清脆的
基因组编辑
Cas9
生物
功能基因组学
计算生物学
CRISPR干扰
引导RNA
基因组工程
拟南芥
基因组
烟草
亚基因组mRNA
合成生物学
遗传学
基因
基因组学
核糖核酸
突变体
作者
Levi G. Lowder,Dengwei Zhang,Nicholas J. Baltes,Joseph Suresh Paul,Xu Tang,Xuelian Zheng,Daniel F. Voytas,Tzung-Fu Hsieh,Yong Zhang,Yiping Qi
出处
期刊:Plant Physiology
[Oxford University Press]
日期:2015-10-01
卷期号:169 (2): 971-985
被引量:461
摘要
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.
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