Truncated and Dual-Type Mismatched crRNA to Enhance Single Nucleotide Polymorphism (SNP)-Specific Detection by CRISPR/Cas12a

Single nucleotide polymorphisms (SNPs) are important genetic markers for disease diagnosis and precision medicine. However, conventional SNP detection methods are often limited by complex workflows, lengthy processing times, and high costs. CRISPR/Cas12a-based molecular diagnostics have emerged as a promising platform for SNP detection because of their rapid response and cost-effectiveness. Nevertheless, the high tolerance of conventional CRISPR RNA (crRNA) toward single-base mismatches limits the specificity of CRISPR/Cas12a-mediated SNP discrimination. To overcome this limitation, a truncated and dual-type mismatched crRNA (TDM-crRNA) was developed by combining a 17-nt spacer, an additional mismatch at position 2 within the seed region, and a wobble base pair at position 14. The performance of TDM-crRNA was systematically evaluated using SNP sites located both within and outside the seed region. Significantly enhanced SNP discrimination specificity was achieved in an amplification-free CRISPR/Cas12a assay, enabling reliable differentiation of both homozygous and heterozygous mutant genotypes. Furthermore, kinetic analysis and free-energy modeling revealed that the improved specificity resulted from reduced R-loop stability on mismatched targets together with an increased energetic barrier to R-loop formation, thereby enhancing the ability of Cas12a to distinguish single-nucleotide variations. Overall, TDM-crRNA provides a simple and effective strategy for improving the SNP recognition specificity of CRISPR/Cas12a and demonstrates considerable potential for SNP detection, genetic disease screening, and precision diagnostics.