Abstract
The CRISPR/Cas9 genome-editing system has emerged as a popular powerful tool for biological research. However, the process of selecting efficiently edited Cas9-free plants is usually laborious and time consuming. Here, we demonstrated P2A to be the most efficient self-cleaving peptide for fusing Cas9 and GFP in Arabidopsis and then used Cas9-P2A-GFP to develop a novel CRISPR/Cas9 system. Additionally, a pair of isocaudomer restriction enzymes were selected to conveniently assemble multiple sgRNAs. In this system, the GFP fluorescence intensity in T1 transgenic plants indicates the expression level of the Cas9 protein, which correlates well with the editing efficiency. Furthermore, Cas9-free plants can be easily selected by examining GFP fluorescence in T2 transgenic plants. The efficient knockout of BRI1, BZR1 and BES1 demonstrated the robustness of our new system. Thus, we designed a novel CRISPR/Cas9 system that can generate Cas9-free multiplex mutants efficiently in Arabidopsis and possibly in other plant species.
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Acknowledgements
We thank Prof. Xing Wang Deng for lab resources, Prof. Jian-Kang Zhu for providing the p35S-Cas9-SK and pAtU6-26-SK plasmids, and Prof. Qi-Jun Chen for providing the pHEE401E vector. This study was supported by the National Key R&D Program of China (2018YFE0204700), the National Natural Science Foundation of China (31621001), the Peking-Tsinghua Center for Life Sciences, and the State Key Laboratory of Protein and Plant Gene Research.
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JW and HC designed the study. JW performed the experiments. JW and HC analyzed the data. JW and HC wrote the manuscript.
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Wang, J., Chen, H. A novel CRISPR/Cas9 system for efficiently generating Cas9-free multiplex mutants in Arabidopsis. aBIOTECH 1, 6–14 (2020). https://doi.org/10.1007/s42994-019-00011-z
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DOI: https://doi.org/10.1007/s42994-019-00011-z