TSA Promotes CRISPR/Cas9 Editing Efficiency and Expression of Cell Division-Related Genes from Plant Protoplasts

• Published in: International journal of molecular sciences Vol. 22; no. 15; p. 7817
• Main Authors: Choi, Seung Hee, Lee, Myoung Hui, Jin, Da Mon, Ju, Su Ji, Ahn, Woo Seok, Jie, Eun Yee, Lee, Ji Min, Lee, Jiyoung, Kim, Cha Young, Kim, Suk Weon
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 22.07.2021; MDPI
• Subjects: Acetylation; Cell Division; chromatin de-condensation; Chromatin remodeling; CRISPR; CRISPR-Cas Systems; Design; Editing; Efficiency; Epigenetics; Gene Editing - methods; Gene expression; Genes; Genome editing; genome editing efficiency; Genome, Plant; Genomes; gRNA; histone acetylation; Histone deacetylase; Histone H3; Histones; Hydroxamic Acids - pharmacology; Lactuca - drug effects; Lactuca - genetics; Lactuca - growth & development; Lactuca - metabolism; Lettuce; Mutation; Nicotiana - drug effects; Nicotiana - genetics; Nicotiana - growth & development; Nicotiana - metabolism; Plant Cells; Plant Proteins - antagonists & inhibitors; Plant Proteins - genetics; Plant Proteins - metabolism; plant protoplasts; Polyethylene glycol; Protein Synthesis Inhibitors - pharmacology; Proteins; Protoplasts; Protoplasts - drug effects; Protoplasts - metabolism; Tobacco; Transfection; Trichostatin A; trichostatin A; chromatin de-condensation; plant protoplasts; tobacco; genome editing efficiency; lettuce; histone acetylation
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms22157817

Trichostatin A (TSA) is a representative histone deacetylase (HDAC) inhibitor that modulates epigenetic gene expression by regulation of chromatin remodeling in cells. To investigate whether the regulation of chromatin de-condensation by TSA can affect the increase in the efficiency of Cas9 protein-gRNA ribonucleoprotein (RNP) indel formation from plant cells, genome editing efficiency using lettuce and tobacco protoplasts was examined after several concentrations of TSA treatments (0, 0.1, 1 and 10 μM). RNP delivery from protoplasts was conducted by conventional polyethylene glycol (PEG) transfection protocols. Interestingly, the indel frequency of the gene from TSA treatments was about 3.3 to 3.8 times higher than DMSO treatment in lettuce protoplasts. The TSA-mediated increase of indel frequency of the gene in lettuce protoplasts occurred in a concentration-dependent manner, although there was not much difference. Similar to lettuce, TSA also increased the indel frequency by 1.5 to 1.8 times in a concentration-dependent manner during genome editing using tobacco protoplasts. The MNase test clearly showed that chromatin accessibility with TSA treatments was higher than that of DMSO treatment. Additionally, TSA treatment significantly increased the level of histone H3 and H4 acetylation from lettuce protoplasts. The qRT-PCR analysis showed that expression of cell division-related genes ( , , , and ) was increased by TSA treatment. These findings could contribute to increasing the efficiency of CRISPR/Cas9-mediated genome editing. Furthermore, this could be applied for the development of useful genome-edited crops using the CRISPR/Cas9 system with plant protoplasts.