Repositorium
Journal Article / 2014
Xing, Hui-Li; Dong, Li; Wang, Zhi-Ping; Zhang, Hai-Yan; Han, Chun-Yan; Liu, Bing; Wang, Xue-Chen; Chen, Qi-Jun
BACKGROUND 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. RESULTS 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. CONCLUSIONS 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.
Techniques
ID | Corresponding Author Country |
Plant Species | GE Technique Sequence Identifier |
Trait Type of Alteration |
Progress in Research Key Topic |
---|---|---|---|---|---|
570 |
Chen, Qi-Jun China |
Arabidopsis thaliana |
CRISPR/Cas9 TRY/CPC/ETC2 |
curled leafes SDN1 |
Basic research Basic research |
571 |
Chen, Qi-Jun China |
Zea mays |
CRISPR/Cas9 HKT1 |
No information SDN1 |
Basic research Basic research |