Tian Mei, Luo Linglong, Jin Baolong, Liu Jianing, Chen Tong, Tang Jinfu, Shen Ye, Zhang Haiyan, Guo Juan, Zhang Huawei, Cui Guanghong, Huang Luqi
State Key Laboratory for Quality Ensurance and Sustainable Use of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijng, China.
National Key Laboratory of Wheat Improvement, Peking University Institute of Advanced Agricultural Sciences, Shandong Laboratory of Advanced Agriculture Sciences in Weifang, Weifang, Shandong, China.
Plant Biotechnol J. 2025 Jun;23(6):2406-2417. doi: 10.1111/pbi.70029. Epub 2025 Mar 26.
The CRISPR/Cas9 system is a powerful tool for genomic editing with significant potential for gene function validation and molecular breeding in medicinal plants. Salvia miltiorrhiza, a model medicinal plant, was among the pioneers to utilize CRISPR/Cas9 technology, though achieving high-efficiency homozygous knockout mutants has been challenging. In this study, the analysis of variations at 241 single-guide RNA (sgRNA) across different reference genomes and experimental materials was conducted first, leading to the identification of the six-generation inbred line bh2-7 as the most suitable reference genome and experimental material for gene editing research in S. miltiorrhiza. Next, five Agrobacterium rhizogenes strains were evaluated for hairy root induction, editing efficiency, and mutation patterns, with C58C1 and K599 emerging as the most effective delivery systems. Using the CRISPR/Cas9 vector pZKD672, 53 target sites were successfully edited, with K599 achieving 71.07% editing efficiency and 36.74% homozygous or biallelic (HOM) efficiency and C58C1 showing 62.27% editing efficiency and 23.61% HOM efficiency. We thus constructed a large-scale mutant library targeting 121 genes with 170 sgRNAs, yielding 1664 homozygous or biallelic mutants. Analysis of 65 low-efficiency target sites revealed that sgRNA mismatches and secondary structures were key factors reducing HOM efficiency, offering insights for future target design. This study establishes an efficient CRISPR/Cas9 system, advancing precision breeding and metabolic engineering research in medicinal plants.
CRISPR/Cas9系统是一种强大的基因组编辑工具，在药用植物的基因功能验证和分子育种方面具有巨大潜力。丹参作为一种模式药用植物，是最早利用CRISPR/Cas9技术的植物之一，不过获得高效的纯合敲除突变体一直具有挑战性。在本研究中，首先对不同参考基因组和实验材料中的241个单向导RNA（sgRNA）的变异进行了分析，从而确定六代自交系bh2-7是丹参基因编辑研究中最合适的参考基因组和实验材料。接下来，评估了五种发根农杆菌菌株的发根诱导、编辑效率和突变模式，结果显示C58C1和K599是最有效的传递系统。使用CRISPR/Cas9载体pZKD672成功编辑了53个靶位点，其中K599的编辑效率达到71.07%，纯合或双等位基因（HOM）效率为36.74%，C58C1的编辑效率为62.27%，HOM效率为23.61%。因此，我们构建了一个针对121个基因的大规模突变体文库，使用170个sgRNA，获得了1664个纯合或双等位基因突变体。对65个低效靶位点的分析表明，sgRNA错配和二级结构是降低HOM效率的关键因素，为未来的靶标设计提供了思路。本研究建立了一个高效的CRISPR/Cas9系统，推动了药用植物的精准育种和代谢工程研究。
