Mirror-synchronized asymmetric CRISPR nanoswitch for single-molecule profiling of multiple circRNAs in different stages of breast cancer

Circular RNAs (circRNAs) represent a class of endogenous noncoding RNAs characterized by their covalently closed circular structures. They have been implicated in significant transcriptional and post-transcriptional regulation of gene expression. Here, we present a one-pot method for the detection of circRNAs based on engineered DNA hairpins and CRISPR-Cas12a signal amplification, which involves signal pre-amplification via coupled probe-mediated hairpin amplification of two palindromic hairpins and Cas12a signal generation via trans-cleavage. We demonstrate that this platform is sensitive (detection limit of 1.07 aM), specific (capable of single-mismatch discrimination), and fast (reaction time of 25 min) and can be used to detect different circRNAs from RNase R-treated RNA (both in vitro and in clinically relevant samples, including correct classification of disease progression). This method enables single-molecule profiling and can be extended to detect other types of nucleic acids.