DNA-guided CRISPR/Cas effector for programable RNA-recognition and cleavage

CRISPR/Cas effectors rely on RNA guides to recognize target nucleic acids. In Cas9 and Cas12a systems, protospacer adjacent motif (PAM) on target DNA engage conserved residues in the Cas protein, thereby accelerating target binding and catalytic activation. Here, we overturn this paradigm by reprograming the canonical RNA-guided DNA-targeting machinery into a DNA-guided RNA-targeting platform. We engineered synthetic CRISPR DNA (crDNA) that emulates the PAM-duplex architecture to form stable deoxyribonucleoprotein complexes with Cas9 or Cas12a, redirecting their specificity towards RNA substrates. Coupling DNA-guided Cas12a with isothermal amplification enables attomolar detection of nucleic acid samples, while DNA-guided Cas9 achieves over 70% knock-down of EGFP expression in HEK293T cells. This DNA-guided paradigm reshapes our understanding of CRISPR targeting and paves the way for new diagnostic and therapeutic applications.