The College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China.
Plant Biotechnol J. 2018 Feb;16(2):415-427. doi: 10.1111/pbi.12781. Epub 2017 Aug 2.
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.
近年来，II 型 CRISPR 系统已成为在包括模式生物和作物植物在内的多种物种中实现定点突变的广泛使用和强大技术。然而，很少有研究使用 CRISPR 系统来操纵植物中的代谢途径。在这里，我们引入了 pYLCRISPR/Cas9 系统，该系统使用一个或两个单靶点向导 RNA 来靶向番茄八氢番茄红素脱氢酶基因。在 T0 再生植物中观察到明显的白化表型，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 系统来操纵番茄中的γ-氨基丁酸（GABA）分流。通过单株转化获得了 53 株基因组编辑植物，这些样本代表了单突变体到四突变体。基因组编辑系中叶片和果实中的 GABA 积累明显增加，四倍体突变体叶片中的 GABA 含量比野生型植物高 19 倍。我们的数据表明，多重 CRISPR/Cas9 系统可用于精确编辑番茄基因组序列并有效创建多位点敲除突变，这为植物代谢工程调控提供了新的思路。
