An Ultrasensitive, One-Pot RNA Detection Method Based on Rationally Engineered Cas9 Nickase-Assisted Isothermal Amplification Reaction

RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR) have revolutionized molecular diagnostics by offering versatile Cas effectors. We previously developed an isothermal amplification reaction method using Cas9 nickase (Cas9 nAR) to detect genomic DNA. However, slow dissociation of Cas9n from nicked double-stranded DNA (dsDNA) substrates dramatically hampers the cooperation between Cas9n and DNA polymerase, leading to low amplification efficiency. Here, we use structure-guided protein engineering to generate a Cas9n variant with faster kinetics and enhanced targeting specificity, and apply it to develop Cas9 nAR version 2 (Cas9 nAR-v2) by deftly merging reverse transcription with nicking-extension-displacement-based amplification for isothermal, one-pot RNA detection. This assay is validated by detecting Salmonella typhimurium 16S rRNA, Escherichia coli O157:H7 16S rRNA, synthetic SARS-CoV-2 genes, and HIV virus RNA, showing a quantitative analysis over a wide, linear range and a detection limit as low as fewer than ten copies of RNA molecules per reaction (20 μL volume). It also shows an excellent nucleotide-mutation discrimination capability in detecting SARS-CoV-2 variants. Furthermore, Cas9 nAR-v2 is compatible with low-cost point-of-care (POC) tests based on fluorescence and lateral-flow readouts. In summary, this method provides a new paradigm for sensitive, direct RNA detection and would spur the exploration of engineered Cas effectors with improved properties for a wide range of biological applications.