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An efficient sorghum protoplast assay for transient gene expression and gene editing by CRISPR/Cas9

PeerJ. 2020; 
Ruirui Meng, Chenchen Wang, Lihua Wang, Yanlong Liu, Qiuwen Zhan, Jiacheng Zheng, Jieqin Li
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DNA Sequencing … The PCR products from the second round were digested by T7 endonuclease I (NEB, UK) at 37° for 30 min. The digested products were detected by 1% agar gel and sequenced by Genscript Company (Nanjing, China). Data analysis … Get A Quote

摘要

Protoplasts are commonly used in genetic and breeding research. In this study, the isolation of sorghum protoplasts was optimized and applied to transient gene expression and editing by CRISPR/Cas9. The protoplast was most viable in 0.5 M mannitol, which was the highest of three concentrations after 48- and 72-hours treatments. Using this method we can derive an average of 1.6×10 cells which vary from 5 to 22 nm in size. The average transfection of the protoplasts was 68.5% using the PEG-mediated method. The subcellular assays located Sobic.002G279100-GFP and GFP proteins in the cell compartments as predicted bioinformatically. Two CRISPR/Cas9 plasmids were transfected into sorghum protoplasts to screen for an... More

关键词

CRISPR/Cas9, Gene editing, Protoplast, Sorghum, Transient gene expression