基于CRISPR/cas9系统高效编辑小鼠Galt基因  

Efficient Editing of Mouse Galt Gene Based on CRISPR/cas9 System

作　　者：岳鹏鹏 郭俊璠 于鸿浩 付灿 王小燕 高进涛[1] YUE Peng-peng;GUO Jun-fan;YU Hong-hao;FU Can;WANG Xiao-yan;GAO Jin-tao(College of Biotechnology,Guilin Medical University,Guilin 541000)

机构地区：[1]桂林医学院生物技术学院,桂林541000

出　　处：《生物技术通报》2020年第8期235-242,共8页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31860302);广西自然科学基金项目(2018JJA140455);广西科技计划项目(基于碱基编辑技术打靶Galt基因构建Ⅰ型半乳糖血症动物模型);广西大学生创新创业训练项目(201810601023);广西中青年教师能力提升项目(30606017005)

摘　　要：GALT基因突变是人类I型半乳糖血症的主要病因。拟通过CRISPR/cas9系统打靶小鼠Galt基因以模拟人GALT基因突变,从而为建立精准模拟I型半乳糖血症的动物模型奠定基础。首先分析了我国I型半乳糖血症GALT基因的致病突变位点,并将其定位在小鼠Glat基因上,作为小鼠Galt基因的拟突变位点,然后根据拟突变位点区域的序列设计了sgRNA导向序列,构建sgRNA表达质粒,将其与cas9表达质粒共转染小鼠3T3细胞,通过嘌呤霉素和杀稻瘟菌素筛选阳性转染细胞,提取阳性细胞基因组DNA,PCR扩增打靶位点的DNA片段,通过TA克隆测序鉴定基因编辑情况并分析编辑效率。结果表明,3个sgRNA导向序列均可以通过CRISPR/Cas9系统高效编辑小鼠Galt基因,编辑效率为100%。GALT gene mutation is the main cause of human type I galactosemia.This paper intends to target the mouse Galt gene through the CRISPR/cas9 system for simulating human GALT gene mutation,so as to lay the foundation for the establishment of an accurate animal model of type I galactosemia.We first analyzed pathogenic mutation sites of the GALT gene of type I galactosemia in China,and designed the sgRNA-directed sequence of the specifically targeted mouse Galt gene that can mimic the pathogenic mutation.Then sgRNA expression plasmid was then constructed and co-transfected with the cas9 expression plasmid into mouse 3T3 cells.The positive transfected cells were selected by puromycin and blasticidin,and the genomic DNA of the positive cells was extracted and the DNA fragment of the target site was amplified by PCR.The gene editing was identified and the editing efficiency was analyzed by TA clone sequencing.The results showed that the three sgRNAdirected sequences designed in this efficiently edited the mouse Galt gene through the CRISPR/Cas9 system,and the editing efficiency was 100%.

关 键 词：sgRNA CRISPR/Cas9系统 GALT 基因编辑 半乳糖血症 

分 类 号：Q78[生物学—分子生物学]