关键词: 白化病, Tyr基因, 基因编辑, CRISPR/Cas9

Abstract: The TYR gene encodes tyrosinase. This gene mutation affects the production of melanin and is the genetic cause of human albinism. In this study, databases such as OMIM, ExAC and ClinVar were used to screen the strong pathogenic mutation site of human TYR gene. The site located on the mouse genome according to human strong pathogenic mutation site was found by protein sequence comparison analysis. Then three guide RNAs (small guide RNAs, sgRNAs) were designed based on the DNA sequence of located site and the principle of CRISPR/Cas9 gene targeting. The construction of sgRNA expression vectors, co-transfection of N2a cells, drug screening, PCR product sequencing and TA clone sequencing analysis were performed for the target efficiency analysis of the three sgRNAs. TA clone sequencing analysis results showed that random insertion or deletion mutations of bases occurred at all three sites, and the mutation efficiency was 100%, indicating that this study successfully constructed a highly efficient CRISPR/Cas9 system targeting mouse Tyr gene for simulating the strong pathogenic mutation of human albinism. The CRISPR/Cas9 gene editing system has laid the foundation for further preparation of Tyr genetically engineered mouse, in-depth study of the pathogenic mechanism of TYR gene mutation and the search for reliable treatment methods.

Key words: albinism; Tyr gene, gene editing, CRISPR/Cas9