基础医学与临床 ›› 2021, Vol. 41 ›› Issue (3): 325-332.

• 研究论文 • 上一篇    下一篇

构建模拟人致病位点突变的靶向小鼠Krt14的CRISPR/Cas9系统

岳鹏鹏,黎成钦,农月娟,陈玲,付灿,于鸿浩   

  1. 桂林医学院
  • 收稿日期:2020-07-01 修回日期:2020-09-28 出版日期:2021-03-05 发布日期:2021-03-01
  • 通讯作者: 于鸿浩 E-mail:geneyhh@126.com
  • 基金资助:
    遗传性多发性外生骨疣家系中EXT基因致病突变位点鉴定及动物模型构建

Establishing of a CRISPR/Cas9 system targeting at mouse Krt14 that stimulates pathogenic site mutations of human

  • Received:2020-07-01 Revised:2020-09-28 Online:2021-03-05 Published:2021-03-01

摘要: 目的 建立可模拟人类KRT14强致病位点突变的高效靶向小鼠Krt14的CRISPR/Cas9系统。方法:本研究通过检索多个数据库筛查人类单纯性大疱性表皮松懈症致病基因KRT14的强致病突变位点,并通过人和小鼠KRT14蛋白序列比对分析将筛查到的强致病位点定位于小鼠基因组上。根据CRISPR/Cas9系统的基因打靶原理,设计了4个靶向小鼠Krt14的sgRNA,并对应构建了4个sgRNA表达质粒。将sgRNA表达质粒分别与Cas9表达质粒共转染小鼠3T3细胞,转染细胞经过药物筛选、PCR产物测序、TA克隆测序等实验验证4个sgRNA的打靶效率。结果 根据筛查结果判定人KRT14蛋白的p.Arg125位点为强致病位点,与小鼠KRT14蛋白的p.Arg131位点相对应;根据小鼠p.Arg131位点的DNA序列,成功构建了4个靶向该位点的sgRNA表达质粒;药筛阳性细胞打靶位点的PCR扩增产物测序表明4个位点均发生了突变;TA克隆测序结果表明4个位点的突变效率分别为70%、90%、65%和100%。结论 根据筛查的人KRT14基因强致病位点,成功构建了靶向小鼠Krt14的高效CRISPR/Cas9系统,为深入研究Krt14基因的功能以及建立Krt14基因编辑小鼠模型探讨疾病的机制和治疗方法奠定基础。

关键词: 单纯性大疱性表皮松懈症, Krt14基因, 基因编辑, CRISPR/Cas9

Abstract: Objective To establish effective CRISPR/Cas9 system targeting mouse Krt14 that can mimic the strong pathogenic site mutation of human KRT14. Methods In this study, databases such as OMIM, ExAC and ClinVar were searched to screen the strong pathogenic sites of human KRT14, the causative gene of human epidermolysis bullosa simplex. Then, the human and mouse KRT14 protein sequence alignment analysis was used to locate the strong pathogenic sites on the mouse genome. Based on the gene targeting principle of the CRISPR/Cas9 system, we designed 4 sgRNAs targeting the mouse Krt14 and constructed 4 expression plasmids. Then, sgRNA expression plasmids and Cas9 expression plasmid were co-transfected into mouse 3T3 cells. PCR product sequencing, TA clone sequencing of the positive cells screened by drugs were performed to verify the target efficiency of 4 sgRNAs. Results According to the screening results, the p.Arg125 site of human KRT14 protein is determined to be a strong pathogenic site, which corresponds to the p.Arg131 site of mouse KRT14 protein. According to the DNA sequence of the mouse p.Arg131 site, 4 sgRNA expression plasmids targeting this site was successfully constructed. Sequencing of PCR amplification products of the target site of drug screen positive cells showed that 4 sites were all mutated. The results of TA cloning sequencing showed that the mutation efficiency of the four sites was respectively 70%, 90%, 65% and 100%. Conclusions The above results indicate that we have successfully constructed efficient CRISPR/Cas9 system targeting the mouse Krt14 based on the highly pathogenic sites of the human KRT14 screened. This study lays the foundation for the in-depth study of Krt14 gene function and the establishment of Krt14 gene editing mouse model to explore the mechanism and treatment of disease.

Key words: epidermolysis bullosa simplex, Krt14 gene, gene editing, CRISPR/Cas9