利用CRISPR/Cas系统快速高效构建血友病乙小鼠模型

doi:10.16288/j.yczz.15-117

遗传 ›› 2015, Vol. 37 ›› Issue (11): 1143-1148.doi: 10.16288/j.yczz.15-117

利用CRISPR/Cas系统快速高效构建血友病乙小鼠模型

汪启翰¹,怀聪¹,孙瑞林²,庄华²,陈红岩¹,费俭²,卢大儒¹

1. 复旦大学生命科学学院,遗传工程国家重点实验室,上海 200433;
2. 上海南方模式生物研究中心,上海 201203

收稿日期:2015-03-22 修回日期:2015-06-29 出版日期:2015-11-20 发布日期:2015-08-31
通讯作者: 卢大儒,博士,教授,研究方向：人类医学分子遗传学。E-mail: drlu@fudan.edu.cn;费俭,博士,教授,研究方向：基因功能的模式生物研究。E-mail：Jfei@tongji.edu.cn E-mail:rlsun@126.com
作者简介:汪启翰,硕士在读,专业方向：生物工程。Tel: 021-51630621;E-mail: zgzjfhwqh@gmail.com;怀聪：博士在读,专业方向：人类医学分子遗传学。E-mail: huaic@fudan.edu.cn;孙瑞林：博士,专业方向：基因功能的模式生物研究。E-mail: rlsun@126.com;汪启翰、怀聪和孙瑞林同为第一作者。
基金资助:
国家自然科学基金项目(编号：31571371)资助

A quick and efficient method to generate hemophilia B mouse models by the CRISPR/Cas system

Qihan Wang¹,Cong Huai¹,Ruilin Sun²,Hua Zhuang²,Hongyan Chen¹,Jian Fei²,Daru Lu¹

1. State Key Laboratory of Genetic Engineering, School of Life Science, Fudan University, Shanghai 200433, China;
2. Shanghai Research Center for Model Organisms, Shanghai 201203, China

Received:2015-03-22 Revised:2015-06-29 Online:2015-11-20 Published:2015-08-31

摘要/Abstract

摘要： 血友病乙是由凝血因子Ⅸ(Factor Ⅸ,FⅨ)缺乏或功能缺陷导致的出血性疾病,为伴X染色体隐性遗传病。小鼠模型对于血友病乙的研究具有十分重要的意义,而基因组编辑技术又为小鼠模型的构建提供了一种快捷而且高效的途径。本文利用CRISPR/Cas系统,在小鼠FⅨ基因第8外显子上选择靶位点,将Cas9 mRNA和带有靶位点的sgRNA显微注射到C57BL/6品系小鼠的受精卵中,获得基因修饰的小鼠。利用高分辨率熔解曲线分析(High resolution melting, HRM)技术进行精确基因分型,并通过测序验证,在60只小鼠中,总共有51只小鼠的靶位点发生了突变,突变率高达85%,其中雄鼠的突变率为79.5%,雌鼠的突变率为95.2%;未检测到非目标位置的基因编辑脱靶。凝血活性实验显示,突变小鼠的FⅨ活性值(Factor Ⅸ coagulant activity, FⅨ: C)是非突变小鼠的6.82%,大大低于非突变小鼠,表明突变小鼠的凝血活性缺失。本研究表明,利用CRISPR/Cas系统成功构建了人类血友病乙遗传病小鼠模型。

关键词: 血友病乙, CRISPR/Cas系统, 基因组编辑, 小鼠模型

Abstract: Hemophilia B, or the Christmas disease, is a common human disease caused by coagulation factor Ⅸ (FⅨ) deficiency. It is an X-linked recessive hereditary disease. Here we obtained FⅨ-knockout mouse strains with phenotype of hemophilia B with the CRISPR/Cas system efficiently. We chose the 8th exon as the target locus, and co-injected codon-optimized Cas9 mRNA with sgRNA of FⅨ into C57BL/6 mice zygotes. We obtained 60 mice in total and genotyped them by high resolution melting (HRM) and sequencing. The results showed the mutation rate was 85.0% in total, and 79.5% and 95.2% in males and females, respectively. No off-targets were detected in the similar locus by HRM. We future measured the FⅨ activity of each mice. The FⅨ: C of mutant mice were significantly below the normal level and reduced to 6.82% of wild-type mice. The activity assay demonstrated that all the mutant mice were lack of FⅨ. In summary, we have generated hemophilia B model mice with extreme efficiency, using the RNA-guided Cas9 nuclease gene editing system.

Key words: hemophilia B, CRISPR/Cas system, genome editing, mouse model

汪启翰,怀聪,孙瑞林,庄华,陈红岩,费俭,卢大儒. 利用CRISPR/Cas系统快速高效构建血友病乙小鼠模型[J]. 遗传, 2015, 37(11): 1143-1148.

Qihan Wang,Cong Huai,Ruilin Sun,Hua Zhuang,Hongyan Chen,Jian Fei,Daru Lu. A quick and efficient method to generate hemophilia B mouse models by the CRISPR/Cas system[J]. HEREDITAS(Beijing), 2015, 37(11): 1143-1148.

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利用CRISPR/Cas系统快速高效构建血友病乙小鼠模型

A quick and efficient method to generate hemophilia B mouse models by the CRISPR/Cas system

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