Abstract
Single nucleotide polymorphisms (SNPs) are associated with many human diseases, so accurate and efficient SNP detection is of great significance for early diagnosis and clinical prognosis. This report proposes a universal and high-fidelity genotyping method in microfluidic point-of-care equipment based on the clustered regularly interspaced short palindromic repeat (CRISPR) system. Briefly, by systematically inserting the protospacer-adjacent-motif (PAM) sequence, we improved the universality of the CRISPR/Cas12a based SNP detection; by removing the complementary ssDNA and introducing an additional nucleotide mismatch, we improved the sensitivity and specificity. We preloaded the CRISPR/Cas12a reagents into the point-of-care biochip for automating the process, increasing the stability and long-term storage. This biochip enables us to rapidly and conveniently detect the genotypes within 20 min. In a practical application, the CRISPR/Cas12a biochip successfully distinguished three genotypes (homozygous wild type; the homozygous mutant type; and the heterozygous mutant type) of the CYP1A1*2 (A4889G, rs1048943), CYP2C19*2 (G681A, rs4244285), CYP2C9*3 (A1075C, rs1057910), and CYP2C19*3 (G636A, rs4986893) genes related to multiple cancers from 17 clinical blood samples. This CRISPR/Cas12a-based SNP genotyping method, being universal, accurate, and sensitive, will have broad applications in molecular diagnostics and clinical research.