利用CRISPR/Cas9技术构建定点突变小鼠品系

doi:10.16288/j.yczz.15-127

遗传 ›› 2015, Vol. 37 ›› Issue (10): 1029-1035.doi: 10.16288/j.yczz.15-127

利用CRISPR/Cas9技术构建定点突变小鼠品系

白敏^1,2, 李崎², 邵艳姣³, 黄元华², 李大力³, 马燕琳²

1. 贵阳中医学院研究生院,贵阳 550002;
2. 海南医学院附属医院,海南省人类生殖与遗传重点实验室,海口 570102;
3. 华东师范大学生命科学学院,上海 200241

收稿日期:2015-03-26 出版日期:2015-10-20 发布日期:2015-10-20
通讯作者: 马燕琳,博士,副教授,研究方向：生殖医学与医学遗传学。E-mail: mayanlinma@hotmail.com。李大力,博士,研究员,研究方向：发育生物学。E-mail: dalidalili@yahoo.com
作者简介:白敏,硕士研究生,专业方向：生殖医学。Tel: 0898-66776091;E-mail: whizzard@163.com
基金资助:
国家重点基础研究发展计划项目(973项目)(编号：2012CB966502),国家国际科技合作专项(编号：2014DFA30180),国家自然科学基金项目(编号：81060175,30860103,81460034,81260032,81060016,31140021),海南省重大科技项目(编号：ZDZX2013003),海南省国际科技合作专项(编号：GJXM20100004,GJXM201106,KJHZ2014-11),人力资源和社会保障部2011年度留学人员科技活动择优资助项目和教育部归国留学人员启动基金项目资助

Generation of site-specific mutant mice using the CRISPR/Cas9 system

Min Bai^1，2， Qi Li²， Yanjiao Shao³， Yuanhua Huang²， Dali Li³， Yanlin Ma²

1. Guiyang College of Traditional Chinese Medicine, Guiyang 550002, China;
2. Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, Hainan Reproductive Medical Center, the Affiliated Hospital of Hainan Medical University, Haikou 570102, China;
3. Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China

Received:2015-03-26 Online:2015-10-20 Published:2015-10-20

摘要/Abstract

摘要： CRISPR/Cas9技术是新近发展起来的对细胞和动物模型进行基因编辑的重要方法。本文利用DNA双链断裂(Double-strand breaks, DSBs)引起的同源重组(Homologous recombination, HR)依赖与非依赖的修复机制,建立基于CRISPR/Cas9核酸酶技术构建定点突变小鼠品系的技术体系。针对赖氨酸特异脱甲基化酶2b(Lysine (K)-specific demethylase 2b, Kdm2b)酶活关键位点对应的基因组DNA序列设计单一导向RNA(Single-guide RNA, sgRNA),通过与Cas9 mRNA共显微注射,分别得到Kdm2b基因发生移码突变的基因失活品系及关键位点氨基酸缺失的酶活突变型小鼠品系。此外,利用HR介导的修复机理,将黄素单加氧酶3(Flavin containing monooxygenases3, Fmo3)基因的sgRNA序列及对应的点突变单链寡脱氧核苷(Single strand oligonucleotides, ssODN)修复模板共注射到小鼠受精卵雄原核。对F₀小鼠基因测序分析显示,成功构建了Fmo3基因移码突变的基因敲除和单碱基定点突变的基因敲入小鼠,这些突变能够稳定遗传给子代。本研究利用CRISPR/Cas9技术,通过同源重组依赖与非依赖两种DNA损伤修复方式,成功构建了特定位点突变的小鼠品系。

关键词: 基因敲除, 基因敲入, 酶活突变, 同源重组

Abstract: The CRISPR/Cas9 system is a recently developed important technology for genome editing in cellular and animal models. Here we established a CRISPR/Cas9-based system of generating site-specific mutant mice using DNA double-strand breaks (DSBs) induced homologous recombination (HR)-dependent or independent repair mechanism. Through co-microinjection of Cas9 mRNA and single-guide RNA (sgRNA) targeting genomic DNA sequence corresponding to enzyme activity of lysine (K)-specific demethylase 2b (Kdm2b), both a frame-shifted Kdm2b null mutant and a Kdm2b enzyme activity disrupted mouse strain were obtained simultaneously. Moreover, sgRNA targeting flavin containing monooxygenases3 (Fmo3) gene and the corresponding single strand oligonucleotides (ssODN) donor template with point mutation were co-injected into the male pronucleus of one-cell mouse embryos stimulated HR-mediated repair mechanism. Genomic sequence analysis of F₀ mice showed that frame-shifted Fmo3 knockout mouse and site-specific Fmo3 knock-in mouse with single base substitution were successfully generated, and these mutations could be stably transmitted to the next generation. Therefore, we successfully generated mouse strains containing site-specific mutations through HR-dependent and -independent DSB repair using the CRISPR/Cas9 system.

Key words: knockout, knock-in, enzyme activity mutant, homologous recombination

白敏, 李崎, 邵艳姣, 黄元华, 李大力, 马燕琳. 利用CRISPR/Cas9技术构建定点突变小鼠品系[J]. 遗传, 2015, 37(10): 1029-1035.

Min Bai， Qi Li， Yanjiao Shao， Yuanhua Huang， Dali Li， Yanlin Ma. Generation of site-specific mutant mice using the CRISPR/Cas9 system[J]. HEREDITAS(Beijing), 2015, 37(10): 1029-1035.

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利用CRISPR/Cas9技术构建定点突变小鼠品系

Generation of site-specific mutant mice using the CRISPR/Cas9 system

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