Detecting Melanocortin 1 Receptor Gene's SNPs by CRISPR/enAsCas12a.

Beyond its powerful genome-editing capabilities, the CRISPR/Cas system has opened up a new era of molecular diagnostics due to its highly specific base recognition and trans-cleavage activity. However, most CRISPR/Cas detection systems are mainly used to detect nucleic acids of bacteria or viruses, while the application of single nucleotide polymorphism (SNP) detection is limited. The SNPs were investigated by CRISPR/enCas12a and are not limited to the protospacer adjacent motif (PAM) sequence in vitro. Specifically, we optimized the reaction conditions, which proved that the enCas12a has a preference for divalent magnesium ion (Mg) and can effectively distinguish the genes with a single base difference in the presence of Mg, and the Melanocortin l receptor () gene with three kinds of SNP sites (T305C, T363C, and G727A) was quantitatively detected. Since the enCas12a is not limited by PAM sequence in vitro, the method shown here can extend this extraordinary CRISPR/enCas12a detection system to other SNP targets, thus providing a general SNP detection toolbox.