Smart-SNPer: Automated crRNA design for ultrasensitive RPA-Cas12a SNP detection

Single-nucleotide polymorphism (SNP) detection is critical for precision medicine but is often hindered by complex workflows in resource-limited settings. Here, we introduce Smart-SNPer (https://smart-crisprer.com), a one-click, structure-guided crRNA design tool that automates the development of highly specific RPA (Recombinase Polymerase Amplification)-Cas12a assays (CORDSv2). The tool achieves 96 % target coverage across major SNP databases while optimizing crRNA folding and off-target avoidance. Using Smart-SNPer, we engineered the CORDSv2-NRAS:g.648C > A assay, which detects 0.1 % mutant alleles and requires only 20 ng of input genomic DNA--enabling low-abundance mutation detection by reducing wild-type DNA competition. In 27 clinical samples (including 1.2 % low-frequency variants), the assay showed 100 % concordance with Next-generation sequencing (NGS), alongside a linear semiquantitative response (R² > 0.9). The entire workflow is field-deployable, delivering visual results within 1 h and minimizing patient sampling burdens. By integrating automated crRNA design, ultrasensitive detection, and user-friendly web access, Smart-SNPer bridges the gap between high-performance SNP diagnostics and point-of-care applications.