RAPID: Evaluation of Cas12a Protospacer Nicking and Chimeric Reporters for PAM-free RNA and DNA diagnostics

CRISPR-Cas nucleases have revolutionized diagnostics and biotechnology by providing programmable specificity. Here, we extend the understanding of Cas12a biology with a screen that, unexpectedly, finds that Cas12a trans cleavage activity can be modulated by nicks in the protospacer in a position-dependent manner. Wanting to explore the impact of non-conventional trans cleavage substrates, we subsequently find that non-specific Cas12a cleavage can be significantly reduced with RNA and chimeric (mixed RNA/DNA) reporter sequences. Exploiting these features, we introduce RAPID ( R NA/DNA A dvanced chimeric, P AM-free, I ntegrated Nicking, D iagnostics), a PAM-independent nucleic acid detection platform. By strategically introducing a nick within the spacer region, RAPID expands Cas12a detection to include target RNAs, which can be ligated in situ to create a hybrid protospacer-target with trans cleavage activity matching conventional Cas12a. We then apply RAPID to detect single point mutations in ssDNA and RNA substrates, a challenge for traditional Cas12 and Cas13 systems. In combination with RT-LAMP, RAPID is used for PAM-free RNA detection in clinical samples, achieving sensitivity down to ∼1 aM and 100% concordance with RT-qPCR.