rapid test for highly sensitive HIV-1 detection. If the R61 phase milestones are achieved, we will further develop and characterize this platform and validate its functionality using clinical HIV specimens during the R33 phase by pursuing the following specific aims: 4) Further develop and characterize the lateral flow CRISPR-Cas13-based rapid HIV-1 test; 5) Validate the functionality of the CRISPR-Cas13-based rapid HIV-1 test using clinical specimens. This approach is innovative because it combines the speed, simplicity and affordability of lateral flow-based diagnostic tests with the high sensitivity of CRISPR-Cas-based nucleic acid detection, and it is significant because it will improve the effectiveness of HIV self-testing for early diagnosis and patient monitoring during ART therapy, thus minimizing unintentional HIV transmission and reducing HIV-associated morbidity and mortality.