生物学杂志 ›› 2021, Vol. 38 ›› Issue (5): 34-.doi: 10.3969/j.issn.2095-1736.2021.05.034

• 研究报告 • 上一篇    下一篇

靶向小鼠Tyr基因的CRISPR/Cas9系统构建及打靶效力分析 #br# #br#

  

  1. 1. 桂林医学院 生物技术学院, 桂林 541199; 2. 桂林医学院 公共卫生学院, 桂林 541199;3.北京体育大学 运动人体科学学院,北京 100084
  • 出版日期:2021-10-18 发布日期:2021-10-20
  • 通讯作者: 于鸿浩,博士,教授,研究方向为分子遗传学,E-mail:geneyhh@126.com
  • 作者简介:岳鹏鹏,博士,副教授,研究方向为基因工程,E-mail:yue_pengpeng@163.com
  • 基金资助:
    国家自然科学基金项目(31860302);广西自然科学基金项目(2018JJA140455);广西中青年教师能力提升项目(2018KY0399)

Construction of CRISPR/Cas9 system targeting mouse Tyr geneand analysis of targeting effectiveness

  1. 1. School of Biotechnology, Guilin Medical College, Guilin 541199, China;
    2. School of Public Health, Guilin Medical College, Guilin 541199, China;
    3. School of Sports and Human Sciences, Beijing Sport University, Beijing 100084, China
  • Online:2021-10-18 Published:2021-10-20

摘要: TYR基因编码酪氨酸酶,该基因变异影响黑色素的生成,是人类白化病的遗传学病因。研究利用OMIM、ExAC和ClinVar等数据库筛查人TYR基因的强致病突变位点,并通过蛋白序列比对分析将该位点定位于小鼠基因组上,然后基于定位位点的DNA序列和CRISPR/Cas9系统基因打靶的原理设计了3个向导RNA(small guide RNA, sgRNA)。通过sgRNA表达载体的构建、N2a细胞的共转染、药物筛选、PCR产物测序,以及TA克隆测序分析等实验步骤完成了3个sgRNA的打靶效力分析。TA克隆测序分析结果显示3个位点均发生了碱基的随机插入或缺失突变,突变效率均为100%,表明研究成功构建了高效的靶向小鼠Tyr基因,并可模拟人类白化病强致病突变的CRISPR/Cas9基因编辑系统,为进一步制备Tyr基因工程小鼠,深入研究Tyr基因变异的致病机制和探寻可靠治疗手段奠定了基础。

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

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