Yuan Cheng, Zeng Jianmin, Liu Yong, Yu Haiqin, Tong Zhijun, Zhang Jianduo, Gao Qian, Wang Zhong, Sui Xueyi, Xiao Bingguang, Huang Changjun
Yunnan Academy of Tobacco Agricultural Sciences, Key Laboratory of Tobacco Biotechnological Breeding, National Tobacco Genetic Engineering Research Center, Kunming, China.
Technology Center, China Tobacco Yunnan Industrial Co. LTD, Kunming, China.
Front Plant Sci. 2024 Mar 4;15:1329697. doi: 10.3389/fpls.2024.1329697. eCollection 2024.
Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) system has been widely applied in cultivated crops, but limited in their wild relatives. is a typical wild species of genus that is globally distributed as a horticultural plant and well-studied as a self-incompatibility model. It also has valuable genes for disease resistance and ornamental traits. However, it lacks an efficient genetic transformation and genome editing system, which hampers its gene function and breeding research. In this study, we developed an optimized hypocotyl-mediated transformation method for CRISPR-Cas9 delivery. The genetic transformation efficiency was significantly improved from approximately 1% to over 80%. We also applied the CRISPR-Cas9 system to target the () gene in and obtained edited plants with PDS mutations with over 50% editing efficiency. To generate self-compatible lines, a polycistronic tRNA-gRNA (PTG) strategy was used to target exonic regions of allelic genes and generate targeted knockouts simultaneously. We demonstrated that our system is feasible, stable, and high-efficiency for genome editing. Our study provides a powerful tool for basic research and genetic improvement of and an example for other wild tobacco species.
成簇规律间隔短回文重复序列（CRISPR）相关蛋白9（CRISPR-Cas9）系统已在栽培作物中广泛应用，但在其野生近缘种中应用有限。[物种名称]是[属名]的一个典型野生种，作为一种园艺植物在全球分布，并作为自交不亲和模型得到了深入研究。它还具有抗病和观赏性状的宝贵基因。然而，它缺乏高效的遗传转化和基因组编辑系统，这阻碍了其基因功能和育种研究。在本研究中，我们开发了一种优化的用于递送CRISPR-Cas9的下胚轴介导转化方法。遗传转化效率从约1%显著提高到80%以上。我们还应用CRISPR-Cas9系统靶向[物种名称]中的[基因名称]基因，并获得了编辑效率超过50%的具有PDS突变的编辑植株。为了产生自交亲和的[物种名称]株系，采用多顺反子tRNA-gRNA（PTG）策略靶向等位[基因名称]基因的外显子区域并同时产生靶向敲除。我们证明了我们的系统对于[物种名称]基因组编辑是可行、稳定且高效的。我们的研究为[物种名称]的基础研究和遗传改良提供了一个强大的工具，并为其他野生烟草物种提供了一个范例。
