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Cas13, a type VI CRISPR effector, targets RNA with high sensitivity through both cis (target RNA) and trans (collateral RNA) cleavage. This property enables the use of fluorescent reporters for sensitive diagnostics. However, Cas13\u2019s heightened sensitivity also leads to reduced specificity due to its susceptibility to single-nucleotide mismatches, potentially causing off-target effects. To overcome this limitation, we developed the first Dual-guide RNA system for Cas13 that improves mismatch discrimination and enhances target specificity. This system employs two distinct RNAs\u2014dcrRNA and dtracrRNA\u2014which cooperatively recognize the target and reduce off-target activity. In vitro experiments demonstrated robust cis- and trans-RNase activity, indicating efficient and specific cleavage. The system accurately detected SARS-CoV-2 RNA, distinguished KRAS G12D and G12C mutations, and differentiated mucocutaneous from cutaneous Leishmania sequences in analytical assays, with clinical validation confirming accurate detection of positive and negative samples. These results highlight the Dual-guide Cas13 platform\u2019s potential for precise, rapid, and reliable RNA detection. Overall, this approach represents a substantial advance over conventional Cas13 systems, offering improved specificity while maintaining clinically relevant sensitivity, and provides a generalizable tool for next-generation molecular diagnostics and precision RNA targeting and regulation.<\/jats:p>","DOI":"10.1093\/nar\/gkag161","type":"journal-article","created":{"date-parts":[[2026,2,24]],"date-time":"2026-02-24T12:39:49Z","timestamp":1771936789000},"source":"Crossref","is-referenced-by-count":7,"title":["A novel Dual-guide CRISPR\u2013Cas13 strategy improves specificity for single-nucleotide variant detection"],"prefix":"10.1093","volume":"54","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2960-8672","authenticated-orcid":false,"given":"Araceli","family":"Aguilar-Gonz\u00e1lez","sequence":"first","affiliation":[{"name":"GENYO, Centre for Genomics and Oncological Research, Pfizer, University of Granada, Andalusian Regional Government, PTS Granada , Avenida de la Ilustraci\u00f3n, 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