CRISPR-Cas13-based rapid HIV-1 test

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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.