title: A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation.
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.
authors: 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.).
authors: Levi G
authors: LG
authors: Lowder
authors: 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.).
authors: Dengwei
authors: D
authors: Zhang
authors: 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.).
authors: Nicholas J
authors: NJ
authors: Baltes
authors: 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.).
authors: Joseph W
authors: http://orcid.org/0000-0003-4241-3498
authors: JW
authors: Paul
authors: 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.).
authors: Xu
authors: X
authors: Tang
authors: 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.).
authors: Xuelian
authors: http://orcid.org/0000-0002-7475-7888
authors: X
authors: Zheng
authors: 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.).
authors: Daniel F
authors: DF
authors: Voytas
authors: 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.).
authors: Tzung-Fu
authors: http://orcid.org/0000-0001-7584-3721
authors: TF
authors: Hsieh
authors: 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.
authors: Yong
authors: http://orcid.org/0000-0003-3704-4835
authors: Y
authors: Zhang
authors: 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.
authors: Yiping
authors: http://orcid.org/0000-0002-9556-6706
authors: Y
authors: Qi
mesh_terms: D017360:Arabidopsis / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics*; D000076987:CRISPR-Associated Protein 9; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D019175:DNA Methylation; D004269:DNA, Bacterial; D004720:Endonucleases / Q000235:genetics*; D018506:Gene Expression Regulation, Plant; D005818:Genetic Engineering / Q000379:methods*; D018745:Genome, Plant; D018392:Genomic Imprinting; D016296:Mutagenesis; D012275:Oryza / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D000094704:RNA, Guide, CRISPR-Cas Systems; D014026:Nicotiana / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D010940:Plant Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; C036483:T-DNA; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases