Split crRNA-motivated amplification-free RNA testing with CRISPR-Cas12a

The CRISPR RNA (crRNA) consists of a conserved repeat RNA (rRNA) and an alterable spacer RNA (sRNA), which can guide the Cas12a effector to recognize and target DNA molecules of interest in both full-length and split fashion. We herein demonstrated the split crRNA can be repurposed for RNA detection through serving the sRNA as an RNA target. Inspired by this phenomenon, we developed a Cas12a-based direct RNA detection method, known as split crRNA-motivated amplification-free RNA testing (SMART). We adopted SMART to detect both short-stranded and long-stranded RNA target using two Cas12a orthologs (LbCas12a and FnCas12a), and it showed more prominent ability in detecting short-stranded RNA than long-stranded RNA. The potential mechanism revealed that RNA overhangs impede the RNA strand and dsDNA activator from accessing the catalytic site in the RuvC domain of the Cas12a effector, compromising the stability of the quaternary complex, and thus reducing the efficiency of SMART. Surprisingly, by simply introducing a short DNA activator, SMART could detect attomolar miRNA targets and femtomolar long-stranded RNA target without the need for additional preamplification or reverse transcription procedures. In addition, SMART showed wonderful discrimination ability toward single-nucleotide mutations. Moreover, the collaboration of SMART with the CRISPR-Cas13a system enabled simultaneous detection of multiplex RNAs. Overall, SMART is a simple, yet potent tool that can be flexibly applied to various short-stranded RNA detection, and holds great potential to be extended to other Cas12 orthologs.