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

Ruirui Meng; Chenchen Wang; Lihua Wang; Yanlong Liu; Qiuwen Zhan; Jiacheng Zheng; Jieqin Li

doi:10.7717/peerj.10077

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

PeerJ. 2020 Oct 13:8:e10077. doi: 10.7717/peerj.10077. eCollection 2020.

Authors

Ruirui Meng^#¹, Chenchen Wang^#¹, Lihua Wang¹, Yanlong Liu¹, Qiuwen Zhan¹, Jiacheng Zheng¹, Jieqin Li¹

Affiliation

¹ College of Agriculture, Anhui Science and Technology University, Fengyang, China.

^# Contributed equally.

Abstract

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.

Keywords: CRISPR/Cas9; Gene editing; Protoplast; Sorghum; Transient gene expression.

Grants and funding

This work was supported by the National Natural Science Foundation of China (31971993), Anhui Provincial Natural Science Fund (2008085MC73), the Key-construction Subject Plan of Anhui Province (WanJiaoMiKe[2014]28), and the youth talent support program of Anhui Science and Technology University (XiaoRenFa[2015]69). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.