A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation1[OPEN]
By Levi G. Lowder, Dengwei Zhang, N. Baltes, Joseph W. Paul, Xu Tang, Xuelian Zheng, Daniel F. Voytas, T. Hsieh, Yong Zhang, Yiping Qi. Plant Physiology, 2015
609 citations recorded by the source corpus.
A CRISPR/Cas9 toolbox enables multiplex genome editing and transcriptional regulation of genes in plants. 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.
Subfields: Insect Science, Molecular Biology, Plant Science
View Paper: https://www.semanticscholar.org/paper/e1c4e4d6f3d6675e45202f8b98c1417bce19528e
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Citations in the corpus
- Past and Future Use of Wild Relatives in Crop Breeding (2017, background): “, chromosomal fragments), are becoming increasingly feasible (Lowder et al., 2015; Ma et al., 2015).”
- CRISPR/Cas9 in Genome Editing and Beyond. (2016, background): “Other work has also expanded the applications of CRISPRi/a to the regulation of gene expression in multicellular organisms, such as Drosophila (193) and plants (194, 195), and to the reactivation of latent reservoirs of HIV-1 for its permanent elimination (196).”
- Genome Editing—Principles and Applications for Functional Genomics Research and Crop Improvement (2017, methodology): “Several approaches have been developed to express multiple sgRNAs, such as Golden Gate assembly, polycistronic tRNA–gRNA system, selfcleaving ribozyme flanked gRNAs and target-adaptor ligation (Gao and Zhao, 2014; Lowder et al., 2015; Ma et al., 2015; Xie et al., 2015).”
- Enhanced Rice Blast Resistance by CRISPR/Cas9-Targeted Mutagenesis of the ERF Transcription Factor Gene OsERF922 (2016, background): “Compared with TALENs, CRISPR/Cas9-expressing vectors are much easier to construct and can be competed in just two or three days [38, 75], whereas the construction of TALENs typically requires over seven days [76–78].”
- CRISPR/Cas Genome Editing and Precision Plant Breeding in Agriculture. (2019, methodology): “(93) recently evaluated simultaneous multigene activation and repression in plants.”