A CRISPR/Cas9 toolkit for multiplex genome editing in plants
By Hui-Li Xing, Li Dong, Zhiping Wang, Hai-Yan Zhang, Chun-Yan Han, Bing Liu, Xue-Chen Wang, Qi-jun Chen. BMC Plant Biology, 2014
1,422 citations recorded by the source corpus.
To accelerate the application of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) system to a variety of plant species, a toolkit with additional plant selectable markers, more gRNA modules, and easier methods for the assembly of one or more gRNA expression cassettes is required. We developed a CRISPR/Cas9 binary vector set based on the pGreen or pCAMBIA backbone, as well as a gRNA (guide RNA) module vector set, as a toolkit for multiplex genome editing in plants. This toolkit requires no restriction enzymes besides BsaI to generate final constructs harboring maize-codon optimized Cas9 and one or more gRNAs with high efficiency in as little as one cloning step. The toolkit was validated using maize protoplasts, transgenic maize lines, and transgenic Arabidopsis lines and was shown to exhibit high efficiency and specificity. More importantly, using this toolkit, targeted mutations of three Arabidopsis genes were detected in transgenic seedlings of the T1 generation. Moreover, the multiple-gene mutations could be inherited by the next generation. We developed a toolkit that facilitates transient or stable expression of the CRISPR/Cas9 system in a variety of plant species, which will facilitate plant research, as it enables high efficiency generation of mutants bearing multiple gene mutations.
Subfields: Molecular Biology
View Paper: https://www.semanticscholar.org/paper/f2192be5852162aee7fd77dcd3b730edf914e71e
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Citations in the corpus
- A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation1[OPEN] (2015, methodology): “While we were preparing our article, Xing et al. (2014) and Ma et al. (2015) reported a CRISPR/Cas9 toolkit for targetedmutagenesis in plants.”
- Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation (2015, methodology): “Generation of transgenic Arabidopsis plants and analysis of mutations We transformed the pHEE2A/B/D1/D2/D3/E/F-TRI, pHEN2C-TRI, pHSE2A-TRI, and pHEE2A-CHLI constructs into Agrobacterium strain GV3101, and transformed pHEN2A/B-TRI into GV3101/pSoup [26].”
- A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. (2015, background): “…current plant CRISPR/Cas9 vector systems can only target one or few genomic sites (Jiang et al., 2013a, 2013b; Li et al., 2013; Mao et al., 2013; Shan et al., 2013a, 2013b; Xie and Yang, 2013; M Fauser et al., 2014; Feng et al., 2014; Xing et al., 2014; Zhang et al., 2014; Zhou et al., 2014).”