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

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• 作者：Wenshan Liu ; Xiaohong Zhu ; Mingguang Lei ; Qingyou Xia&#8230
• 关键词：CRISPR/Cas9 ; Targeted gene editing ; Genome engineering ; Arabidopsis thaliana
• 刊名：Chinese Science Bulletin
• 出版年：2015
• 出版时间：August 2015
• 年：2015
• 卷：60
• 期：15
• 页码：1332-1347
• 全文大小：2,026 KB
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• 作者单位：Wenshan Liu (1) (2) (4)
  Xiaohong Zhu (2)
  Mingguang Lei (3)
  Qingyou Xia (4)
  Jose Ramon Botella (5)
  Jian-Kang Zhu (2) (3)
  Yanfei Mao (2)
  
  1. School of Life Sciences, Chongqing University, Chongqing, 400044, China
  2. Shanghai Center for Plant Stress Biology, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai, 200032, China
  4. State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, 400715, China
  3. Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN, 47907, USA
  5. School of Agriculture and Food Sciences, University of Queensland, Brisbane, QLD, 4072, Australia
  
• 刊物主题：Science, general; Life Sciences, general; Physics, general; Chemistry/Food Science, general; Earth Sciences, general; Engineering, general;
• 出版者：Springer Berlin Heidelberg
• ISSN：1861-9541

文摘

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.