Programmable RNA Detection with CRISPR-Cas12a

CRISPR/Cas12a RNA-guided complexes are widely utilized for diagnostic purposes through nucleic acid detection, followed by Cas12a collateral DNA cleavage properties. However, the recognition and cleavage of RNA substrates by Class II Type V enzymes requires reverse transcription of RNA to DNA, due to Cas enzyme specificity. In this report, we demonstrate that the simultaneous addition of two truncated activators mimicking a full-length target can efficiently initiate trans-cleavage activity of three Cas12a orthologs. From this discovery, we have found that the PAM-proximal “seed” region of the crRNA exclusively recognizes DNA for trans-cleavage, while the PAM-distal region of the crRNA can tolerate both RNA and DNA substrates for LbCas12a, AsCas12a, and Er Cas12a effector proteins. Harnessing these properties, we have developed a “split activator” method named ‘Split Activators for Highly Accessible RNA Analysis’ (SAHARA) in which we are able to detect RNA sequences by merely supplying a short (12 nt) ssDNA or a PAM containing dsDNA to the seed region. For the first time, SAHARA allows for reverse transcription-free detection of RNA using Cas12a. The mechanism has been proven to detect picomolar concentrations of synthetic RNA resembling a polypeptide precursor gene in the Hepatitis C Virus (HCV) and miRNA-155. Our research provides valuable insights into the nucleic acid requirements and configurations for the activation of trans-cleavage activity in CRISPR/Cas12a and simultaneous detection of both DNA and RNA substrates.