Single-tube Lambda exonuclease-mediated LbuCas13a detect of ssDNA for single-nucleotide polymorphisms genotyping

Single-nucleotide polymorphisms (SNPs) are critically important in genetics, disease association studies, and personalized medicine because they affect gene function, phenotypes, and drug response. Traditional methods like sequencing and PCR are time-consuming and often lack robust single-base resolution. Recent studies have shown that the Cas13a enzyme from Leptotrichia buccalis (LbuCas13a) can recognize single-stranded DNA (ssDNA) with exceptional specificity. Building on this, we have developed a rapid, isothermal, one-tube SNP genotyping platform (termed SNP-Specific Enzyme-based Nucleic acid Sensor for Efficient detection SNP-SENSE) that integrates recombinase polymerase amplification (RPA), Lambda exonuclease treatment, and LbuCas13a detection. In this assay, RPA amplifies the target allele CYP2C19∗2 (rs4244285, c.681G > A) CYP2C19∗3 (rs4986893, c.636G > A) and CYP2C19∗17 (rs12248560, c.806C > T), Lambda exonuclease converts the phosphorylated RPA product into ssDNA, and LbuCas13a-crRNA complexes cleave a fluorescent RNA reporter only in the presence of the perfectly matched target. This workflow operates at a constant temperature (∼39 °C) and completes in about 30 min. The method achieves single-nucleotide specificity, accurately distinguishing wild-type, heterozygous, and mutant genotypes. In tests with human whole blood samples, SNP-SENSE genotyped CYP2C19 alleles with 100 % concordance to Sanger sequencing. This single-pot platform offers a fast and precise tool for point-of-care genetic testing, supporting personalized treatment strategies and advancing precision medicine.