A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation
A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation
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用于多重植物基因组编辑和转录调控的 CRISPR/Cas9 工具箱
DOI：
10.1104/pp.15.00636
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发表时间：
2015-10-01
期刊：
影响因子：
7.4
通讯作者：
Qi, Yiping
中科院分区：
文献类型：
--
作者：
Lowder, Levi G.;Zhang, Dengwei;Qi, Yiping
用于基因组操作的基于成簇规则间隔短回文重复序列（CRISPR）/CRISPR相关蛋白9（Cas9）的试剂的相对容易性、速度和生物学范围正在彻底改变分子生物科学的几乎所有领域，包括功能基因组学、遗传学、应用生物医学研究和农业生物技术。然而，在工厂系统中，目前存在许多障碍，限制了这项技术充分发挥其潜力。例如，仍然需要大量的植物分子生物学专业知识和努力来产生允许多个不同基因组基因座的同时编辑，特别是转录调控或多重化的功能表达构建体，这是CRISPR/Cas9相对于其他基因组编辑系统的显著优势。为了简化和促进基于CRISPR/Cas9的技术在植物研究中的快速和大规模使用，我们开发并实施了一个全面的分子工具箱，用于植物中多方面的CRISPR/Cas9应用。该工具箱为研究人员提供了一种方案和试剂，可以使用Golden Gate和Gateway克隆方法快速有效地为单子叶植物和双子叶植物组装功能性CRISPR/Cas9转移DNA构建体。它具有全套功能，包括多重基因编辑和植物内源基因的转录激活或抑制。我们报告的功能和有效性，这个工具箱中的模式植物，如烟草（本氏烟草），拟南芥（拟南芥），水稻（水稻），展示其实用性的基础和应用植物研究。
The relative ease, speed, and biological scope of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated Protein9 (Cas9)-based reagents for genomic manipulations are revolutionizing virtually all areas of molecular biosciences, including functional genomics, genetics, applied biomedical research, and agricultural biotechnology. In plant systems, however, a number of hurdles currently exist that limit this technology from reaching its full potential. For example, significant plant molecular biology expertise and effort is still required to generate functional expression constructs that allow simultaneous editing, and especially transcriptional regulation, of multiple different genomic loci or multiplexing, which is a significant advantage of CRISPR/Cas9 versus other genome-editing systems. To streamline and facilitate rapid and wide-scale use of CRISPR/Cas9-based technologies for plant research, we developed and implemented a comprehensive molecular toolbox for multifaceted CRISPR/Cas9 applications in plants. This toolbox provides researchers with a protocol and reagents to quickly and efficiently assemble functional CRISPR/Cas9 transfer DNA constructs for monocots and dicots using Golden Gate and Gateway cloning methods. It comes with a full suite of capabilities, including multiplexed gene editing and transcriptional activation or repression of plant endogenous genes. We report the functionality and effectiveness of this toolbox in model plants such as tobacco (Nicotiana benthamiana), Arabidopsis (Arabidopsis thaliana), and rice (Oryza sativa), demonstrating its utility for basic and applied plant research.