Ren Chong, Liu Yanfei, Guo Yuchen, Duan Wei, Fan Peige, Li Shaohua, Liang Zhenchang
Beijing Key Laboratory of Grape Science and Enology, and CAS Key Laboratory of Plant Resources, Institute of Botany, The Innovative Academy of Seed Design, The Chinese Academy of Sciences, 100093, Beijing, People's Republic of China.
University of Chinese Academy of Sciences, 100049, Beijing, People's Republic of China.
Hortic Res. 2021 Mar 1;8(1):52. doi: 10.1038/s41438-021-00489-z.
The efficacy of the CRISPR/Cas9 system in grapevine (Vitis vinifera L.) has been documented, but the optimization of this system, as well as CRISPR/Cas9-mediated multiplex genome editing, has not been explored in this species. Herein, we identified four VvU3 and VvU6 promoters and two ubiquitin (UBQ) promoters in grapevine and demonstrated that the use of the identified VvU3/U6 and UBQ2 promoters could significantly increase the editing efficiency in grape by improving the expression of sgRNA and Cas9, respectively. Furthermore, we conducted multiplex genome editing using the optimized CRISPR/Cas9 vector that contained the conventional multiple sgRNA expression cassettes or the polycistronic tRNA-sgRNA cassette (PTG) by targeting the sugar-related tonoplastic monosaccharide transporter (TMT) family members TMT1 and TMT2, and the overall editing efficiencies were higher than 10%. The simultaneous editing of TMT1 and TMT2 resulted in reduced sugar levels, which indicated the role of these two genes in sugar accumulation in grapes. Moreover, the activities of the VvU3, VvU6, and UBQ2 promoters in tobacco genome editing were demonstrated by editing the phytoene desaturase (PDS) gene in Nicotiana benthamiana leaves. Our study provides materials for the optimization of the CRISPR/Cas9 system. To our knowledge, our simultaneous editing of the grape TMT family genes TMT1 and TMT2 constitutes the first example of multiplex genome editing in grape. The multiplex editing systems described in this manuscript expand the toolbox of grape genome editing, which would facilitate basic research and molecular breeding in grapevine.
CRISPR/Cas9系统在葡萄（Vitis vinifera L.）中的功效已有文献记载，但该系统的优化以及CRISPR/Cas9介导的多重基因组编辑在该物种中尚未得到探索。在此，我们在葡萄中鉴定出四个VvU3和VvU6启动子以及两个泛素（UBQ）启动子，并证明使用鉴定出的VvU3/U6和UBQ2启动子可分别通过提高sgRNA和Cas9的表达，显著提高葡萄的编辑效率。此外，我们使用优化的CRISPR/Cas9载体进行多重基因组编辑，该载体包含传统的多个sgRNA表达盒或多顺反子tRNA-sgRNA盒（PTG），通过靶向与糖相关的液泡膜单糖转运蛋白（TMT）家族成员TMT1和TMT2，总体编辑效率高于10%。同时编辑TMT1和TMT2导致糖水平降低，这表明这两个基因在葡萄糖积累中的作用。此外，通过编辑本氏烟草叶片中的八氢番茄红素去饱和酶（PDS）基因，证明了VvU3、VvU6和UBQ2启动子在烟草基因组编辑中的活性。我们的研究为CRISPR/Cas9系统的优化提供了材料。据我们所知，我们同时编辑葡萄TMT家族基因TMT1和TMT2构成了葡萄多重基因组编辑的首个实例。本手稿中描述的多重编辑系统扩展了葡萄基因组编辑的工具箱，这将有助于葡萄的基础研究和分子育种。
