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.
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acknowleDGMents We thank the members of the Gao laboratory for their support. This work was funded by grants from the National Natural Science Foundation of China (no. 31271795) and the Ministry of Agriculture of China (nos. 2014ZX0801003B, 2013ZX08002-004, 2013ZX08002-005 and 2013ZX08010-002).