Plant Biotechnology Applications of Zinc Finger Technology

  • Stephen Novak
Part of the Methods in Molecular Biology book series (MIMB, volume 1864)


With ever-increasing genomic information combined with modern tools for genome modification, we are entering a new era of plant biotechnology. One major tool used for genome modification is the zinc finger nuclease (ZFN). Here, we discuss how ZFNs have proven useful in many genome modification applications. In order to remove the function of a gene or genes, targeted mutagenesis using ZFNs has been readily demonstrated creating numerous gene knockouts, and gene deletion has been demonstrated with removal of gene segments both native and transgenic up to 9 Mb. Applications for gain of function have also been demonstrated. Precision gene editing using ZFNs has resulted in the development of herbicide tolerance, and numerous forms of targeted gene addition have been exhibited. In addition to genome modification, this chapter also describes the use of zinc finger protein transcription factors (ZFP-TFs) for gene regulation in order to provide useful modification of gene expression resulting in altered phenotypes.

Key words

Zinc finger nuclease (ZFN) Zinc finger protein (ZFP) Zinc finger protein transcription factors (ZFP-TFs) Targeted mutagenesis Gene editing Gene targeting Gene deletion Gene regulation Double-strand breaks (DSBs) Homologous recombination (HR) Nonhomologous end joining (NHEJ) 


