Transgene‐free CRISPR/Cas9‐mediated gene editing through protoplast‐to‐plant regeneration enhances active compounds in Salvia miltiorrhiza

作者：Chen‐Tran Hsu, Chi‐Chou Chiu, Pao‐Yuan Hsiao, Chih‐Yu Lin, Chih‐Yu Lin, Sy‐Chyi Cheng, Yao‐Cheng Lin, Yuliang Yang, Fu‐Hui Wu, Horng‐Jyh Harn, Shinn‐Zong Lin, Choun‐Sea Lin, Choun‐Sea Lin · 发表于：Plant Biotechnology Journal · 年份：2024 · DOI：10.1111/pbi.14285 · 被引用次数：41 · 研究领域：CRISPR and Genetic Engineering、Photosynthetic Processes and Mechanisms、Plant biochemistry and biosynthesis

Salvia miltiorrhiza (red sage, Chinese pinyin; danshen) is used in Eastern medicine to treat cardiovascular diseases. S. miltiorrhiza contains water-soluble and lipid-soluble bioactive compounds, including phenolic acids and diterpenoid tanshinones, respectively (Shi et al., 2021); the latter gives its root surface a red colour (Skała and Wysokińska, 2005). Several studies have sought to inactivate specific biosynthetic or transcription factor genes related to the bioactive compounds in S. miltiorrhiza by introducing a clustered regularly interspaced short palindromic repeat (CRISPR)-/CRISPR-associated nuclease 9 (Cas9)-based genome editing cassette via Agrobacterium-mediated hairy root transformation (Deng et al., 2020). Nevertheless, chimaeras in transformation and removing transgenes in plants with high-genetic heterozygosity like S. miltiorrhiza present significant challenges (Su et al., 2023). Here, we established a protoplast regeneration system for S. miltiorrhiza using either in vitro-assembled sgRNA-Cas9 ribonucleoprotein (RNP) complexes or plasmids carrying CRISPR/Cas9 system genes to target one or multiple sites for editing the genes through a single transfection event. As transcription factors regulating entire metabolic pathways are generally recognized as valuable tools for engineering elevated metabolite levels (Broun and Somerville, 2001), seven transcription factor genes—MYB28, MYB36, MYB39, MYB100, basic leucine zipper 1 (bZIP1), bZIP2 and MYB98—were selecte...