trans Single-Stranded DNA Cleavage via CRISPR/Cas14a1 Activated by Target RNA without Destruction

As a CRISPR-Cas system (clustered regularly interspaced short palindromic repeats and CRISPR associated proteins), Cas14a1 can cis/trans cleave single-stranded DNA (ssDNA). Here, we describe an unreported capacity of Cas14a1: RNA can trigger the trans ssDNA cleavage. This Cas14a1-based RNA-activated detection platform (Amplification, Transcription, Cas14a1-based RNA-activated trans ssDNA cleavage, ATCas-RNA) has an outstanding specificity for the detection of target RNAs with point mutation resolution, which is better than that of the Cas14a1-based ssDNA-activation. Using ATCas-RNA via a fluorophore quencher-labeled ssDNA reporter (FQ), we were able to detect 1 aM pathogenic nucleic acid within 1 h, and achieve 100 % accuracy with 25 milk samples. This platform can serve as a new tool for high-efficiency nucleic acid diagnostics. Importantly, this work can expand our understanding of Cas14a1 and inspire further mechanisms and applications of Class-2 Cas systems.