Autocatalytic Cas13a biosensor enabled by RNA-nanocircles for ultrasensitive RNA detection

Cas13a-based diagnostic systems have been widely utilized for the detection of RNA targets. However, without preamplification such systems are difficult to realize ultrasensitive (RT-PCR level) single pot RNA detection. Here, we found that double strand RNA can effectively activate the trans-cleavage activity of Cas13a RNP, while the cleavage rates of dsRNA by activated Cas13a RNP are very low. In addition, specially designed RNA-Nanocircle has limited ability to activate Cas13a RNP, but this activation is restored once the circular structures are cleaved and become linear. Based on this original method to control trans-cleavage activity of Cas13a RNP, we developed a Cas13a autocatalytic biosensing system assisted by RNA-Nanocircles, which allows one target RNA to activate numerous Cas13a RNPs. With this approach we show ultrasensitive detection of 1aM of synthetic RNA targets without preamplification within 15 min. The clinical utility of this biosensor was validated by monitoring miRNA-21 levels in plasma samples from colorectal cancer patients. This innovative approach highlights the potential of Cas13a-based biosensors in precision oncology, offering a rapid, non-invasive, and ultrasensitive method for RNA biomarker detection in liquid biopsies.