GLOW-CRISPR: A G-Quadruplex Loop-Engineered Optical Workflow Combined with CRISPR-Cas12a System for SARS-CoV-2 Detection

Abstract SARS-CoV-2 remains a serious public health threat, highlighting the need for rapid and accessible nucleic-acid diagnostics for decentralized testing. CRISPR-Cas systems are promising platforms for viral diagnostics because of their high specificity and compatibility with isothermal workflows. However, many CRISPR-based assays still rely on labelled reporters, dedicated fluorescence instrumentation, or single-mode outputs that increase cost and limit field deployment. Herein, we developed a label-free GLOW-CRISPR assay (a G-quadruplex loop-engineered optical workflow combined with the CRISPR-Cas12a system) to detect SARS-CoV-2. The assay provides fluorescence and colorimetric readouts in separate endpoint workflows through loop-engineered G4 reporters. Upon recognition of the SARS-CoV-2 target, activated Cas12a cleaves the loop-engineered G4 reporters, thereby suppressing both fluorescence and colorimetric signals. Using optimized conditions, the GLOW-CRISPR assay achieved limits of detection of 7.7 nM and 2.9 nM for fluorescence and colorimetric readouts, respectively. It also showed no detectable cross-reactivity with non-target respiratory viruses and correctly classified patient-derived nasopharyngeal samples in concordance with RT-qPCR results. Collectively, these results establish G4 loop engineering as a general strategy for label-free CRISPR-Cas12a signal transduction without probe labelling or specialized instrumentation. By expanding optical readout flexibility while reducing assay cost and complexity, the GLOW-CRISPR assay provides a practical framework for decentralized molecular diagnostics in resource-limited settings.