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

PeerJ

article

Ruirui Meng¹  Chenchen Wang¹  Lihua Wang¹  Yanlong Liu¹  Qiuwen Zhan¹  Jiacheng Zheng¹  Jieqin Li¹ 

[1] College of Agriculture, Anhui Science and Technology University

关键词: Sorghum;    Protoplast;    Transient gene expression;    Gene editing;    CRISPR/Cas9;   

DOI  :  10.7717/peerj.10077

学科分类：社会科学、人文和艺术（综合）

来源: Inra

【 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 1.6×106 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 appropriate sgRNA for gene editing. One plasmid can correctly edit the target region using a single protoplast cell as template DNA. Our results indicated that the protoplast assays as optimized are suitable for transient gene expression and sgRNA screening in CRISPR/Cas9 gene editing procedures.

【 授权许可】

CC BY