Multiplexed Nucleic Acid Detection Using Sequence-Specific Cleavage Mediated by Repair Endonucleases

Rapid, portable detection of multiple nucleic acid targets is essential for infectious disease surveillance and precision oncology. CRISPR-based diagnostics have set a high bar for sensitivity and single-base specificity, yet their reliance on collateral nuclease activity complicates multiplexing, integration with amplification, and point-of-care deployment. Here, we present TIMBER (templated incision mediated by endonucleases of repair), a non-CRISPR platform that achieves comparable performance without nonspecific nuclease activity. TIMBER uses a repair endonuclease to cleave probes containing abasic sites only when they are hybridized to a matched target. The system exhibits strong specificity, enabling single-nucleotide polymorphism discrimination. TIMBER provides an analytical limit of detection of 12 pM without preamplification or one copy per μL (1.6 aM) with preamplification through RT-PCR or RT-LAMP, with results observable by visual fluorescence or on lateral flow. We deploy TIMBER to detect SARS-CoV-2 in clinical saliva samples and further demonstrate multiplex detection of four targets, enabling identification of EGFR mutations in lung cancer samples. This approach offers a flexible, rapid, and low-instrument-ready solution for diverse nucleic acid diagnostics, from viral detection to cancer genotyping.