Shanghai Center for Plant Stress Biology and Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, 200032, Shanghai, China.
University of Chinese Academy of Sciences, 100049, Beijing, China.
Nat Commun. 2018 May 17;9(1):1967. doi: 10.1038/s41467-018-04416-0.
Homologous recombination-based gene targeting is a powerful tool for precise genome modification and has been widely used in organisms ranging from yeast to higher organisms such as Drosophila and mouse. However, gene targeting in higher plants, including the most widely used model plant Arabidopsis thaliana, remains challenging. Here we report a sequential transformation method for gene targeting in Arabidopsis. We find that parental lines expressing the bacterial endonuclease Cas9 from the egg cell- and early embryo-specific DD45 gene promoter can improve the frequency of single-guide RNA-targeted gene knock-ins and sequence replacements via homologous recombination at several endogenous sites in the Arabidopsis genome. These heritable gene targeting can be identified by regular PCR. Our approach enables routine and fine manipulation of the Arabidopsis genome.
基于同源重组的基因靶向是一种精确基因组修饰的强大工具，已广泛应用于从酵母到果蝇和小鼠等高等生物的研究中。然而，包括最广泛使用的模式植物拟南芥在内的高等植物的基因靶向仍然具有挑战性。在这里，我们报道了一种在拟南芥中进行基因靶向的序贯转化方法。我们发现，在 DD45 基因启动子的卵母细胞和早期胚胎特异性表达的细菌内切酶 Cas9 的亲本系中，可以提高几个内源位点的单引导 RNA 靶向基因敲入和序列替换的同源重组频率。这些可遗传的基因靶向可以通过常规 PCR 进行鉴定。我们的方法可以实现对拟南芥基因组的常规和精细操作。
