| CRISPR/Cas9介导烟草多基因编辑体系的应用 |
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| 引用本文： | 谢小东,高军平,李泽锋,张剑锋,魏攀,罗朝鹏,王晨,武明珠,翟妞,杨军.CRISPR/Cas9介导烟草多基因编辑体系的应用[J].中国烟草学报,2019,25(4):72-80. |
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| 作者姓名： | 谢小东 高军平 李泽锋 张剑锋 魏攀 罗朝鹏 王晨 武明珠 翟妞 杨军 |
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| 作者单位： | 1.中国烟草总公司郑州烟草研究院, 国家烟草基因研究中心, 郑州高新技术产业开发区枫杨街2号 450001 |
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| 基金项目： | 郑州烟草研究院院长科技发展基金项目“烟草多基因CRISPRCas9编辑体系构建及应用研究”902017CA0210国家烟草专卖局资助项目“烟草基因功能元件全景图构建及关键基因挖掘与调控节点研究”110201601033（JY-07） |
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| 摘 要： | CRISPR/Cas9是一种新型的基因组定向编辑技术，近年来在烟草基因组的定向编辑中得到了广泛应用。CRISPR/Cas9介导的多基因编辑技术在许多物种中表现出很大的潜力，为了探索CRISPR/Cas9介导烟草多基因编辑的技术体系，本文针对烟草PCS1、HMA2、eIF4E、TOM3、TOM1 5个不同性状相关的基因构建了多靶点敲除的CRISPR/Cas9系统，并借助农杆菌介导的遗传转化技术转化烟草品种K326。对阳性植株的筛选、靶位点基因组的PCR扩增与测序分析表明，构建的CRISPR/Cas9多基因编辑系统成功转化到烟草中，能够同时靶向突变5个基因。5个基因同时突变的检出率为53.8%，单基因的突变检出率在76.9%与92.3%之间。进一步的脱靶检测分析显示，在所预测脱靶的候选位点上均未发生脱靶现象。本文构建的CRISPR/Cas9多基因编辑系统可将多个基因进行有效突变，为烟草基因功能研究和基于CRISPR/Cas9技术的多性状改良奠定了基础。
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| 关 键 词： | 烟草 CRISPR/Cas9 多基因 突变 |
| 收稿时间： | 2019-02-13 |
Application of multigene editing system mediated by CRISPR/Cas9 to Nicotiana tabacum |
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| Affiliation: | 1.China Tobacco Gene Research Center, Zhengzhou Tobacco Research Institute of CNTC, Zhengzhou 450001, China2.Technology Center, China Tobacco Hunan Industrial Co., Ltd., Changsha 410008, China |
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| Abstract: | CRISPR/Cas9 is a new type of directional genome editing technology, which has been widely used in targeted editing of tobacco genome in recent years. CRISPR/Cas9-mediated multi-gene editing techniques have shown great potential in gene functional research and genetic improvement on many species. In order to explore the technical system of CRISPR/Cas9-mediated multi-gene editing in tobacco, a multi-target CRISPR/Cas9 system was constructed for five genes (PCS1, HMA2, eIF4E, TOM3 and TOM1) associated with different traits, and then transformed into Nicotiana tabacum variety K326 by using agrobacterium-mediated genetic transformation technology. Through screening of the positive plants, PCR amplifcation and sequencing analysis of genome DNA of target sites, it is shown that the established multi-target CRISPR/Cas9 system can be transformed into tobacco, allowing simultaneous mutation of 5 target genes. The positive rate of simultaneous mutation of 5 genes was 53.8%. The detection rate of a single gene mutation was between 76.9% and 92.3%. Further evaluation and analysis of the off-targets showed that no off-targets occurred at the predicted candidate sites. This indicated that the multi-gene CRISPR/Cas9 editing system constructed in this paper could effectively mutate multiple genes, which laid a foundation for the study of tobacco gene function and the improvement of multi-traits based on CRISPR/Cas9 technology. |
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