Transgene-free CRISPR/Cas9-mediated gene editing through protoplast-to-plant regeneration enhances active compounds in Salvia miltiorrhiza
Salvia miltiorrhiza is used in Eastern medicine to treat cardiovascular diseases which contains water‐soluble and lipid‐soluble bioactive compounds, including phenolic acids and diterpenoid tanshinones, respectively; the latter gives its root surface a red color. Several studies have sought to inactivate genes related to the bioactive compounds in S. miltiorrhiza by introducing CRISPR/Cas‐based genome editing cassette via Agrobacterium‐mediated hairy root transformation. Nevertheless, chimaeras in transformation and removing transgenes in plants with high‐genetic heterozygosity like S. miltiorrhiza present significant challenges. We established a protoplast regeneration system for S. miltiorrhiza using either 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. Seven transcription factor genes-MYB28, MYB36, MYB39, MYB100, basic leucine zipper 1 (bZIP1), bZIP2 and MYB98 were selected as targets for mutagenesis. bZIP1 is a negative regulator of tanshinone biosynthesis and a positive regulator of phenolic acid biosynthesis, whereas bZIP2 is a negative regulator of phenolic acid biosynthesis. Protoplast regenerated plants harboring knockout mutations of either gene had deeper red roots, with higher levels of lipid‐soluble compounds. We demonstrated high‐efficiency gene editing in various transcription factor genes in S. miltiorrhiza through a single transfection event using transgene‐free CRISPR/Cas9 reagents in protoplasts, and regeneration of knockout plants through a newly established system, which requires 6 months from transfected protoplasts to whole plants.Co-researchers:Chen-Tran Hsu, Chi-Chou Chiu, Pao-Yuan Hsiao, Chih-Yu Lin, Sychyi Cheng, Yao-Cheng Lin, Yu-Liang Yang, Fu-Hui Wu, Horng-Jyh Harn, Shinn-Zong Lin