Negishi Katsuya, Endo Masaki, Endo Tomoko, Nishitani Chikako
Institute of Fruit Tree and Tea Science, National Agriculture and Food Research Organization, 2-1 Fujimoto, Tsukuba, Ibaraki 305-8605, Japan.
Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, 3-1-3 Kannnondai, Tsukuba, Ibaraki 305-8604, Japan.
Plant Biotechnol (Tokyo). 2024 Dec 25;41(4):425-436. doi: 10.5511/plantbiotechnology.24.0903a.
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been used for genome editing in various fruit trees, including apple ( × ). In previous studies, transfer DNA (T-DNA) expressing genome editing tools, Cas9 (SpCas9) and single guide RNA (sgRNA), was stably integrated into the apple genome via -mediated transformation. However, due to self-incompatibility, long generation period, and the high heterozygosity of apple, removing only the integrated T-DNA from the apple genome by crossbreeding while maintaining the introduced varietal trait is difficult. Therefore, an efficient SpCas9-sgRNA delivery system without transgene insertion is required for genome editing of apple. In this study, we used geminivirus-derived replicons (GVRs) for the transient expression of genome editing tools. Small DNA vectors were deconstructed by splitting the elements necessary for the production of GVRs from bean yellow dwarf virus into two vectors. Production of GVRs using these vectors was demonstrated in and apple cells. Genome editing was improved by using the GVR-producing vectors with genome editing tools in protoplasts. The use of the GVR-producing vectors for SpCas9 and sgRNA delivery into apple leaves improved the expression levels of SpCas9 and sgRNA, enabling the detection of targeted mutations introduced in the endogenous apple genome. These findings demonstrate the utility of GVRs in genome editing via transient gene expression in apple. It can be expected that our GVR-based genome editing technology has potential utility for transgene-free genome editing in apple.
成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关蛋白9（Cas9）系统已被用于包括苹果（×）在内的各种果树的基因组编辑。在先前的研究中，表达基因组编辑工具Cas9（SpCas9）和单向导RNA（sgRNA）的转移DNA（T-DNA）通过介导的转化稳定整合到苹果基因组中。然而，由于苹果的自交不亲和性、生长周期长以及高度杂合性，通过杂交仅从苹果基因组中去除整合的T-DNA同时保持引入的品种性状是困难的。因此，苹果基因组编辑需要一种高效的无转基因插入的SpCas9-sgRNA递送系统。在本研究中，我们使用双生病毒衍生的复制子（GVR）来瞬时表达基因组编辑工具。通过将菜豆黄花叶病毒产生GVR所需的元件拆分成两个载体，构建了小DNA载体。在烟草和苹果细胞中证明了使用这些载体产生GVR。在烟草原生质体中使用产生GVR的载体与基因组编辑工具提高了基因组编辑效率。使用产生GVR的载体将SpCas9和sgRNA递送至苹果叶片中提高了SpCas9和sgRNA的表达水平，从而能够检测内源性苹果基因组中引入的靶向突变。这些发现证明了GVR在苹果中通过瞬时基因表达进行基因组编辑的实用性。可以预期，我们基于GVR的基因组编辑技术在苹果的无转基因基因组编辑中具有潜在的应用价值。
