A novel method for high-frequency genome editing in rice, using the CRISPR/Cas9 system
2017
Jung, Y.J., Hankyong National University, Ansung, Republic of Korea | Bae, S., Hankyong National University, Ansung, Republic of Korea | Lee, G.J., Chungnam National University, Daejeon, Republic of Korea | Seo, P.J., Sungkyunkwan University, Suwon, Republic of Korea | Cho, Y.G., Chungbuk National University, Cheongju, Republic of Korea | Kang, K.K., Hankyong National University, Ansung, Republic of Korea
The CRISPR/Cas9 is a core technology that can result in a paradigm for breeding new varieties. This study describes in detail the sgRNA design, vector construction, and the development of a transgenic plant and its molecular analysis, and demonstrates how gene editing technology through the CRISPR/Cas9 system can be applied easily and accurately. CRISPR/Cas9 facilitates targeted gene editing through RNA-guided DNA cleavage, followed by cellular DNA repair mechanisms that introduce sequence changes at the site of cleavage. It also allows the generation of heritable-targeted gene mutations and corrections. Here, we present detailed procedures involved in the CRISPR/Cas9 system to acquire faster, easier and more cost-efficient gene edited transgenic rice. The protocol described here establishes the strategies and steps for the selection of targets, design of sgRNA, vector construction, and analysis of the transgenic lines. The same principles can be used to customize the versatile CRISPR/Cas9 system, for application to other plant species.
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