Saskatoon Research and Development Centre, Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N OX2, Canada.
Int J Mol Sci. 2022 May 17;23(10):5597. doi: 10.3390/ijms23105597.
Transgenic or genetically modified crops have great potential in modern agriculture but still suffer from heavy regulations worldwide due to biosafety concerns. As a promising alternative route, cisgenic crops have received higher public acceptance and better reviews by governing authorities. To serve the purpose of cisgenic plant breeding, we have developed a CRISPR/Cas9-based vector system, which is capable of delivering target gene-of-interest (GOI) into recipient plants while removing undesired genetic traces in the plants. The new system features a controllable auto-excision feature, which is realized by a core design of embedded multi-clonal sequence and the use of inducible promoters controlling the expression of Cas9 nuclease. In the current proof-of-concept study in (L.) Heynh., we have successfully incorporated a GOI into the plant and removed the selection marker and CRISPR/Cas9 components from the final product. Following the designed workflow, we have demonstrated that novel cisgenic plant germplasms with desired traits could be developed within one to two generations. Further characterizations of the vector system have shown that heat treatment at 37 °C could significantly improve the editing efficiency (up to 100%), and no off-target mutations were identified in the background. This novel vector system is the first CRISPR/Cas9-based genome editing tool for cisgenic plant breeding and should prove powerful for other similar applications in the bright future of precision molecular breeding.
转基因或基因修饰作物在现代农业中有巨大的潜力，但由于对生物安全的担忧，在全球范围内仍受到严格的监管。作为一种有前途的替代途径，基因编辑作物因其较高的公众接受度和管理部门的较好评价而受到青睐。为了满足基因编辑植物育种的目的，我们开发了一种基于 CRISPR/Cas9 的载体系统，该系统能够将目标基因（GOI）导入受体植物，同时去除植物中不需要的遗传痕迹。该新系统具有可控的自动切除特性，这是通过嵌入式多克隆序列的核心设计和使用诱导启动子控制 Cas9 核酸酶的表达来实现的。在目前在 （L.）Heynh.中的概念验证研究中，我们已经成功地将一个 GOI 整合到植物中，并从最终产物中去除了选择标记和 CRISPR/Cas9 组件。按照设计的工作流程，我们已经证明，具有所需特性的新型基因编辑植物种质可以在一到两代内开发出来。对载体系统的进一步特征分析表明，37°C 的热处理可以显著提高编辑效率（高达 100%），并且在 背景中没有发现脱靶突变。这个新型的载体系统是第一个用于基因编辑作物育种的基于 CRISPR/Cas9 的基因组编辑工具，应该在精准分子育种的光明未来中为其他类似的应用提供强大的支持。
