Kuwabara Chikako, Miki Ryuji, Maruyama Nobuyuki, Yasui Masanori, Hamada Haruyasu, Nagira Yozo, Hirayama Yumiko, Ackley Wataru, Li Feng, Imai Ryozo, Taoka Naoaki, Yamada Tetsuya
Graduate School of Agriculture, Hokkaido University, Kita 9, Nishi 9, Kita-ku, Sapporo, Hokkaido 060-8589, Japan.
Agri-Bio Research Center, Kaneka Corporation, 700 Higashibara, Iwata, Shizuoka 438-0802, Japan.
Plant Physiol. 2024 Dec 2;196(4):2320-2329. doi: 10.1093/plphys/kiae491.
Here, we report a smart genome editing system for soybean (Glycine max) using the in planta bombardment-ribonucleoprotein (iPB-RNP) method without introducing foreign DNA or requiring traditional tissue culture processes such as embryogenesis and organogenesis. Shoot apical meristem (SAM) of embryonic axes was used as the target tissue for genome editing because the SAM in soybean mature seeds has stem cells and specific cell layers that develop germ cells during the reproductive growth stage. In the iPB-RNP method, the RNP complex of the CRISPR/Cas9 system was directly delivered into SAM stem cells via particle bombardment, and genome-edited plants were generated from these SAMs. Soybean allergenic gene Gly m Bd 30K was targeted in this study. Many E0 (the first generation of genome-edited) plants in this experiment harbored mutant alleles at the targeted locus. Editing frequency of inducing mutations transmissible to the E1 generation was approximately 0.4% to 4.6% of all E0 plants utilized in various soybean varieties. Furthermore, simultaneous mutagenesis by iPB-RNP method was also successfully performed at other loci. Our results offer a practical approach for both plant regeneration and DNA-free genome editing achieved by delivering RNP into the SAM of dicotyledonous plants.
在此，我们报道了一种用于大豆（Glycine max）的智能基因组编辑系统，该系统采用植物体内轰击核糖核蛋白（iPB-RNP）方法，无需引入外源DNA，也无需诸如胚胎发生和器官发生等传统组织培养过程。胚轴的茎尖分生组织（SAM）被用作基因组编辑的靶组织，因为大豆成熟种子中的SAM具有干细胞和在生殖生长阶段发育生殖细胞的特定细胞层。在iPB-RNP方法中，CRISPR/Cas9系统的核糖核蛋白复合物通过粒子轰击直接导入SAM干细胞，并从这些SAM中产生基因组编辑植物。本研究以大豆过敏基因Gly m Bd 30K为靶点。该实验中的许多E0（第一代基因组编辑）植物在靶位点携带突变等位基因。在不同大豆品种中，诱导可传递至E1代的突变的编辑频率约为所有使用的E0植物的0.4%至4.6%。此外，通过iPB-RNP方法在其他位点也成功实现了同时诱变。我们的结果为通过将核糖核蛋白导入双子叶植物的SAM实现植物再生和无DNA基因组编辑提供了一种实用方法。
