基于CRISPR/Cas9技术构建原位敲入亨廷顿病(HD)小鼠模型  

Establishing a in-situ-knock-in mouse model of Huntington's Disease(HD)by using CRISPR/Cas9 technology

作　　者：彭怡 王柏彬 付彬[1] 徐兴然[1] Peng Yi;Wang Baibin;Fu Bin;Xu Xingran＊(College of Pharmaceutical Science,Southwest University,Chongqing 400716,China)

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30期24-29,33,共7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:探究CRISPR/Cas9技术在建立亨廷顿病(Huntington’s Disease,HD)原位敲入小鼠模型中的运用。方法:首先,设计gRNA靶点序列(Htt gRNA)并检测其体外Cas9酶切活性,选择活性较好的一对备用。其次,利用全基因合成具有150Q(150个CAG重复)的Donor序列。然后将Cas9 mRNA,Htt-L3 gRNA,Htt-R3 gRNA和Donor DNA混合均匀进行胚胎显微注射,再将胚胎移植到受体小鼠中备孕。待小鼠出生后,鉴定F0代及其子代F1,F2,F3代的基因型。结果:F0,F1,F2及F3代小鼠基因组上均出现150Q的准确敲入,且子代稳定遗传。结论:在不改变小鼠HD基因表达调控的基础上,仅改变其特定致病基因的长度,利用CRISPR/Cas9技术成功构建HD原位敲入小鼠模型,为进一步模拟人类HD缓慢且迟发的发病过程及后续临床治疗研究拓展了HD模型。Objective： To explore the use of CRISPR/Cas9 technology in the establishment of a in-situ-knock-in mouse model of Huntington’s Disease. Methods： Firstly, the gRNA target sequences（Htt gRNA）are designed and Cas9 digestive activities are tested in vitro. A pair of gRNA target sequences with better activity are selected. Secondly, the Donor sequence with 150Q（150 CAG repeats）is synthesized by using gene synthesis. Then, Cas9 mRNA, Htt-L3 gRNA, Htt-R3 gRNA and Donor DNA are uniformly mixed and injected into the embryo, and the embryo is transplanted into the recipient mice. After the mice are born, the genotypes of F0, F1, F2, and F3 generation are identified. Results：150Q occur in the mouse genomes of F0, F1, F2 and F3 generation, and the descendants are stably inherited.Conclusion： Only the length of specific pathogenic gene is changed without changing the mouse HD gene expression,and the HD in-situ-knock-in mouse model is successfully constructed by CRISPR/Cas9 technology. This model is expanded to further simulate the slowly delayed onset process and clinical treatment studies of HD.

关 键 词：亨廷顿病 CRISPR/Cas9 原位敲入 小鼠模型 150Q 

分 类 号：Q789[生物学—分子生物学]