Journal of Plant Research

, Volume 131, Issue 1, pp 179–189 | Cite as

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

  • Hilda Tiricz
  • Bettina Nagy
  • Györgyi Ferenc
  • Katalin Török
  • István Nagy
  • Dénes DuditsEmail author
  • Ferhan Ayaydin
Regular Paper


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.


Gene targeting Maize GFP Oligonucleotide Chromatin 



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).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.


  1. Andrieu-Soler C, Casas M, Faussat A-M, Gandolphe C, Doat M et al (2005) Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs. Nucl Acids Res 33:3733–3742. doi: 10.1093/nar/gki686 CrossRefPubMedPubMedCentralGoogle Scholar
  2. Beetham PR, Kipp PB, Sawycky XL, Arntzen CJ, May GD (1999) A tool for functional plant genomics: chimeric RNA/DNA oligonucleotides cause in vivo gene-specific mutations. Proc Natl Acad Sci USA 96:8774–8778CrossRefPubMedPubMedCentralGoogle Scholar
  3. Bond DM, Dennis ES, Pogson BJ, Finnegan EJ (2009) Histone acetylation, Vernalization Insensitive 3, Flowering Locus C, and the vernalization response. Mol Plant 2:724–737. doi: 10.1093/mp/ssp021 CrossRefPubMedGoogle Scholar
  4. Bonner M, Kmiec EB (2009) DNA breakage associated with targeted gene alteration directed by DNA oligonucleotides. Mutat Res 669:85–94. doi: 10.1016/j.mrfmmm.2009.05.004 CrossRefPubMedPubMedCentralGoogle Scholar
  5. Brachman EE, Kmiec EB (2005) Gene repair in mammalian cells is stimulated by the elongation of S phase and transient stalling of replication forks. DNA Rep (Amst) 4:445–457. doi: 10.1016/j.dnarep.2004.11.007 CrossRefGoogle Scholar
  6. Breyer D et al (2009) Genetic modification through oligonucleotide-mediated mutagenesis. A GMO regulatory challenge? Environ Biosaf Res 8:57–64. doi: 10.1051/ebr/2009007 CrossRefGoogle Scholar
  7. Christensen AH, Quail PH (1996) Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgen Res 5:213–218CrossRefGoogle Scholar
  8. Chua YL, Watson LA, Gray JC (2003) The transcriptional enhancer of the pea plastocyanin gene associates with the nuclear matrix and regulates gene expression through histone acetylation. Plant Cell 15:1468–1479CrossRefPubMedPubMedCentralGoogle Scholar
  9. Delcuve GP, Khan DH, Davie JR (2012) Roles of histone deacetylases in epigenetic regulation: emerging paradigms from studies with inhibitors. Clin Epigenet 4:5. doi: 10.1186/1868-7083-4-5 CrossRefGoogle Scholar
  10. Dong C, Beetham P, Vincent K, Sharp P (2006) Oligonucleotide-directed gene repair in wheat using a transient plasmid gene repair assay system. Plant Cell Rep 25:457–465. doi: 10.1007/s00299-005-0098-x CrossRefPubMedGoogle Scholar
  11. Earley KW, Shook MS, Brower-Toland B, Hicks L, Pikaard CS (2007) In vitro specificities of Arabidopsis co-activator histone acetyltransferases: implications for histone hyperacetylation in gene activation. Plant J 52:615–626. doi: 10.1111/j.1365-313X.2007.03264.x CrossRefPubMedGoogle Scholar
  12. Engstrom JU, Kmiec EB (2007) Manipulation of cell cycle progression can counteract the apparent loss of correction frequency following oligonucleotide-directed gene repair. BMC Mol Biol 8:9. doi: 10.1186/1471-2199-8-9 CrossRefPubMedPubMedCentralGoogle Scholar
  13. Engstrom JU, Kmiec EB (2008) DNA replication, cell cycle progression and the targeted gene repair reaction. Cell Cycle 7:1402–1414. doi: 10.4161/cc.7.10.5826 CrossRefPubMedGoogle Scholar
  14. Fogarty RD, McKean SC, White PJ, Atley LM, Werther GA, Wraight CJ (2002) Sequence dependence of C5-propynyl-dU,dC-phosphorothioate oligonucleotide inhibition of the human IGF-I receptor: mRNA, protein, and cell growth. Antisense Nucl Acid Drug Dev 12:369–377. doi: 10.1089/108729002321082447 CrossRefGoogle Scholar
