# Harnessing the RNA-Binding Properties of Cas13a for HIV-1 Self-Testing > **NIH NIH R33** · J. DAVID GLADSTONE INSTITUTES · 2021 · $884,506 ## Abstract 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 In unpublished preliminary results, the Ott/Doudna Labs also show that recombinant Cas13a in combination with HIV-1-specific guide RNAs (crRNAs) enables sensitive detection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 ... ## Key facts - **NIH application ID:** 10423661 - **Project number:** 4R33AI140465-04 - **Recipient organization:** J. DAVID GLADSTONE INSTITUTES - **Principal Investigator:** Melanie Maria Ott - **Activity code:** R33 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2021 - **Award amount:** $884,506 - **Award type:** 4N - **Project period:** 2019-08-01 → 2023-07-31 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/10423661 ## Citation > US National Institutes of Health, RePORTER application 10423661, Harnessing the RNA-Binding Properties of Cas13a for HIV-1 Self-Testing (4R33AI140465-04). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10423661. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*