Intrinsic RNA Targeting Triggers Indiscriminate DNase Activity of CRISPR‐Cas12a

The CRISPR‐Cas12a system has emerged as a powerful tool for nucleic acid‐based molecular diagnostics. However, it has long been believed to be effective only on DNA targets. Here, we investigate the intrinsic RNA‐enabled trans‐cleavage activity of AsCas12a and LbCas12a and discover that they can be directly activated by fully complementary RNA targets, although LbCas12a exhibits weaker trans‐cleavage activity than AsCas12a on single‐stranded DNA and RNA substrates. Remarkably, we find that the RNA‐activated Cas12a exhibits higher specificity compared to DNA activation. Based on these findings, we develop the “Universal Nuclease for Identification of Virus Empowered by RNA‐sensing” (UNIVERSE) assay for nucleic acid testing. We incorporate a T7 transcription step into this assay, thereby eliminating the requirement for a protospacer adjacent motif (PAM) sequence in the target. Additionally, we successfully detect multiple PAM‐less targets in HIV clinical samples that are undetectable by the conventional Cas12a assay, demonstrating random target selection with the UNIVERSE assay. We further validate the clinical utility of the UNIVERSE assay by testing both HIV RNA and HPV 16 DNA in clinical samples. We envision that the intrinsic RNA targeting capability may bring a paradigm shift in Cas12a‐based nucleic acid detection and further enhance the understanding of CRISPR‐Cas biochemistry.