  15. Gilles AF, Averof M (2014) Functional genetics for all: engineered nucleases, CRISPR and the gene editing revolution. EvoDevo 5:43. doi: 10.1186/2041-9139-5-43 CrossRefPubMedPubMedCentralGoogle Scholar
  16. Hu Y, Parekh-Olmedo H, Drury M, Skogen M, Kmiec EB (2005) Reaction parameters of targeted gene repair in mammalian cells. Mol Biotechnol 29:197–210. doi: 10.1385/MB:29:3:197 CrossRefPubMedGoogle Scholar
  17. Huen MS, Li XT, Lu LY, Watt RM, Liu DP, Huang JD (2006) The involvement of replication in single stranded oligonucleotide-mediated gene repair. Nucl Acids Res 34:6183–6194. doi: 10.1093/nar/gkl852 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Huen MS, Lu LY, Liu DP, Huang JD (2007) Active transcription promotes single-stranded oligonucleotide mediated gene repair. Biochem Biophys Res Commun 353:33–39. doi: 10.1016/j.bbrc.2006.11.146 CrossRefPubMedGoogle Scholar
  19. Kim H, Kim JS (2014) A guide to genome engineering with programmable nucleases. Nat Rev Genet 15:321–334. doi: 10.1038/nrg3686 CrossRefPubMedGoogle Scholar
  20. Kochevenko A, Willmitzer L (2003) Chimeric RNA/DNA oligonucleotide-based site-specific modification of the tobacco acetolactate syntase gene. Plant Physiol 132:174–184. doi: 10.1104/pp.102.016857 CrossRefPubMedPubMedCentralGoogle Scholar
  21. Morocz S, Donn G, Nerneth J, Dudits D (1990) An improved system to obtain fertile regenerants via maize protoplasts isolated from a highly embryogenic suspension culture. Theor Appl Genet 80:721–726. doi: 10.1007/BF00224183 CrossRefPubMedGoogle Scholar
  22. Okuzaki A, Toriyama K (2004) Chimeric RNA/DNA oligonucleotide-directed gene targeting in rice. Plant Cell Rep 22:509–512. doi: 10.1007/s00299-003-0698-2 CrossRefPubMedGoogle Scholar
  23. Parekh-Olmedo H, Engstrom JU, Kmiec EB (2003) The effect of hydroxyurea and trichostatin a on targeted nucleotide exchange in yeast and Mammalian cells. Ann NY Acad Sci 1002:43–55CrossRefPubMedGoogle Scholar
  24. Paul AL, Ferl RJ (1993) Osmium tetroxide footprinting of a scaffold attachment region in the maize Adh1 promoter. Plant Mol Biol 22:1145–1151CrossRefPubMedGoogle Scholar
  25. Paul AL, Ferl RJ (1998) Higher order chromatin structures in maize and Arabidopsis. Plant Cell 10:1349–1359CrossRefPubMedPubMedCentralGoogle Scholar
  26. Puchta H, Fauser F (2014) Synthetic nucleases for genome engineering in plants: prospects for a bright future. Plant J 78:727–741. doi: 10.1111/tpj.12338 CrossRefPubMedGoogle Scholar
  27. Radecke S, Radecke F, Cathomen T, Schwarz K (2010) Zinc-finger nuclease-induced gene repair with oligodeoxynucleotides: wanted and unwanted target locus modifications. Mol Ther 18:743–753. doi: 10.1038/mt.2009.304 CrossRefPubMedPubMedCentralGoogle Scholar
  28. Rivera-Torres N, Kmiec EB (2016) Genetic spell-checking: gene editing using single-stranded DNA oligonucleotides. Plant Biotechnol J 14:463–470. doi: 10.1111/pbi.12473 CrossRefPubMedGoogle Scholar
  29. Ruiter R, van den Brande I, Stals E, Delaure S, Cornelissen M, D’Halluin K (2003) Spontaneous mutation frequency in plants obscures the effect of chimeraplasty. Plant Mol Biol 53:675–689. doi: 10.1023/B:PLAN.0000019111.96107.01 CrossRefPubMedGoogle Scholar
  30. Ruxton GD (2006) The unequal variance t-test is an underused alternative to Student’s t-test and the Mann–Whitney U test. Behav Ecol 17:688–690. doi: 10.1093/beheco/ark016 CrossRefGoogle Scholar
  31. Sargent RG, Kim S, Gruenert DC (2011) Oligo/polynucleotide-based gene modification: strategies and therapeutic potential. Oligonucleotides 21:55–75. doi: 10.1089/oli.2010.0273 CrossRefPubMedPubMedCentralGoogle Scholar
  32. Sauer NJ, Mozoruk J, Miller RB, Warburg ZJ, Walker KA et al (2016a) Oligonucleotide-directed mutagenesis for precision gene editing. Plant Biotechnol J 14:496–502. doi: 10.1111/pbi.12496 CrossRefPubMedGoogle Scholar
