Optimizing the CRISPR/Cas9 system for genome editing in grape by using grape promoters.
Optimizing the CRISPR/Cas9 system for genome editing in grape by using grape promoters.
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利用葡萄启动子优化葡萄基因组编辑 CRISPR/Cas9 系统
DOI：
10.1038/s41438-021-00489-z
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发表时间：
2021-03-01
影响因子：
8.7
通讯作者：
Liang Z
中科院分区：
文献类型：
--
作者：
Ren C;Liu Y;Guo Y;Duan W;Fan P;Li S;Liang Z
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尚未在该物种中进行探索。本文在葡萄中鉴定出4个VvU3和VvU6启动子以及2个UBQ启动子，并证明使用鉴定出的VvU3/U6启动子和UBQ2启动子可以通过分别提高sgRNA和Cas9的表达，显著提高葡萄的编辑效率。此外，我们利用优化后的CRISPR/Cas9载体，针对糖相关tonoplastic单糖转运体（TMT）家族成员TMT1和TMT2，对传统的多个sgRNA表达盒或多顺反子tRNA-sgRNA表达盒（PTG）进行了多重基因组编辑，总体编辑效率均高于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中糖积累中的作用。此外，通过编辑烟叶中植物烯去饱和酶（PDS）基因，证实了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第一个例子。本文所描述的多重编辑系统扩充了葡萄基因组编辑的工具箱，为葡萄的基础研究和分子育种提供了便利。
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.
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通过改进 sgRNA 表达优化棉花 CRISPR/Cas9 基因组编辑
影响因子：
5.1
作者：
Long L;Guo DD;Gao W;Yang WW;Hou LP;Ma XN;Miao YC;Botella JR;Song CP
通讯作者：
Song CP
影响因子：
46.9
作者：
Doench JG;Fusi N;Sullender M;Hegde M;Vaimberg EW;Donovan KF;Smith I;Tothova Z;Wilen C;Orchard R;Virgin HW;Listgarten J;Root DE
通讯作者：
Root DE
影响因子：
64.5
作者：
Hsu PD;Lander ES;Zhang F
通讯作者：
Zhang F
影响因子：
27.5
作者：
Ma, Xingliang;Zhang, Qunyu;Liu, Yao-Guang
通讯作者：
Liu, Yao-Guang
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle