SPARC: An Orthogonal Cas12a/Cas13a Dual-Channel CRISPR Platform for Reliable SNV Identification and Mutation Confirmation

Rapid and reliable detection of single nucleotide variants (SNVs) is essential for accurate pathogen diagnostics, genetic mutation screening, and personalized medicine. However, existing CRISPR-based nucleic acid detection platforms frequently suffer from ambiguous signal interpretation, specificity limitations, and complex assay workflows. Herein, we introduce SPARC (specific and precise mutation recognition with Cas12a/Cas13a), a novel orthogonal dual-channel CRISPR assay that significantly enhances the SNV detection reliability. SPARC integrates Acidaminococcus sp. Cas12a (AsCas12a), which specifically detects a conserved region as an internal reference, with our recently identified DNA-activated Leptotrichia buccalis Cas13a (LbuCas13a), which exhibits exceptionally high intrinsic SNV specificity without requiring engineered crRNA mismatches. The orthogonal design uniquely resolves the common diagnostic ambiguity between genuine SNVs and target absence. Combined with recombinase polymerase amplification (RPA) and T7 exonuclease digestion, the SPARC platform achieved a sensitivity as low as 1 aM. We demonstrated the platform's robust clinical applicability through successful detection and accurate differentiation of hepatitis B virus (HBV) and clinically significant YMDD resistance mutations. This work presents an innovative and versatile CRISPR-based solution, highlighting substantial potential for advancing clinical diagnostics and precision medicine.