植物CRISPRCas9基因组编辑系统与突变分析.pdf

植物CRISPR/Cas9 基因组编辑系统与突变分析 1,2 1,2 马兴亮 ，刘耀光 1. 亚热带农业生物资源保护与利用国家重点实验室，广州510642； 2. 华南农业大学生命科学学院，广州510642； 摘要：基因组定点编辑技术通过可编码核酸酶切割基因组特定位点，进而诱导基因组定点突变。CRISPR/Cas9 （Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 ）基因组编辑系统由Cas9 核酸酶以及sgRNA（Single guide RNA ）组成，与其他可编码核酸酶系统如锌指核酸酶（Zinc finger nuclease, ZFNs ） 和类转录激活因子效应物核酸酶 （Transcription activator-like effector nuclease, TALENs ）相比具有更简便的操作 性和更高的基因组定点编辑效率。目前在植物中已有多例应用CRISPR/Cas9 系统进行基因组编辑的报道。本文 从Cas9 基因与sgRNA 表达载体的构建策略，获得基因组定点编辑突变体的转化方法、突变的效率和特征、突 变的检测方法等方面进行了总结，最后对植物中利用CRISPR/Cas9 系统进行基因组编辑存在的问题以及发展前 景进行了讨论。 关键词：植物；CRISPR/Cas9 ；靶向突变；基因型鉴定 CRISPR/Cas9-based genome editing systems and the analysis of targeted genome mutations in plants Xingliang Ma1,2，Yaoguang Liu1,2 1.State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangzhou 510642, China; 2.College of Life Sciences, South China Agricultural University, Guangzhou 510642, China Abstract: Targeted genomic editing technologies use programmable DNA nucleases to cleave genomic target sites, thus inducing targeted mutations in the genomes. The newly prevailed clustered regularly interspaced short palindromic repeats/CRISPR -associated protein 9 (CRISPR/Cas9) system that consists of the Cas9 nuclease and single guide RNA (sgRNA) has the advantages of simplicity and high efficiency as compared to other programmable DNA nuclease systems such as zinc finger nucleases (ZFNs) and transcription activator like effector nucleases (TALENs). Currently, a number of cases have been reported on the application of the CRISPR/Cas9 genomic editing technology in plants. In this review, we summarize the strategies for preparing the Cas9 and sgRNA expression 收稿日期：2015-09-15；修回日期：2015-12-03 基金项目：转基因植物研究与产业化专项（编号：2014ZX08010-001 和 2014ZX08009-002 ）资助[Supported by Transgenic Research Program (2014ZX08010-001, 2014ZX08009-002)