You Jun, Li Donghua, Yang Li, Dossou Senouwa Segla Koffi, Zhou Rong, Zhang Yanxin, Wang Linhai
Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture and Rural Affairs, Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan, China.
Front Plant Sci. 2022 Jul 11;13:935825. doi: 10.3389/fpls.2022.935825. eCollection 2022.
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been widely utilized for targeted genome modification in a wide range of species. It is a powerful genome editing technology, providing significant benefits for gene functional research and molecular breeding. However, to date, no study has applied this genome editing tool to sesame ( L.), one of the most ancient and important oil crops used widely in diverse industries such as food and medicine. Herein, the CRISPR/Cas9 system along with hairy root transformation was used to induce targeted mutagenesis in sesame. Two single guide RNAs (sgRNAs) were designed to target two sesame cytochrome P450 genes ( and ), which are the key biosynthetic gene of sesamin and sesamolin, respectively. Sequencing data illustrated the expected InDel mutations at the target sites, with 90.63 and 93.33% mutation frequency in and , respectively. The most common editing event was single nucleotide deletion and insertion. Sequencing of potential off-target sites of -sgRNA showed no off-target events in cases of three mismatches. High-performance liquid chromatography analysis showed that sesamin and sesamolin biosynthesis was effectively disrupted in the mutated hairy roots, confirming the crucial role of and in sesame lignan biosynthesis. These results demonstrated that targeted mutagenesis was efficiently created by the CRISPR/Cas9 system, and CRISPR/Cas9 coupled with hairy root transformation is an effective tool for assessing gene functions in sesame.
成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关蛋白9（Cas9）系统已被广泛用于多种物种的靶向基因组修饰。它是一种强大的基因组编辑技术，为基因功能研究和分子育种带来了显著益处。然而，迄今为止，尚无研究将这种基因组编辑工具应用于芝麻（Sesamum indicum L.），芝麻是最古老且重要的油料作物之一，广泛应用于食品和医药等多个行业。在此，利用CRISPR/Cas9系统结合发根转化在芝麻中诱导靶向诱变。设计了两个单向导RNA（sgRNA）分别靶向两个芝麻细胞色素P450基因（CYP81Q1和CYP71A1），它们分别是芝麻素和芝麻林素的关键生物合成基因。测序数据表明在靶位点出现了预期的插入缺失突变，CYP81Q1和CYP71A1中的突变频率分别为90.63%和93.33%。最常见的编辑事件是单核苷酸缺失和插入。对CYP81Q1-sgRNA潜在脱靶位点的测序表明，在三个错配的情况下未发生脱靶事件。高效液相色谱分析表明，突变发根中芝麻素和芝麻林素的生物合成被有效破坏，证实了CYP81Q1和CYP7IA1在芝麻木脂素生物合成中的关键作用。这些结果表明，CRISPR/Cas9系统有效地产生了靶向诱变，并且CRISPR/Cas9与发根转化相结合是评估芝麻基因功能的有效工具。