  33. Sauer NJ, Narváez-Vásquez J, Mozoruk J, Miller RB, Warburg ZJ et al (2016b) Oligonucleotide-mediated genome editing provides precision and function to engineered nucleases and antibiotics in plants. Plant Physiol 170:1917–1928. doi: 10.1104/pp.15.01696 CrossRefPubMedPubMedCentralGoogle Scholar
  34. Schrader C, Schielke A, Ellerbroek L, Johne R (2012) PCR inhibitors—occurrence, properties and removal. J Appl Microbiol 113:1014–1026CrossRefPubMedGoogle Scholar
  35. Shu H, Gruissem W, Hennig L (2013) Measuring Arabidopsis chromatin accessibility using DNase I-polymerase chain reaction and DNase I-chip assays. Plant Physiol 162:1794–1801. doi: 10.1104/pp.113.220400 CrossRefPubMedPubMedCentralGoogle Scholar
  36. Strouse B, Bialk P, Niamat RA, Rivera-Torres N, Kmiec EB (2014) Combinatorial gene editing in mammalian cells using ssODNs and TALENs. Sci Rep 4:3791. doi: 10.1038/srep03791 CrossRefPubMedPubMedCentralGoogle Scholar
  37. Svitashev S, Young JK, Schwartz C, Gao H, Falco SC, Cigan AM (2015) Targeted mutagenesis, precise gene editing, and site-specific gene insertion in Maize using Cas9 and guide RNA. Plant Physiol 169:931–945. doi: 10.1104/pp.15.00793 CrossRefPubMedPubMedCentralGoogle Scholar
  38. Voytas DF (2013) Plant genome engineering with sequence-specific nucleases. Annu Rev Plant Biol 64:327–350. doi: 10.1146/annurev-arplant-042811-105552 CrossRefPubMedGoogle Scholar
  39. Voytas DF, Gao C (2014) Precision genome engineering and agriculture: opportunities and regulatory challenges. PLoS Biol 12:e1001877. doi: 10.1371/journal.pbio.1001877 CrossRefPubMedPubMedCentralGoogle Scholar
  40. Wang M, Liu Y, Zhang C, Liu J, Liu X et al (2015) Gene editing by co-transformation of TALEN and chimeric RNA/DNA oligonucleotides on the rice OsEPSPS gene and the inheritance of mutations. PLoS One 10:e0122755. doi: 10.1371/journal.pone.0122755 CrossRefPubMedPubMedCentralGoogle Scholar
  41. Wefers B, Meyer M, Ortiz O, Hrabe de Angelis M, Hansen J, Wurst W, Kuhn R (2013) Direct production of mouse disease models by embryo microinjection of TALENs and oligodeoxynucleotides. Proc Natl Acad Sci USA 110:3782–3787. doi: 10.1073/pnas.1218721110 CrossRefPubMedPubMedCentralGoogle Scholar
  42. Yao Q, Cong L, Chang JL, Li KX, Yang GX, He GY (2006) Low copy number gene transfer and stable expression in a commercial wheat cultivar via particle bombardment. J Exp Bot 57:3737–3746. doi: 10.1093/jxb/erl145 CrossRefPubMedGoogle Scholar
  43. Zhu T, Peterson DJ, Tagliani L, St Clair G, Baszczynski CL, Bowen B (1999) Targeted manipulation of maize genes in vivo using chimeric RNA/DNA oligonucleotides. Proc Natl Acad Sci USA 96:8768–8773CrossRefPubMedPubMedCentralGoogle Scholar
  44. Zhu T, Mettenburg K, Peterson DJ, Tagliani L, Baszczynski CL (2000) Engineering herbicide-resistant maize using chimeric RNA/DNA oligonucleotides. Nat Biotechnol 18:555–558. doi: 10.1038/75435 CrossRefPubMedGoogle Scholar

Copyright information

© The Botanical Society of Japan and Springer Japan KK 2017

Authors and Affiliations

  • Hilda Tiricz
    • 1
  • Bettina Nagy
    • 1
  • Györgyi Ferenc
    • 1
  • Katalin Török
    • 1
  • István Nagy
    • 2
    • 3
  • Dénes Dudits
    • 1
    Email author
  • Ferhan Ayaydin
    • 1
    • 4
  1. 1.Institute of Plant Biology, Biological Research CentreHungarian Academy of SciencesSzegedHungary
  2. 2.Institute of Biochemistry, Biological Research CentreHungarian Academy of SciencesSzegedHungary
  3. 3.SeqOmics Biotechnology Ltd.MórahalomHungary
  4. 4.Laboratory of Cellular Imaging, Biological Research CentreHungarian Academy of SciencesSzegedHungary

Personalised recommendations