Relaxed chromatin induced by histone deacetylase inhibitors improves the oligonucleotide-directed gene editing in plant cells. 2018

Hilda Tiricz, and Bettina Nagy, and Györgyi Ferenc, and Katalin Török, and István Nagy, and Dénes Dudits, and Ferhan Ayaydin
Institute of Plant Biology, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary.

Improving efficiency of oligonucleotide-directed mutagenesis (ODM) is a prerequisite for wide application of this gene-editing approach in plant science and breeding. Here we have tested histone deacetylase inhibitor treatments for induction of relaxed chromatin and for increasing the efficiency of ODM in cultured maize cells. For phenotypic assay we produced transgenic maize cell lines expressing the non-functional Green Fluorescent Protein (mGFP) gene carrying a TAG stop codon. These transgenic cells were bombarded with corrective oligonucleotide as editing reagent to recover GFP expression. Repair of green fluorescent protein function was monitored by confocal fluorescence microscopy and flow cytometry was used for quantification of correction events. Sequencing PCR fragments of the GFP gene from corrected cells indicated a nucleotide exchange in the stop codon (TAG) from T to G nucleotide that resulted in the restoration of GFP function. We show that pretreatment of maize cells with sodium butyrate (5-10 mM) and nicotinamide (1-5 mM) as known inhibitors of histone deacetylases can cause elevated chromatin sensitivity to DNase I that was visualized in agarose gels and confirmed by the reduced presence of intact PCR template for the inserted exogenous mGFP gene. Maize cells with more relaxed chromatin could serve as an improved recipient for targeted nucleotide exchange as indicated by an average of 2.67- to 3.62-fold increase in GFP-positive cells. Our results stimulate further studies on the role of the condition of the recipient cells in ODM and testing the application of chromatin modifying agents in other, programmable nuclease-based genome-editing techniques in higher plants.

UI MeSH Term Description Entries
D009841 Oligonucleotides Polymers made up of a few (2-20) nucleotides. In molecular genetics, they refer to a short sequence synthesized to match a region where a mutation is known to occur, and then used as a probe (OLIGONUCLEOTIDE PROBES). (Dorland, 28th ed) Oligonucleotide
D010940 Plant Proteins Proteins found in plants (flowers, herbs, shrubs, trees, etc.). The concept does not include proteins found in vegetables for which PLANT PROTEINS, DIETARY is available. Plant Protein,Protein, Plant,Proteins, Plant
D002843 Chromatin The material of CHROMOSOMES. It is a complex of DNA; HISTONES; and nonhistone proteins (CHROMOSOMAL PROTEINS, NON-HISTONE) found within the nucleus of a cell. Chromatins
D003313 Zea mays A plant species of the family POACEAE. It is a tall grass grown for its EDIBLE GRAIN, corn, used as food and animal FODDER. Corn,Indian Corn,Maize,Teosinte,Zea,Corn, Indian
D000072669 Gene Editing Genetic engineering or molecular biology techniques that involve DNA REPAIR mechanisms for incorporating site-specific modifications into a cell's genome. Base Editing,Genome Editing,Editing, Base,Editing, Gene,Editing, Genome
D056572 Histone Deacetylase Inhibitors Compounds that inhibit HISTONE DEACETYLASES. This class of drugs may influence gene expression by increasing the level of acetylated HISTONES in specific CHROMATIN domains. HDAC Inhibitor,HDAC Inhibitors,Histone Deacetylase Inhibitor,Deacetylase Inhibitor, Histone,Deacetylase Inhibitors, Histone,Inhibitor, HDAC,Inhibitor, Histone Deacetylase,Inhibitors, HDAC,Inhibitors, Histone Deacetylase
D059828 Plant Cells Basic functional unit of plants. Cell, Plant,Cells, Plant,Plant Cell

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