RAPID: evaluation of Cas12a protospacer nicking and chimeric reporters for PAM-independent 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 and building on emerging protospacer adjacent motif (PAM)-independent Cas12a diagnostics that use engineered DNA activators and split-guide architectures, we introduce RAPID (RNA/DNA Advanced chimeric, PAM-independent, Integrated Nicking, Diagnostics), a nick-tuned, PAM-duplex-mediated platform for PAM-independent RNA and DNA detection. 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-independent RNA detection in clinical samples, achieving sensitivity down to ∼1 aM and 100% concordance with RT-qPCR for samples with Ct ≤ 33.