Ma Xingliang, Liu Yao-Guang
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangzhou, China.
College of Life Sciences, South China Agricultural University, Guangzhou, China.
Curr Protoc Mol Biol. 2016 Jul 1;115:31.6.1-31.6.21. doi: 10.1002/cpmb.10.
The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome targeting system has been applied to a variety of organisms, including plants. Compared to other genome-targeting technologies such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), the CRISPR/Cas9 system is easier to use and has much higher editing efficiency. In addition, multiple "single guide RNAs" (sgRNAs) with different target sequences can be designed to direct the Cas9 protein to multiple genomic sites for simultaneous multiplex editing. Here, we present a procedure for highly efficient multiplex genome targeting in monocot and dicot plants using a versatile and robust CRISPR/Cas9 vector system, emphasizing the construction of binary constructs with multiple sgRNA expression cassettes in one round of cloning using Golden Gate ligation. We also describe the genotyping of targeted mutations in transgenic plants by direct Sanger sequencing followed by decoding of superimposed sequencing chromatograms containing biallelic or heterozygous mutations using the Web-based tool DSDecode. © 2016 by John Wiley & Sons, Inc.
成簇规律间隔短回文重复序列（CRISPR）/Cas9介导的基因组靶向系统已应用于包括植物在内的多种生物。与其他基因组靶向技术如锌指核酸酶（ZFNs）和转录激活样效应因子核酸酶（TALENs）相比，CRISPR/Cas9系统使用起来更简便，编辑效率也高得多。此外，可以设计多个具有不同靶序列的“单向导RNA”（sgRNAs），以引导Cas9蛋白靶向多个基因组位点，实现同步多重编辑。在此，我们介绍一种使用通用且强大的CRISPR/Cas9载体系统在单子叶和双子叶植物中进行高效多重基因组靶向的方法，重点阐述通过金门连接在一轮克隆中构建具有多个sgRNA表达盒的二元构建体。我们还描述了通过直接桑格测序对转基因植物中的靶向突变进行基因分型，随后使用基于网络的工具DSDecode对包含双等位基因或杂合突变的叠加测序色谱图进行解码。© 2016约翰威立国际出版公司
