Ratiometric nonfluorescent CRISPR assay utilizing Cas12a-induced plasmid supercoil relaxation

Most CRISPR-based biosensors rely on labeled reporter molecules and expensive equipment for signal readout. A recent approach quantifies analyte concentration by sizing λ DNA reporters via gel electrophoresis, providing a simple solution for label-free detection. Here, we report an alternative strategy for label-free CRISPR-Cas12a, which relies on Cas12a trans-nicking induced supercoil relaxation of dsDNA plasmid reporters to generate a robust and ratiometric readout. The ratiometric CRISPR (rCRISPR) measures the relative percentage of supercoiled plasmid DNA to the relaxed circular DNA by gel electrophoresis for more accurate target concentration quantification. This simple method is two orders of magnitude more sensitive than the typical fluorescent reporter. This self-referenced strategy solves the potential application limitations of previously demonstrated DNA sizing-based CRISPR-Dx without compromising the sensitivity. Finally, we demonstrated the applicability of rCRISPR for detecting various model DNA targets such as HPV 16 and real AAV samples, highlighting its feasibility for point-of-care CRISPR-Dx applications.