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

Abstract 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.