Corteva Agriscience, Johnston, IA, USA.
Plant Biotechnol J. 2021 Oct;19(10):2000-2010. doi: 10.1111/pbi.13613. Epub 2021 Jul 2.
CRISPR-Cas is a powerful DNA double-strand break technology with wide-ranging applications in plant genome modification. However, the efficiency of genome editing depends on various factors including plant genetic transformation processes and types of modifications desired. Agrobacterium infection is the preferred method of transformation and delivery of editing components into the plant cell. While this method has been successfully used to generate gene knockouts in multiple crops, precise nucleotide replacement and especially gene insertion into a pre-defined genomic location remain highly challenging. Here, we report an efficient, selectable marker-free site-specific gene insertion in maize using Agrobacterium infection. Advancements in maize transformation and new vector design enabled increase of targeted insertion frequencies by two orders of magnitude in comparison to conventional Agrobacterium-mediated delivery. Importantly, these advancements allowed not only a significant improvement of the frequency, but also of the quality of generated events. These results further enable the application of genome editing for trait product development in a wide variety of crop species amenable to Agrobacterium-mediated transformation.
CRISPR-Cas 是一种强大的 DNA 双链断裂技术，在植物基因组修饰中具有广泛的应用。然而，基因组编辑的效率取决于多种因素，包括植物遗传转化过程和所需的修饰类型。农杆菌感染是转化和将编辑组件递送到植物细胞的首选方法。虽然这种方法已成功用于在多种作物中产生基因敲除，但精确的核苷酸替换，特别是基因插入到预定义的基因组位置仍然极具挑战性。在这里，我们报告了一种使用农杆菌感染在玉米中进行高效、无选择标记的定点基因插入的方法。玉米转化的进展和新载体设计使得靶向插入频率比传统的农杆菌介导的递送提高了两个数量级。重要的是，这些进展不仅显著提高了频率，而且提高了生成事件的质量。这些结果进一步使基因组编辑能够应用于适合农杆菌介导转化的各种作物物种的性状产品开发。
