(27ax) Nonfluorescent Ratiometric Sensing Utilizing CRISPR-Cas12a-Induced DNA Supercoil Relaxation

Currently, most CRISPR-based biosensors rely on colorimetric, fluorescent, or electrochemical signaling mechanisms. Recent demonstration of molecular sizing-based approach could be an alternative nonoptical detection method utilizing the size reduction of long λ DNA substrate upon target recognition. However, precise determination of band shift for long DNA reporters (e.g., λ DNA) is challenging because it requires an intact reference λ DNA reporter run on the same gel and the positions of bands from multiple lanes are subject to various other interferences other than the molecular size, such as gel distortion. To overcome this limitation, here we utilized pUC19 plasmid DNA as the nonoptical, ratiometric CRISPR-Cas12a reporter, which includes an internal reference band for self calibration. Instead of utilizing the reporter's size change, we evaluated the conformation change of pUC19 plasmids induced by trans-activated Cas12a. We found that the nonspecific nicking effect of Cas12a could unwind negatively supercoiled pUC19 and turn it into the relaxed conformation. In the presence of target, we observed that supercoiled pUC19 decreased, and the amount of relaxed conformation increased with time (Fig. 1). The relative presence of supercoiled DNA to the relaxed circular DNA was analyzed by the gel electrophoresis to get a distinctive ratiometric signal for target concentration quantification. Utilizing this strategy, we detected ssDNA target with an LOD of ~2.5 pM without any preamplification step. This method can complement with previous molecular sizing-based CRISPR-Cas12a detection scheme and further enrich the nonoptical signaling mechanism of CRISPR-Dx for potential point-of-use applications.