靶向小鼠KRT5基因的CRISPR/Cas9系统设计与打靶效力分析  

CRISPR/Cas9 system design and target effectiveness verification targeting KRT5 gene of mice

作　　者：王蓉 陈圆圆 潘向和 李鑫 于鸿浩 岳鹏鹏 WANG Rong;CHEN Yuanyuan;PAN Xianghe;LI Xin;YU Honghao;YUE Pengpeng(School of Intelligent Medicine&Biotechnology,Guilin Medical University,Guilin 541199;Key Laboratory of Biochemistry&Molecular Biology of Guangxi Institutions of Higher Learning,Guilin Medical University,Guilin 541199;Dept.of Radiology,the 2^(nd) Affiliated Hospital of Guilin Medical University,Guilin 541199,China)

机构地区：[1]桂林医学院智能医学与生物技术学院,广西桂林541199 [2]桂林医学院广西高校生物化学与分子生物学重点实验室,广西桂林541199 [3]桂林医学院第二附属医院放射科,广西桂林541199

出　　处：《华夏医学》2022年第5期1-7,共7页Acta Medicinae Sinica

基　　金：国家自然科学基金项目(31860302);广西科技基地和人才专项(2019AC20329)。

摘　　要：目的:单纯型大疱性表皮松解症(EBS)是一种单基因遗传性皮肤疾病,KRT5基因突变是其主要的遗传学病因。本研究在综合分析人KRT5基因的致病突变位点后,在其附近设计、合成3条sgRNA序列,并构建靶向小鼠KRT5基因的CRISPR/Cas9系统,以模拟人KRT5基因的致病突变。方法:分析数据库中人KRT5基因的致病突变位点,设计3条小鼠拟突变位点sgRNA序列;构建pGL3-U6-KRT5-sgRNA表达质粒,然后采用Lipofectamine3000转染试剂将pGL3-U6-KRT5-sgRNA表达质粒与pST1374-NLS-flag-linker-Cas9表达质粒共同转染入小鼠N2a细胞,再以嘌呤霉素和杀稻瘟菌素进行筛选;采用PCR产物测序和TA克隆测序鉴定突变序列,分析打靶效力。结果:成功构建了3个pGL3-U6-KRT5-sgRNA表达质粒,将其转入小鼠N2a细胞后,打靶KRT5基因的效力最高为64.29%,最低为16.67%。结论:设计的CRISPR/Cas9系统可以有效打靶小鼠KRT5基因,为后续建立KRT5基因编辑的小鼠模型,探讨KRT5基因在EBS发生、发展中的作用和机制奠定基础。Objective: Epidermolysis bullosa simplex(EBS) is a monogenic hereditary skin disease, and KRT5 gene mutation is the main genetic cause. After a comprehensive analysis of the pathogenic mutation sites of the human KRT5 gene in this study, three sgRNA sequences targeting the mouse KRT5 gene were designed and synthesized nearby to mimic the pathogenic mutations of the human KRT5 gene, and the corresponding CRISPR/Cas9 system was constructed so as to validate mimic disease-causing mutations in the human KRT5 gene. Methods: The pathogenic mutation site of human KRT5 was analyzed through the database, three sgRNA sequences near the mutagenic site of mice was designed, and pGL3-U6-KRT5-sgRNA expression plasmid was constructed. Next, the pGL3-U6-KRT5-sgRNA expression plasmid and the pST1374-NLS-flag-linker-Cas9 expression plasmid were co-transfected into mouse N2 a cells through the Lipofectamine3000 transfection reagent kit, and then puromycin and blasticidal were used for screening. Finally, PCR product sequencing and TA cloning sequencing methods were used to analyze the targeting efficiency. Results: The pGL3-U6-KRT5-sgRNA expression plasmid was successfully constructed and co-transinfected into mouse N2 a cells, with the highest and lowest efficacy of targeting KRT5 gene were 64.29% and 16.67% respectively. Conclusion: The designed CRISPR/Cas9 system can effectively target the mouse KRT5 gene, which lays a foundation for the subsequent establishment of a KRT5 gene-edited mouse model and for exploring the role and mechanism of KRT5 gene in the occurrence and development of EBS.

关 键 词：CRISPR/Cas9 基因编辑 单纯型大疱性表皮松解症 KRT5 

分 类 号：R393[医药卫生—基础医学]