Application of CRISPR/Cas9-Mediated Gene Editing in Tomato
Application of CRISPR/Cas9-Mediated Gene Editing in Tomato
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DOI：
10.1007/978-1-4939-8991-1_13
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发表时间：
2019-01-01
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影响因子：
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通讯作者：
Van Eck, Joyce
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文献类型：
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作者：
Reem, Nathan T.;Van Eck, Joyce
CRISPR-/ cas9介导的基因编辑已经在包括番茄在内的许多粮食作物中得到证实。番茄（Solanum lycopersicum）是一种重要的粮食作物，也是一种模式植物，被广泛用于研究基因功能，特别是与果实生物学有关的研究。这种双重目的与现成的资源（突变种群、基因组序列、转化方法）相结合，使番茄成为基因编辑的理想候选者。本实验室常规使用的CRISPR/Cas9系统已应用于多种番茄基因型和野生种茄（Solanum pimpinellifolium）。载体系统是基于金门克隆技术。将含有新霉素磷酸转移酶II （NPTII）选择性标记基因的卡带、CaMV 35S启动子驱动的人类密码子优化Cas9和拟南芥U6聚合酶启动子控制的引导RNA （gRNA）组装成T-DNA载体。通常，我们设计的CRISPR/Cas9构建体每个基因靶标包含两个grna。然而，我们已经成功地包含了多达8个grna，同时靶向多个基因和区域。将CRISPR-/ cas9设计的构建体导入番茄，采用基于农杆菌侵染幼嫩子叶的转化方法，以及基于NPTII基因存在的卡那霉素培养基选择。本章详细介绍了CRISPR/Cas9构建和基因型分析的方法（基于pcr的扩增子测序和T7内切酶）。
CRISPR-/Cas9-mediated gene editing has been demonstrated in a number of food crops including tomato. Tomato (Solanum lycopersicum) is both an important food crop and a model plant species that has been used extensively for studying gene function, especially as it relates to fruit biology. This duality in purpose combined with readily available resources (mutant populations, genome sequences, transformation methodology) makes tomato an ideal candidate for gene editing. The CRISPR/Cas9 system routinely used in our laboratory has been applied to various tomato genotypes and the wild species, Solanum pimpinellifolium. The vector system is based on Golden Gate cloning techniques. Cassettes that contain the neomycin phosphotransferase II (NPTII) selectable marker gene that confers resistance to kanamycin, a human codon-optimized Cas9 driven by the CaMV 35S promoter, and guide RNA (gRNA) under control of the Arabidopsis U6 polymerase promoter are assembled into a T-DNA vector. Generally, we design CRISPR/Cas9 constructs that contain two gRNAs per gene target. However, we have been successful with inclusion of up to eight gRNAs to simultaneously target multiple genes and regions. Introduction of CRISPR-/Cas9-designed constructs into tomato is accomplished by transformation methodology based on Agrobacterium tumefaciens infection of young cotyledon sections and selection on kanamycin-containing medium based on the presence of the NPTII gene. The approaches for the development of CRISPR/Cas9 constructs and genotypic analyses (PCR-based amplicon sequencing and T7 endonuclease) are detailed in this chapter.