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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 \u223c1 aM and 100% concordance with RT-qPCR for samples with Ct\u00a0\u2264\u00a033.<\/jats:p>","DOI":"10.1093\/nar\/gkag748","type":"journal-article","created":{"date-parts":[[2026,7,31]],"date-time":"2026-07-31T11:48:20Z","timestamp":1785498500000},"source":"Crossref","is-referenced-by-count":1,"title":["RAPID: evaluation of Cas12a protospacer nicking and chimeric reporters for PAM-independent RNA and DNA diagnostics"],"prefix":"10.1093","volume":"54","author":[{"given":"Idorenyin A","family":"Iwe","sequence":"first","affiliation":[{"name":"Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto , Toronto, ON M5S 3M2 ,","place":["Canada"]},{"name":"Department of Mechanical and Industrial Engineering, University of Toronto , Toronto, ON M5S 1A1 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