A Novel Dual-Guide CRISPR-Cas13 Strategy Improves Specificity for Single-Nucleotide Variant Detection

The emergence of CRISPR–Cas systems has transformed nucleic acid detection and manipulation. Cas13, a type VI CRISPR effector, targets RNA with high sensitivity through both cis (target RNA) and trans (collateral RNA) cleavage. This property enables the use of fluorescent reporters for sensitive diagnostics. However, Cas13′s heightened sensitivity also leads to reduced specificity due to its susceptibility to single-nucleotide mismatches, potentially causing off-target effects. To overcome this limitation, we developed the first dual-guide RNA system for Cas13 that enhances mismatch discrimination and improves target specificity. This system employs two distinct RNAs (dcrRNA and dtracrRNA) which hybridise to refine target recognition and activation. In vitro experiments demonstrated robust cis- and trans–RNase activity, indicating efficient and specific cleavage. The system accurately detected SARS–CoV–2 RNA, demonstrating its potential for pathogen diagnostics, and successfully discriminated between KRAS G12D and G12C mutations, clinically relevant single-nucleotide variants in cancer diagnosis. These results highlight the dual–guide Cas13 platform′s potential for precise, rapid, and reliable RNA detection. Overall, this approach represents a significant advance over conventional Cas13 systems, offering improved specificity without compromising sensitivity. Its versatility makes it a promising tool for next-generation molecular diagnostics and precision gene editing applications.