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Plant Physiol. 2015 Oct;169(2):971-85. doi: 10.1104/pp.15.00636. Epub 2015 Aug 21.

A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation.

Author information

1
Department of Biology, East Carolina University, Greenville, North Carolina 27858 (L.G.L., J.W.P., Y.Q.);Department of Biotechnology, School of Life Sciences and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China (D.Z., X.T., X.Z., Y.Z.);Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455 (N.J.B., D.F.V.); andDepartment of Plant and Microbial Biology and Plants for Human Health Institute, North Carolina State University, North Carolina Research Campus, Kannapolis, North Carolina 28081 (T.-F.H.).
2
Department of Biology, East Carolina University, Greenville, North Carolina 27858 (L.G.L., J.W.P., Y.Q.);Department of Biotechnology, School of Life Sciences and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China (D.Z., X.T., X.Z., Y.Z.);Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455 (N.J.B., D.F.V.); andDepartment of Plant and Microbial Biology and Plants for Human Health Institute, North Carolina State University, North Carolina Research Campus, Kannapolis, North Carolina 28081 (T.-F.H.) qiy@ecu.edu zhangyong916@uestc.edu.cn.

Abstract

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.

PMID:
26297141
PMCID:
PMC4587453
DOI:
10.1104/pp.15.00636
[Indexed for MEDLINE]
Free PMC Article

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