DNA-guided CRISPR/Cas12 for cellular RNA targeting

CRISPR-Cas nucleases are transforming genome editing and diagnostics but have beenlimited to RNA-guided systems. Here we present psiDNA, a DNA-based guide for Cas12enzymes, engineered for specific and efficient RNA targeting. psiDNA functionally mimicsa crRNA but with a reverse orientation, enables target RNA-Cas12 assembly, and activatestrans-cleavage on ssDNA. psiDNAs are effective in sensing short and long RNAs anddemonstrated 100% accuracy for detecting HCV RNA in clinical samples. We discoveredthat psiDNAs can guide certain Cas12 enzymes for RNA targeting inside human cells,enhancing mRNA decay via ribosome stalling and enabling multiplex knockdown ofmultiple RNA transcripts with 70-95% efficiency. Compared to RfxCas13d, a popular RNAtargeting platform, psiDNA-Cas12a complexes exhibit 6- to 18-fold reduction in off-targeteffects, as confirmed by RNA-Seq profiling. Furthermore, fusing Cas12a to RNase Henabled targeted degradation of specific transcripts using engineered DNA guides, whilefusion with METTL3 facilitated site-specific N6-methyladenosine modification of targetRNAs. Together, our findings position psiDNA guides as a safe, economic, and highlyadaptable toolkit that pushes Cas12 systems beyond traditional genome-editing anddiagnostic roles, empowering precise, programmable control of cellular transcriptomes andtheir epitranscriptomic marks.