An efficient sorghum protoplast assay for transient gene expression and gene editing by CRISPR/Cas9
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...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
PeerJ Inc.
2020-10-01
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| Series: | PeerJ |
| Subjects: | |
| Online Access: | https://peerj.com/articles/10077.pdf |
| Summary: | 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. |
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| ISSN: | 2167-8359 |