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

Wei Yang; Dagang Tao; Bingrong Xu; Yueting Zheng; Shuhong Zhao

doi:10.3390/genes14020394

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

Genes (Basel). 2023 Feb 2;14(2):394. doi: 10.3390/genes14020394.

Authors

Wei Yang^{1

2}, Dagang Tao^{1

2}, Bingrong Xu^{1

2}, Yueting Zheng³, Shuhong Zhao^{1

2}

Affiliations

¹ Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education & Key Lab of Swine Genetics and Breeding of Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, China.
² Guangdong Laboratory of Lingnan Modern Agriculture, Guangzhou 510642, China.
³ Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture and Rural Affairs and Key Laboratory of Animal Growth and Development of Henan Province, College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China.

Abstract

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 MC1R SNPs were investigated by CRISPR/enAsCas12a and are not limited to the protospacer adjacent motif (PAM) sequence in vitro. Specifically, we optimized the reaction conditions, which proved that the enAsCas12a has a preference for divalent magnesium ion (Mg²⁺) and can effectively distinguish the genes with a single base difference in the presence of Mg²⁺, and the Melanocortin l receptor (MC1R) gene with three kinds of SNP sites (T305C, T363C, and G727A) was quantitatively detected. Since the enAsCas12a is not limited by PAM sequence in vitro, the method shown here can extend this extraordinary CRISPR/enAsCas12a detection system to other SNP targets, thus providing a general SNP detection toolbox.

Keywords: CRISPR/enAsCas12a; MC1R; SNP; molecular diagnostics.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Bacteria / genetics
CRISPR-Cas Systems
Gene Editing / methods
Polymorphism, Single Nucleotide*
Receptor, Melanocortin, Type 1* / genetics

Substances

Receptor, Melanocortin, Type 1

Grants and funding

This research was funded by the Laboratory of Lingnan Modern Agriculture Project (No. NZ2021005), the Natural Science Foundation of China (32072685) and the Fund of Modern Industrial Technology System of Pigs (CARS-35).