利用CRISPR/Cas9基因编辑技术建立亨廷顿病(HD)细胞模型  

Establishing a cell model of Huntington's Disease(HD) using CRISPR/Cas9 gene editing technology

作　　者：付彬[1] 彭怡 徐兴然[1] Fu Bin, Peng Yi, Xu Xingran(College of Pharmacy, Southwest-University, Chongqing 400716, Chin)

机构地区：[1]西南大学药学院,重庆400716

出　　处：《江苏科技信息》2018年第17期38-42,共5页Jiangsu Science and Technology Information

基　　金：国家自然科学基金资助项目;项目编号:31472170

摘　　要：目的:探索CRISPR/Cas9基因编辑技术在构建亨廷顿病(Huntington's Disease,HD)细胞模型中的应用。方法:借助CRISPR/Cas9靶点设计网站对IT15基因进行分析并设计gRNA靶点序列,检测gRNA靶点序列的体外Cas9酶切活性。选择体外活性较好的一对gRNA靶点序列并装载到VK001-05打靶质粒上,根据靶点位置设计并构建50个CAG(Poly Q)重复且有EGFP和PuroR标记的Donor质粒。将打靶质粒和Donor质粒混合并借助转染试剂转染CATH-a细胞,提取转染后细胞基因组DNA鉴定gRNA靶点位置的基因型。结果:转染后CATH-a细胞的gRNA靶点位置准确无误地敲入50个CAG重复碱基。结论:研究进一步拓展了CRISPR/Cas9基因编辑技术在构建遗传性基因突变疾病模型方面的应用,同时也为未来临床治疗该类疾病提供了参考基础和研究平台。Objective：To explore the application of CRISPR/Cas9 gene editing technology in the construction of Huntington＇s Disease（HD）cell model.Methods：The IT15 gene was analyzed using the CRISPR/Cas9 target design site and the gRNA target sequence was designed,and the in vitro Cas9 cleavage activity of the gRNA target sequence was examined.A pair of gRNA target sequences with better activity in vitro was selected and loaded onto the VK001-05 target plasmid.Based on the target position,50 CAG（Poly Q）repeats were constructed and the Donor plasmid with EGFP and Puro R markers was constructed.The targeting plasmid and Donor plasmid were mixed and transfected with CATH-a cells by means of transfection reagent.After transfection,the genome DNA of the transfected cells was used to identify the genotype at the gRNA target site.Results：After transfection,the position of the gRNA target in CATH-a cells was accurately knocked into 50 CAG repeat bases.Conclusion：This study has further expanded the application of CRISPR/Cas9 gene editing technology in the construction of inherited genetic mutation disease models.It also provides a reference basis and research platform for future clinical treatment of such diseases.

关 键 词：CRISPR/Cas9 亨廷顿病 CATH-a细胞 POLY Q 

分 类 号：Q291[生物学—细胞生物学]