Single nucleotide polymorphisms genotyping via an ultrasensitive CRISPR-based assay

Single-nucleotide polymorphisms (SNPs) are crucial for dissecting genotype-phenotype associations, clinical diagnosis, and agricultural traits prediction. While CRISPR-based diagnostic tools enable rapid nucleic acid detection, their SNP discrimination ability is constrained by protospacer adjacent motif (PAM) requirements and inherent mismatch tolerance. Here, we found that PCR-generated single-stranded DNA (ssDNA) triggers trans-cleavage activity of Cas12a in a PAM‑free manner, and together with split crRNA enables effective SNP discrimination. Based on these, we developed SNIPER (Single Nucleotide Identification and Precision Evaluation Reporter), a PAM-free strategy optimized specifically for SNP discrimination. SNIPER successfully detects functional SNPs across 106 genetic loci spanning viruses, prokaryotes, and eukaryotes. Compared to previously reported method, SNIPER achieves robust discrimination even at low target concentrations, while SHERLOCK and SUREST failed to efficiently distinguish the given SNPs. SNIPER is compatible with both DNA and RNA targets, and can be formulated as lyophilized pellets of Cas12a and crRNA, thus can served as a versatile and reliable tool for disease control and prevention, clinical diagnosis, and crop germplasm trait prediction.