CRISPR/Cas14a-SNPT: An engineered sgRNA mismatch-enabled platform for ultrasensitive SNP typing in hereditary G6PD deficiency.

Single-nucleotide polymorphism (SNP) detection plays a critical role in early screening and genotyping of genetic diseases. In this study, hereditary glucose-6-phosphate dehydrogenase (G6PD) deficiency was used as a model to develop a CRISPR/Cas14a-based SNP genotyping platform (CRISPR/Cas14a-SNPT). The platform integrates recombinase polymerase amplification (RPA) signal amplification with optimized sgRNA design. By introducing a shortened optimized sgRNA (op-sgRNA) to enhance Cas14a activity, together with engineered sgRNA mismatch regulation, the system enables rapid, highly sensitive, and high-resolution SNP detection. The results demonstrated a limit of detection of 1 copy/μL for plasmid templates, and reliable discrimination of variant allele frequencies as low as 0.1%. In clinical validation, the method successfully identified three hotspot mutations (c.95A > G, c.1388G > A, and c.1376G > T) in 13 human blood samples, showing excellent agreement with pyrosequencing results. Furthermore, molecular dynamics simulations revealed the regulatory mechanism of Cas14a mismatch recognition at the molecular level, suggesting that additional mismatches induce duplex relaxation and structural compensation effects, thereby enhancing target discrimination specificity. This study provides an efficient and practical strategy for accurate genotyping and prenatal carrier screening of G6PD deficiency-associated SNPs, demonstrating strong potential for rapid detection of clinically relevant SNPs.