Shanghai Center for Plant Stress Biology and Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, China.
Shandong Shunfeng Biotechnology Co. Ltd, Jinan, China.
Nat Biotechnol. 2020 Dec;38(12):1402-1407. doi: 10.1038/s41587-020-0581-5. Epub 2020 Jul 6.
CRISPR-Cas9 methods have been applied to generate random insertions and deletions, large deletions, targeted insertions or replacements of short sequences, and precise base changes in plants. However, versatile methods for targeted insertion or replacement of long sequences and genes, which are needed for functional genomics studies and trait improvement in crops, are few and largely depend on the use of selection markers. Building on methods developed in mammalian cells, we used chemically modified donor DNA and CRISPR-Cas9 to insert sequences of up to 2,049 base pairs (bp), including enhancers and promoters, into the rice genome at an efficiency of 25%. We also report a method for gene replacement that relies on homology-directed repair, chemically modified donor DNA and the presence of tandem repeats at target sites, achieving replacement with up to 130-bp sequences at 6.1% efficiency.
CRISPR-Cas9 方法已被应用于生成随机插入和缺失、大片段缺失、靶向短序列插入或替换以及植物的精确碱基改变。然而，用于功能基因组学研究和作物性状改良的长序列和基因的靶向插入或替换的多功能方法很少，并且在很大程度上依赖于选择标记的使用。基于在哺乳动物细胞中开发的方法，我们使用化学修饰的供体 DNA 和 CRISPR-Cas9 将长达 2049 个碱基对 (bp) 的序列，包括增强子和启动子，插入到水稻基因组中，效率为 25%。我们还报告了一种基因替换方法，该方法依赖于同源定向修复、化学修饰的供体 DNA 和靶位点处串联重复的存在，在 6.1%的效率下实现了长达 130bp 序列的替换。
