HIV Viral Load Testing Using CRISPR Cascade Signal Amplification - Abstract The HIV/AIDS epidemic poses a major threat to public health, affecting 40 million people worldwide. Early diagnosis of acute HIV infection during the seroconversion window facilitates early intervention. Monitoring HIV viral load frequently during antiretroviral treatment (ART) is essential to confirm treatment effectiveness and detect viral rebound. HIV viral load testing, which quantifies HIV viral RNA (circulating virus) in plasma, is the gold standard and the most effective method for ART monitoring and acute HIV detection. However, standard HIV viral load testing methods rely on expensive equipment and well-trained personnel, limiting their use to centralized laboratories and hospital environments. Although several portable HIV RNA diagnostic platforms are commercially available, they still require relatively expensive (> $10K) benchtop instruments and trained technicians, rendering them unsuitable for point-of-care applications. Here, we propose to develop a rapid, affordable, and sensitive CRISPR cascade signal amplification approach for HIV viral load testing in clinical samples. We will investigate the allosteric regulation mechanism of CRISPR activation by HIV RNA with various secondary structures and sequences. Furthermore, we will develop a CRISPR cascade signal amplification assay for HIV detection at room temperature. To simplify the detection operation, we will incorporate the assay into a microfluidic platform to create an accessible diagnostic tool. We will validate the clinical application of this microfluidic diagnostic approach in collaboration with clinicians. If successful, this simple and cost-effective CRISPR cascade signal amplification method will enable acute HIV diagnosis and viral load testing at resource- limited settings.
