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Science Bulletin

, Volume 60, Issue 15, pp 1332–1347 | Cite as

A detailed procedure for CRISPR/Cas9-mediated gene editing in Arabidopsis thaliana

  • Wenshan Liu
  • Xiaohong Zhu
  • Mingguang Lei
  • Qingyou XiaEmail author
  • Jose Ramon Botella
  • Jian-Kang Zhu
  • Yanfei MaoEmail author
Article Life & Medical Sciences

Abstract

The newly developed CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system has emerged as an efficient tool for genome-editing, providing an alternative to classical mutagenesis and transgenic methods to study gene function and improve crop traits. CRISPR/Cas facilitates targeted gene editing through RNA-guided DNA cleavage followed by cellular DNA repair mechanisms that introduce sequence changes at the site of cleavage. Here we describe a detailed procedure for our previously developed and highly efficient CRISPR/Cas9 method that allows the generation of heritable-targeted gene mutations and corrections in Arabidopsis. This protocol describes the strategies and steps for the selection of targets, design of single-guide RNA (sgRNA), vector construction and analysis of transgenic lines. We also offer a method to target two loci simultaneously using vectors containing two different sgRNAs. The principles described in this protocol can be applied to other plant species to generate stably inherited DNA modifications.

Keywords

CRISPR/Cas9 Targeted gene editing Genome engineering Arabidopsis thaliana 

摘要

CRISPR是一类来源于细菌的“规律间隔成簇短回文重复序列”,通过与Cas9蛋白形成二元复合体来识别特定的DNA序列。近年来的研究表明,经过改造的CRISPR/Cas9系统可以在植物体内实现对目标基因的高效编辑,从而有望取代经典的基因突变技术和转基因技术来满足基因功能研究和作物品种研发的需要。定制后的CRISPR/Cas9复合体可以根据给定的序列,以碱基配对的方式结合到目标位点上,并造成双链DNA断裂。这种严重的DNA损伤,会激活细胞内源的DNA损伤修复通路,在缺乏模板的情况下,这类修复往往是不正确的,极易导致断裂位置的碱基改变。本文详细描述了如何利用已有的CRISPR/Cas9系统,在拟南芥中进行可遗传的基因定点修饰的策略和具体步骤, 包括靶位点的选择、引导RNA的设计、载体构建和转基因植物的检测和分析。另外,我们也提供了在原有质粒的基础上,构建双敲载体的方法。本实验方案所涉及的原理和策略也同样可以移植到其他植物品种上,来实现可遗传的基因修饰。

Notes

Acknowledgments

The work was supported by the Chinese Academy of Sciences and China Scholarship Council (201206050103).

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11434_2015_848_MOESM1_ESM.docx (16 kb)
Supplementary material 1 (DOCX 16 kb)

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Copyright information

© Science China Press and Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Wenshan Liu
    • 1
    • 2
    • 4
  • Xiaohong Zhu
    • 2
  • Mingguang Lei
    • 3
  • Qingyou Xia
    • 4
    Email author
  • Jose Ramon Botella
    • 5
  • Jian-Kang Zhu
    • 2
    • 3
  • Yanfei Mao
    • 2
    Email author
  1. 1.School of Life SciencesChongqing UniversityChongqingChina
  2. 2.Shanghai Center for Plant Stress Biology, Shanghai Institutes of Biological SciencesChinese Academy of SciencesShanghaiChina
  3. 3.Department of Horticulture and Landscape ArchitecturePurdue UniversityWest LafayetteUSA
  4. 4.State Key Laboratory of Silkworm Genome BiologySouthwest UniversityChongqingChina
  5. 5.School of Agriculture and Food SciencesUniversity of QueenslandBrisbaneAustralia

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