CRISPR/Cas13a-based single-nucleotide polymorphism detection for reliable determination of ABO blood group genotypes

The ABO blood group plays an important role in blood transfusion, linkage analysis, individual identification, etc. Serologic methods of blood typing are gold standards for the time being, which require stable typing antisera and fresh blood samples and are labor intensive. At present, reliable determination of ABO blood group genotypes based on single-nucleotide polymorphisms (SNPs) among A, B, and O alleles remains necessary. Thus, in this work, CRISPR/Cas13a-mediated genotyping for the ABO blood group by detecting SNPs between different alleles was proposed. The ABO*O.01.01(c.261delG) allele (G for the A/B allele and del for the O allele) and ABO*B.01(c.796C > A) allele (C for the A/O allele and A for the B allele) were selected to determine the six genotypes (AA, AO, BB, BO, OO, and AB) of the ABO blood group. Multiplex PCR was adapted to simultaneously amplify the two loci. CRISPR/Cas13a was then used to specifically differentiate ABO*O.01.01(c.261delG) and ABO*B.01(c.796C > A) of A, B, and O alleles. Highly accurate determination of different genotypes was achieved with a limit of detection of 50 pg per reaction within 60 min. The reliability of this method was further validated based on its applicability in detecting buccal swab samples with six genotypes. The results were compared with those of serological and sequencing methods, with 100% accuracy. Thus, the CRISPR/Cas13a-mediated assay shows great application potential in the reliable identification of ABO blood group genotypes in a wide range of samples, eliminating the need to collect fresh blood samples in the traditional method.