(27al) Programmable RNA Detection with CRISPR-Cas12a

CRISPR/Cas12a RNA-guided complexes are commonly employed for diagnostic purposes by detecting nucleic acids, which is followed by Cas12a collateral DNA cleavage. However, Class II Type V enzymes are unable to directly recognize and cleave RNA substrates, requiring reverse transcription of RNA to DNA. In this study, we illustrate that the addition of two truncated activators, simulating a full-length target, can effectively initiate trans-cleavage activity of three Cas12a orthologs simultaneously. Through this discovery, we have found that the PAM-proximal "seed" region of the crRNA only identifies DNA for trans-cleavage, while the PAM-distal region of the crRNA can tolerate both RNA and DNA substrates for Lb, As, and Er Cas12a effector proteins. Utilizing these characteristics, we have developed a "split activator" method called 'Split Activators for Highly Accessible RNA Analysis' (SAHARA), where we can identify RNA sequences by merely providing a short (12 nt) ssDNA or a PAM-containing dsDNA to the seed region. SAHARA permits reverse transcription-free RNA detection using Cas12a for the first time. The mechanism can identify picomolar concentrations of synthetic RNA resembling a polypeptide precursor gene in Hepatitis C Virus (HCV) and miRNA-155. Our study provides valuable insights into the nucleic acid needs and configurations for the activation of trans-cleavage activity in CRISPR/Cas12a, and the concurrent detection of both DNA and RNA substrates.