Genome editing in rice and wheat using the CRISPR/Cas system
Genome editing in rice and wheat using the CRISPR/Cas system
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DOI：
10.1038/nprot.2014.157
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发表时间：
2014-10-01
期刊：
影响因子：
14.8
通讯作者：
Gao, Caixia
中科院分区：
文献类型：
--
作者：
Shan, Qiwei;Wang, Yanpeng;Gao, Caixia
靶向基因组编辑核酸酶，如锌指核酸酶(ZFN)和转录激活因子样核酸酶(TALENS)，是理解基因功能和培育有价值的植物新性状的有力工具。簇状规则散布短回文重复序列(CRISPR)/CAS系统是近年来出现的一种高效、通用的基于核酸酶的基因组工程方法。在这个系统中，只需要改变单引导RNA(SgRNA)中的20个核苷酸的靶向序列来靶向不同的基因。克隆策略的简单性和对潜在靶点的极少限制使CRISPR/CAS系统非常有吸引力。在这里，我们描述了一种选择靶点的分步方案，以及用于序列特异性CRISPR/Cas介导的突变和水稻和小麦基因靶向的sgRNAs的设计、构建、验证和使用。CRISPR/Cas系统提供了一种在1-2周内快速基因打靶的简单方法，在13-17周内就可以产生突变的水稻植株。
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.