Regulating CRISPR/Cas12a trans-cleavage on the hairpin DNA–MB nanointerface for enhanced multiplexed sensing application

RNA-programmed clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated systems (Cas) have garnered considerable attention in gene editing and molecular diagnostics. However, the precise control over the catalytic activity and cleavage site of Cas12a, a typical subtype of the Cas family, as well as its combination with downstream dynamic DNA technology remains challenging. Here, it was demonstrated that the cleavage activity and site can be modulated by adjusting the root, loop and stem of hairpin DNA reporters attached onto magnetic beads (MBs). The accelerated and controlled Cas12a trans-cleavage on the MB-hairpin DNA nanointerfaces is leveraged to trigger downstream hybridization chain reaction (HCR). Capitalizing on fluorescence (FL) labeling or double-stranded DNA (dsDNA) intercalators (SYBR green I or Ru-dppz), multiplexed detection (label or label-free, single-mode FL or dual-mode FL/ICP-MS) can be realized. Taking porcine pseudorabies virus (PRV) DNA as a model target, the potential application of this approach was demonstrated in clinical diagnosis, which achieves 100% accuracy that rivals the gold standard PCR. This methodology holds promise in a wide array of biological applications.