title: Genome editing in rice and wheat using the CRISPR/Cas system.
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.
authors.affiliation: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology (IGDB), Chinese Academy of Sciences (CAS), Beijing, China.
authors.forename: Qiwei
authors.initials: Q
authors.lastname: Shan
authors.affiliation: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology (IGDB), Chinese Academy of Sciences (CAS), Beijing, China.
authors.forename: Yanpeng
authors.initials: Y
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology (IGDB), Chinese Academy of Sciences (CAS), Beijing, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.affiliation: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology (IGDB), Chinese Academy of Sciences (CAS), Beijing, China.
authors.forename: Caixia
authors.initials: C
authors.lastname: Gao
mesh_terms: D019470:Biolistics / Q000379:methods; D003001:Cloning, Molecular; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004260:DNA Repair / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D018745:Genome, Plant; D023281:Genomics / Q000379:methods*; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009154:Mutation; D012275:Oryza / Q000235:genetics*; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction / Q000379:methods; D011523:Protoplasts / Q000502:physiology; D014908:Triticum / Q000235:genetics*