Friday, May 9, 2025
5/9/2025
A rapid CRISPR-based self-testing platform for early detection of HIV - PROJECT ABSTRACT/SUMMARY In 2018 alone, the WHO reported 1.7 million new cases and estimated 770,000 HIV-related deaths due to gaps in HIV services. Early detection of HIV would allow quicker intervention and can significantly reduce the risk of death and further transmission. Therefore, this proposal aims to develop an innovative engineered CRISPR/Cas-based automated self-testing diagnostic platform for early detection of HIV RNA. The type V and VI CRISPR/Cas systems when bound with their specific target sequence, activate a secondary collateral nuclease activity that can rapidly cleave single-stranded nucleic acids in a non-specific multiple-turnover manner. By monitoring the collateral activity of CRISPR/Cas systems using a FRET-based reporter, picomolar concentrations of a target can be detected. Jain lab (PI) discovered that 3'-end extensions of CRISPR RNAs with a short 7-nt DNA, drastically enhances the collateral nuclease activity of LbCas12a by 3.2-fold and termed it ENHANCE. By applying ENHANCE femtomolar concentrations (700 fM) of HIV-1 target gene was detected in 30 min without requiring any target pre-amplification (Nat. Comms., 2020 & Methods, 2021). The Jain lab developed a lyophilized version of ENHANCE v2, which is stable up to 30 days at room temperature and in combination with an isothermal DNA amplification step, achieved a limit of detection of 15 copies/µL of SARS- CoV-2 RNA with ~97% sensitivity and ~97% specificity in patient samples within 50 minutes (Comms. Med., 2022). The PI recently developed a thermophilic BrCas12b-based single-pot SPADE assay to detect SARS- CoV-2 in saliva using a point-of-care device (eBioMed., 2022) and an engineered BrCas12b-based SPLENDID assay to detect HCV RNA in serum extracted with magnetic beads (Cell Rep. Med.-in revision, 2023). Based on the preliminary data, the exploratory R61 phase will have the following specific goals. 1) To enhance sensitivity and specificity of CRISPR/Cas systems to detect 1000 copies of RNA in 1 mL of plasma without the need for an isothermal DNA amplification step. 2) To develop an automated integrated microfluidic device for sample preparation, extraction, and detection of a HIV-1 target by naked eye at 1000 target copies/mL concentration. 3) To perform a pilot study with the optimized device for validating HIV-1 RNA detection in clinical plasma samples from healthy (n=60), and HIV-infected (n=60) with 95% accuracy. Only after achieving these milestones, the project will transition into the R33 phase with the following specific aims: 1) To recruit and survey 200 participants for user acceptability and usability of the device. 2) To perform clinical assessment of the self- testing platform with 200 participants. This integrated approach will have all the components as defined by the ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free and Deliverable to end-users) criteria by WHO. The development of this rapid self-testing platform would allow quicker interventions, reduced outbreak and ultimately reduced deaths in people infected with HIV.
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