DNA-guided CRISPR/Cas12 for RNA targeting

CRISPR-Cas nucleases are transforming genome editing, RNA editing, and diagnostics but have been limited to RNA-guided systems. We present {Psi}DNA, a DNA-based guide for Cas12 enzymes, engineered for specific and efficient RNA targeting. {Psi}DNA mimics a crRNA but with a reverse orientation, enabling stable Cas12-RNA assembly and activating trans-cleavage without RNA components. {Psi}DNAs are effective in sensing short and long RNAs and demonstrated 100% accuracy for detecting HCV RNA in clinical samples. We discovered that {Psi}DNAs can guide certain Cas12 enzymes for RNA targeting in cells, enhancing mRNA degradation via ribosome stalling and enabling multiplex knockdown of multiple RNA transcripts. This study establishes {Psi}DNA as a robust alternative to RNA guides, augmenting the potential of CRISPR-Cas12 for diagnostic applications and targeted RNA modulation in cellular environments.