Multiplexed CRISPR/Cas9-mediated metabolic engineering of γ-aminobutyric acid levels in Solanum lycopersicum.
Multiplexed CRISPR/Cas9-mediated metabolic engineering of γ-aminobutyric acid levels in Solanum lycopersicum.
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番茄中 γ-氨基丁酸水平的多重 CRISPR/Cas9 介导的代谢工程
DOI：
10.1111/pbi.12781
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发表时间：
2018-03
影响因子：
13.8
通讯作者：
Zhu H
中科院分区：
文献类型：
--
作者：
Li R;Li R;Li X;Fu D;Zhu B;Tian H;Luo Y;Zhu H
近年来，II型CRISPR系统已成为一种广泛使用的和稳健的技术，用于在包括模式植物和作物植物在内的各种物种中实施定点诱变。然而，很少有研究使用CRISPR系统操纵植物中的代谢途径。在这里，我们引入了具有一个或两个单位点指导RNA的pYLCRISPR/Cas9系统，以靶向番茄八氢番茄红素去饱和酶基因。在T0再生植株中观察到明显的白化病表型，并且超过61%的所需靶位点被编辑。此外，我们使用靶向五个关键基因的多重pYLCRISPR/Cas9系统操纵番茄中的γ-氨基丁酸（GABA）分流。在单个植物转化后获得了53个基因组编辑的植物，并且这些样品代表单个至四个突变体。基因组编辑株系的叶片和果实中的GABA积累显著增强，四重突变体叶片中的GABA含量比野生型植物高19倍。我们的数据表明，多重CRISPR/Cas9系统可以用于精确编辑番茄基因组序列，并有效地创建多位点敲除突变，这可能为植物代谢工程调控提供新的思路。
In recent years, the type II CRISPR system has become a widely used and robust technique to implement site‐directed mutagenesis in a variety of species including model and crop plants. However, few studies manipulated metabolic pathways in plants using the CRISPR system. Here, we introduced the pYLCRISPR/Cas9 system with one or two single‐site guide RNAs to target the tomato phytoene desaturase gene. An obvious albino phenotype was observed in T0 regenerated plants, and more than 61% of the desired target sites were edited. Furthermore, we manipulated the γ‐aminobutyric acid (GABA) shunt in tomatoes using a multiplex pYLCRISPR/Cas9 system that targeted five key genes. Fifty‐three genome‐edited plants were obtained following single plant transformation, and these samples represented single to quadruple mutants. The GABA accumulation in both the leaves and fruits of genomically edited lines was significantly enhanced, and the GABA content in the leaves of quadruple mutants was 19‐fold higher than that in wild‐type plants. Our data demonstrate that the multiplex CRISPR/Cas9 system can be exploited to precisely edit tomato genomic sequences and effectively create multisite knockout mutations, which could shed new light on plant metabolic engineering regulations.
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影响因子：
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