Márquez-Costa, RosaMontagud-Martínez, RoserMarques, Maria CarmenAlbert, EliseoNavarro, DavidDaròs, José-AntonioRuiz, RaúlRodrigo Tarrega, Guillermo2024-06-112024-06-112023-05-190003-2700https://riunet.upv.es/handle/10251/204991[EN] Recurrent disease outbreaks caused by different viruses, including the novel respiratory virus SARS-CoV-2, are challenging our society at a global scale; so versatile virus detection methods would enable a calculated and faster response. Here, we present a novel nucleic acid detection strategy based on CRISPR-Cas9, whose mode of action relies on strand displacement rather than on collateral catalysis, using the Streptococcus pyogenes Cas9 nuclease. Given a pre-amplification process, a suitable molecular beacon interacts with the ternary CRISPR complex upon targeting to produce a fluorescent signal. We show that SARS-CoV-2 DNA amplicons generated from patient samples can be detected with CRISPR-Cas9. We also show that CRISPR-Cas9 allows the simultaneous detection of different DNA amplicons with the same nuclease, either to detect different SARS-CoV-2 regions or different respiratory viruses. Furthermore, we demonstrate that engineered DNA logic circuits can process different SARS-CoV-2 signals detected by the CRISPR complexes. Collectively, this CRISPR-Cas9 R-loop usage for molecular beacon opening (COLUMBO) platform allows a multiplexed detection in a single tube, complements the existing CRISPR-based methods, and displays diagnostic and biocomputing potential.Reconocimiento (by)Nucleic-acid detectionAmplificationSars-Cov-2DesignArtículo10.1021/acs.analchem.3c01041Abierto37204239PMC10255568