(157e) Nonfluorescent CRISPR-Cas12a Biosensor By Sizing ? DNA

CRISPR-based biosensors have shown great potential in the diagnostic field. One current limitation is that these CRISPR biosensors often require colorimetric, fluorescent, or electrochemical reporters which are expensive and/or rely on sophisticated readout devices to acquire signals. Here, we developed a simple and inexpensive CRISPR-Cas12a-based assay platform to detect short single-stranded (ss) DNA targets using double-stranded (ds) λ DNA as a novel reporter molecule. In this detection platform, the target was mixed with LbCas12a-gRNA in a microcentrifuge tube. LbCas12a became trans-activated, and λ DNA was then added to the reaction mixture. λ DNA was chopped into fragments and visualized in gel electrophoresis (Figure 1). The physical size change of λ DNA was quantitatively correlated to the analyte concentrations. This assay was built on the new discovery that ds λ DNA can be trans-cleaved by the Cas12a effector. We hypothesize that the presence of D loops and short ss loop structures along the λ DNA sequence are the keys to the massive trans-nuclease activity of LbCas12a toward λ DNA. In addition, a strong binding affinity between Cas12a and λ DNA was also observed, which might promote the trans-cleavage activity. Using this nonfluorescent assay, we demonstrated a limit of detection (LOD) of 220 fM for synthetic ssDNA targets without any preamplification steps, which is ~100 times more sensitive when compared with the conventional CRISPR-Cas12a assay based on fluorescent reports (e.g., fluorophore-quencher reporters). This concept of utilizing the physical size change of λ DNA unlocks the possibility of using a variety of dsDNA as nonoptical CRISPR reporters.