Muto Naoki, Matsumoto Takashi
Department of Bioscience, Faculty of Life Sciences, Tokyo University of Agriculture, Tokyo, Japan.
Front Plant Sci. 2022 Oct 13;13:951660. doi: 10.3389/fpls.2022.951660. eCollection 2022.
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a powerful tool widely used for genome editing in various organisms, including plants. It introduces and facilitates the study of rare genetic mutations in a short time and is a potent tool to assist in plant molecular breeding. Radish ( L.) is an important Brassicaceae vegetable cultivated and consumed worldwide. However, the application of the CRISPR/Cas9 system is limited by the absence of an efficient transformation system in radish. This study aimed to establish a CRISPR/Cas9 system in radish employing the -mediated genetic transformation system reported recently. For this purpose, we performed genome editing using the CRISPR/Cas9 system targeting the () orthologs, and , that induces leaf trichome formation in radish. A Cas9/single guide RNA (sgRNA) vector with a common sgRNA corresponding to and was transferred. A total of eight T plants were analyzed, of which six (editing efficiency 75%) had a mutated , five (62.5%) had a mutated , and five showed mutations in both and . Most mutations in T plants were short (<3 bp) deletions or insertions, causing frameshift mutations that might produce non-functional proteins. Chimeric mutations were detected in several T generation plants. In the T generation, the hairless phenotype was observed only in plants with knockout mutations in both and . The majority of mutant alleles in T plants, with the exception of the chimeric mutant plants detected, were stably inherited in the T generation. In conclusion, we successfully knocked out and using the CRISPR/Cas9 system and demonstrated that both and independently contribute to the induction of leaf trichome formation in radish. In this study, genome-edited plants without T-DNA, which are useful as breeding material, were obtained. The findings prove the feasibility of genome editing in radish using a CRISPR/Cas9 system that could accelerate its molecular breeding to improve agronomically desirable traits.
成簇规律间隔短回回回回文重复序列（CRISPR）/CRISPR相关蛋白9（Cas9）是一种强大的工具，广泛用于包括植物在内的各种生物体的基因组编辑。它能在短时间内引入并促进对罕见基因突变的研究，是协助植物分子育种的有力工具。萝卜（L.）是一种重要的十字花科蔬菜，在全球范围内种植和食用。然而，CRISPR/Cas9系统的应用受到萝卜缺乏高效转化系统的限制。本研究旨在利用最近报道的介导遗传转化系统在萝卜中建立CRISPR/Cas9系统。为此，我们使用靶向萝卜中诱导叶毛状体形成的直系同源基因和的CRISPR/Cas9系统进行基因组编辑。转移了一个带有对应于和的共同单向导RNA（sgRNA）的Cas9/sgRNA载体。共分析了8株T代植株，其中6株（编辑效率75%）的发生了突变，5株（62.5%）的发生了突变，5株的和均发生了突变。T代植株中的大多数突变是短（<3 bp）缺失或插入，导致移码突变，可能产生无功能的蛋白质。在几株T代植株中检测到嵌合突变。在T代中，仅在和均发生敲除突变的植株中观察到无毛表型。除检测到的嵌合突变植株外，T代植株中的大多数突变等位基因在T代中稳定遗传。总之，我们成功地使用CRISPR/Cas9系统敲除了和，并证明和均独立地促进萝卜叶毛状体的形成。在本研究中，获得了不含T-DNA的基因组编辑植株，可作为育种材料。这些发现证明了使用CRISPR/Cas9系统对萝卜进行基因组编辑的可行性，这可以加速其分子育种以改善农艺性状。
