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PMID: 25232936 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Genome editing in rice and wheat using the CRISPR/Cas system.

Nature protocols ·Vol. 9 ·No. 10 ·2014-10-00 ·Pages 2395-410

Shan Q, Wang Y, Li J, Gao C

Abstract

Targeted genome editing nucleases, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), are powerful tools for understanding gene function and for developing valuable new traits in plants. The clustered regularly interspersed short palindromic repeats (CRISPR)/Cas system has recently emerged as an alternative nuclease-based method for efficient and versatile genome engineering. In this system, only the 20-nt targeting sequence within the single-guide RNA (sgRNA) needs to be changed to target different genes. The simplicity of the cloning strategy and the few limitations on potential target sites make the CRISPR/Cas system very appealing. Here we describe a stepwise protocol for the selection of target sites, as well as the design, construction, verification and use of sgRNAs for sequence-specific CRISPR/Cas-mediated mutagenesis and gene targeting in rice and wheat. The CRISPR/Cas system provides a straightforward method for rapid gene targeting within 1-2 weeks in protoplasts, and mutated rice plants can be generated within 13-17 weeks.

MeSH Terms
Biolistics/methods Cloning, Molecular Clustered Regularly Interspaced Short Palindromic Repeats DNA Repair/genetics Gene Expression Regulation, Plant Genome, Plant Genomics/methods Mutagenesis, Site-Directed/methods Mutation Oryza/genetics Plants, Genetically Modified Polymerase Chain Reaction/methods Protoplasts/physiology Triticum/genetics
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Shan Qiwei
State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology (IGDB), Chinese Academy of Sciences (CAS), Beijing, China.
Wang Yanpeng
State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology (IGDB), Chinese Academy of Sciences (CAS), Beijing, China.
Li Jun
State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology (IGDB), Chinese Academy of Sciences (CAS), Beijing, China.
Gao Caixia
State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology (IGDB), Chinese Academy of Sciences (CAS), Beijing, China.
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Article Info
Journal
Nature protocols
Abbr.
Nat Protoc
ISSN
1750-2799
DOI
10.1038/nprot.2014.157
Published
2014-10-00
Epub
2014-00-18
Pages
2395-410
Language
English
Region
England
NLM ID
101284307
Subset
IM
Corrections
CommentIn
PMID: 29388938
External Links
PubMed source DOI: 10.1038/nprot.2014.157 View journal details
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