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

Liu, Wenshan, Zhu, Xiaohong, Lei, Mingguang, Xia, Qingyou, Botella, Jose Ramon, Zhu, Jian-Kang and Mao, Yanfei (2015) A detailed procedure for CRISPR/Cas9-mediated gene editing in Arabidopsis thaliana. Science Bulletin, 60 15: 1332-1347. doi:10.1007/s11434-015-0848-2


Author Liu, Wenshan
Zhu, Xiaohong
Lei, Mingguang
Xia, Qingyou
Botella, Jose Ramon
Zhu, Jian-Kang
Mao, Yanfei
Title A detailed procedure for CRISPR/Cas9-mediated gene editing in Arabidopsis thaliana
Formatted title
A detailed procedure for CRISPR/Cas9-mediated gene editing in Arabidopsis thaliana
Journal name Science Bulletin   Check publisher's open access policy
ISSN 2095-9273
2095-9281
Publication date 2015-08-01
Year available 2015
Sub-type Article (original research)
DOI 10.1007/s11434-015-0848-2
Open Access Status Not yet assessed
Volume 60
Issue 15
Start page 1332
End page 1347
Total pages 16
Place of publication Beijing, China
Publisher Science in China Press
Language eng
Subject 1000 General
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.
Formatted 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.
Keyword CRISPR/Cas9
Targeted gene editing
Genome engineering
Arabidopsis thaliana
Q-Index Code C1
Q-Index Status Confirmed Code
Grant ID 201206050103
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: School of Agriculture and Food Sciences
Official 2016 Collection
 
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