Abstract
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.
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Acknowledgements
The authors thank Dr. Chongmei Dong (Plant Breeding Institute, University of Sydney, Australia) for providing the GFP vector constructs and Sándor Mórocz (Cereal Research Institute, Szeged, Hungary) for making the maize cell suspension culture available for transformation and Edit Kotogány for help with flow cytometry analyses. This publication is dedicated to the memory of the late Dr. Sándor Bottka, who inspired the use of synthetic oligonucleotides in our plant research. This work was supported by the National Research, Development and Innovation Office, NKFIH (Grant no. K116318 to FA) and János Bolyai Research Scholarship of the Hungarian Academy of Sciences (to IN).
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Tiricz, H., Nagy, B., Ferenc, G. et al. Relaxed chromatin induced by histone deacetylase inhibitors improves the oligonucleotide-directed gene editing in plant cells. J Plant Res 131, 179–189 (2018). https://doi.org/10.1007/s10265-017-0975-8
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DOI: https://doi.org/10.1007/s10265-017-0975-8