Olivares Felipe, Loyola Rodrigo, Olmedo Blanca, Miccono María de Los Ángeles, Aguirre Carlos, Vergara Ricardo, Riquelme Danae, Madrid Gabriela, Plantat Philippe, Mora Roxana, Espinoza Daniel, Prieto Humberto
Biotechnology Laboratory, La Platina Research Station, National Institute of Agriculture Research, Santiago, Chile.
Phytopathology Laboratory, La Platina Research Station, National Institute of Agriculture Research, Santiago, Chile.
Front Plant Sci. 2021 Dec 23;12:791030. doi: 10.3389/fpls.2021.791030. eCollection 2021.
The woody nature of grapevine ( L.) has hindered the development of efficient gene editing strategies to improve this species. The lack of highly efficient gene transfer techniques, which, furthermore, are applied in multicellular explants such as somatic embryos, are additional technical handicaps to gene editing in the vine. The inclusion of geminivirus-based replicons in regular T-DNA vectors can enhance the expression of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) elements, thus enabling the use of these multicellular explants as starting materials. In this study, we used (BeYDV)-derived replicon vectors to express the key components of CRISPR/Cas9 system and evaluate their editing capability in individuals derived from -mediated gene transfer experiments of 'Thompson Seedless' somatic embryos. Preliminary assays using a BeYDV-derived vector for reporter gene expression demonstrated marker visualization in embryos for up to 33 days post-infiltration. A universal BeYDV-based vector (pGMV-U) was assembled to produce all CRISPR/Cas9 components with up to four independent guide RNA (gRNA) expression cassettes. With a focus on fungal tolerance, we used gRNA pairs to address considerably large deletions of putative grape susceptibility genes, including (), (), (), and (). The editing functionality of gRNA pairs in pGMV-U was evaluated by grapevine leaf agroinfiltration assays, thus enabling longer-term embryo transformations. These experiments allowed for the establishment of greenhouse individuals exhibiting a double-cut edited status for all targeted genes under different allele-editing conditions. After approximately 18 months, the edited grapevine plants were preliminary evaluated regarding its resistance to and . Assays have shown that a transgene-free double-cut edited line exhibits over 90% reduction in symptoms triggered by powdery mildew infection. These results point to the use of geminivirus-based replicons for gene editing in grapevine and other relevant fruit species.
葡萄（Vitis vinifera L.）的木质特性阻碍了高效基因编辑策略的发展，从而影响了该物种的改良。此外，缺乏高效的基因转移技术，且这些技术应用于多细胞外植体（如体细胞胚）时存在困难，这是葡萄基因编辑的其他技术障碍。在常规T-DNA载体中加入基于双生病毒的复制子，可以增强成簇规律间隔短回文重复序列/CRISPR相关蛋白9（CRISPR/Cas9）元件的表达，从而能够将这些多细胞外植体用作起始材料。在本研究中，我们使用源自甜菜黄矮病毒（BeYDV）的复制子载体来表达CRISPR/Cas9系统的关键组件，并评估其在“汤普森无核”体细胞胚的农杆菌介导基因转移实验所获得的个体中的编辑能力。使用源自BeYDV的载体进行报告基因表达的初步试验表明，在浸润后长达33天的胚胎中可观察到标记物。构建了一种通用的基于BeYDV的载体（pGMV-U），以产生带有多达四个独立向导RNA（gRNA）表达盒的所有CRISPR/Cas9组件。以真菌耐受性为重点，我们使用gRNA对来处理假定的葡萄易感基因的大量缺失，包括VvNAC1、VvWRKY1、VvWRKY2和VvWRKY3。通过葡萄叶片农杆菌浸润试验评估了pGMV-U中gRNA对的编辑功能，从而实现了长期的胚胎转化。这些实验使得能够建立在不同等位基因编辑条件下所有靶向基因均呈现双切口编辑状态的温室个体。大约18个月后，对编辑后的葡萄植株对白粉病和霜霉病的抗性进行了初步评估。试验表明，一条无转基因的双切口编辑品系在白粉病感染引发的症状方面减少了90%以上。这些结果表明基于双生病毒的复制子可用于葡萄及其他相关水果物种的基因编辑。
