CRISPR/Cas9-mediated gene targeting in Arabidopsis using sequential transformation

Daisuke Miki (), Wenxin Zhang, Wenjie Zeng, Zhengyan Feng and Jian-Kang Zhu ()
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Daisuke Miki: Chinese Academy of Sciences
Wenxin Zhang: Chinese Academy of Sciences
Wenjie Zeng: Chinese Academy of Sciences
Zhengyan Feng: Chinese Academy of Sciences
Jian-Kang Zhu: Chinese Academy of Sciences

Nature Communications, 2018, vol. 9, issue 1, 1-9

Abstract: Abstract 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.

Date: 2018
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DOI: 10.1038/s41467-018-04416-0

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