  1. 1.
    Voytas DF (2013) Plant genome engineering with sequence-specific nucleases. In: Merchant SS (ed) Annual review of plant biology, vol 64. pp 327–350CrossRefGoogle Scholar
  2. 2.
    Petolino JF (2015) Genome editing in plants via designed zinc finger nucleases. In Vitro Cell Dev Biol-Plant 51(1):1–8CrossRefGoogle Scholar
  3. 3.
    Cardi T, Stewart CN (2016) Progress of targeted genome modification approaches in higher plants. Plant Cell Rep 35(7):1401–1416CrossRefGoogle Scholar
  4. 4.
    Kim YG, Cha J, Chandrasegaran S (1996) Hybrid restriction enzymes: Zinc finger fusions to Fok I cleavage domain. Proc Natl Acad Sci U S A 93(3):1156–1160CrossRefGoogle Scholar
  5. 5.
    Smith J, Berg JM, Chandrasegaran S (1999) A detailed study of the substrate specificity of a chimeric restriction enzyme. Nucleic Acids Res 27(2):674–681CrossRefGoogle Scholar
  6. 6.
    Smith J, Bibikova M, Whitby FG, Reddy AR, Chandrasegaran S, Carroll D (2000) Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains. Nucleic Acids Res 28(17):3361–3369CrossRefGoogle Scholar
  7. 7.
    Bibikova M, Carroll D, Segal DJ, Trautman JK, Smith J, Kim YG, Chandrasegaran S (2001) Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol Cell Biol 21(1):289–297CrossRefGoogle Scholar
  8. 8.
    Choulika A, Perrin A, Dujon B, Nicolas JF (1995) Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of saccharomyces-cerevisiae. Mol Cell Biol 15(4):1968–1973CrossRefGoogle Scholar
  9. 9.
    Lieber MR (2010) The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu Rev Biochem 79:181–211CrossRefGoogle Scholar
  10. 10.
    Pabo CO, Peisach E, Grant RA (2001) Design and selection of novel Cys(2)His(2) zinc finger proteins. Annu Rev Biochem 70:313–340CrossRefGoogle Scholar
  11. 11.
    Isalan M, Choo Y (2001) Engineering nucleic acid-binding proteins by phage display. DNA-Protein Interactions: Principles and Protocols:417–429Google Scholar
  12. 12.
    Beerli RR, Barbas CF (2002) Engineering polydactyl zinc-finger transcription factors. Nat Biotechnol 20(2):135–141CrossRefGoogle Scholar
  13. 13.
    Urnov FD, Rebar EJ, Holmes MC, Zhang HS, Gregory PD (2010) Genome editing with engineered zinc finger nucleases. Nat Rev Genet 11(9):636–646CrossRefGoogle Scholar
  14. 14.
    Moore M, Klug A, Choo Y (2001) Improved DNA binding specificity from polyzinc finger peptides by using strings of two-finger units. Proc Natl Acad Sci 98(4):1437–1441CrossRefGoogle Scholar
  15. 15.
    Miller JC, Holmes MC, Wang J, Guschin DY, Lee Y-L, Rupniewski I, Beausejour CM, Waite AJ, Wang NS, Kim KA et al (2007) An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol 25(7):778–785CrossRefGoogle Scholar
  16. 16.
    Bibikova M, Golic M, Golic KG, Carroll D (2002) Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics 161(3):1169–1175PubMedPubMedCentralGoogle Scholar
  17. 17.
    Bibikova M, Beumer K, Trautman JK, Carroll D (2003) Enhancing gene targeting with designed zinc finger nucleases. Science 300(5620):764–764CrossRefGoogle Scholar
  18. 18.
    Porteus MH, Carroll D (2005) Gene targeting using zinc finger nucleases. Nat Biotechnol 23(8):967–973CrossRefGoogle Scholar
  19. 19.
    Lloyd A, Plaisier CL, Carroll D, Drews GN (2005) Targeted mutagenesis using zinc-finger nucleases in Arabidopsis. Proc Natl Acad Sci U S A 102(6):2232–2237CrossRefGoogle Scholar
  20. 20.
    Maeder ML, Thibodeau-Beganny S, Osiak A, Wright DA, Anthony RM, Eichtinger M, Jiang T, Foley JE, Winfrey RJ, Townsend JA et al (2008) Rapid “open-source” engineering of customized zinc-finger nucleases for highly efficient gene modification. Mol Cell 31(2):294–301CrossRefGoogle Scholar
  21. 21.
    Marton I, Zuker A, Shklarman E, Zeevi V, Tovkach A, Roffe S, Ovadis M, Tzfira T, Vainstein A (2010) Nontransgenic genome modification in plant cells. Plant Physiol 154(3):1079–1087CrossRefGoogle Scholar
  22. 22.
    Osakabe K, Osakabe Y, Toki S (2010) Site-directed mutagenesis in Arabidopsis using custom-designed zinc finger nucleases. Proc Natl Acad Sci U S A 107(26):12034–12039CrossRefGoogle Scholar
  23. 23.
    Zhang F, Maeder ML, Unger-Wallace E, Hoshaw JP, Reyon D, Christian M, Li X, Pierick CJ, Dobbs D, Peterson T et al (2010) High frequency targeted mutagenesis in Arabidopsis thaliana using zinc finger nucleases. Proc Natl Acad Sci U S A 107(26):12028–12033CrossRefGoogle Scholar
  24. 24.
    Shukla VK, Doyon Y, Miller JC, DeKelver RC, Moehle EA, Worden SE, Mitchell JC, Arnold NL, Gopalan S, Meng X et al (2009) Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature 459(7245):437–U156CrossRefGoogle Scholar
  25. 25.
    Curtin SJ, Zhang F, Sander JD, Haun WJ, Starker C, Baltes NJ, Reyon D, Dahlborg EJ, Goodwin MJ, Coffman AP et al (2011) Targeted mutagenesis of duplicated genes in soybean with zinc-finger nucleases. Plant Physiol 156(2):466–473CrossRefGoogle Scholar
  26. 26.
    Gupta MP, Palta AM, Stephen N, Fyodor U, Sunita G (2013) Engineered zinc finger proteins targeting 5-enolpyruvyl shikimate-3-phosphate synthase genes. In: USPTO (ed) USPTO. Dow Agrosciences, LLC, Sangamo Bio Sciences, Inc., United States of AmericaGoogle Scholar
  27. 27.
    Shukla V, Gupta M, Urnov F, Guschin D, De Both MJ, Bundock P, Sastry-Dent L (2016) Targeted modification of malate dehydrogenase. In: USPTO (ed) USPTO. Dow Agrosciences LLC, Sangamo Biosciences, Inc., United States of AmericaGoogle Scholar
  28. 28.
    Peer R, Rivlin G, Golobovitch S, Lapidot M, Gal-On A, Vainstein A, Tzfira T, Flaishman MA (2015) Targeted mutagenesis using zinc-finger nucleases in perennial fruit trees. Planta 241(4):941–951CrossRefGoogle Scholar
  29. 29.
    Lu HW, Klocko AL, Dow M, Ma C, Amarasinghe V, Strauss SH (2016) Low frequency of zinc-finger nuclease-induced mutagenesis in Populus. Mol Breed 36(9):121CrossRefGoogle Scholar
  30. 30.
    Townsend JA, Wright DA, Winfrey RJ, Fu FL, Maeder ML, Joung JK, Voytas DF (2009) High-frequency modification of plant genes using engineered zinc-finger nucleases. Nature 459(7245):442–U161CrossRefGoogle Scholar
  31. 31.
    de Pater S, Pinas JE, Hooykaas PJJ, van der Zaal BJ (2013) ZFN-mediated gene targeting of the Arabidopsis protoporphyrinogen oxidase gene through Agrobacterium-mediated floral dip transformation. Plant Biotechnol J 11(4):510–515CrossRefGoogle Scholar
  32. 32.
    Wright DA, Townsend JA, Winfrey RJ Jr, Irwin PA, Rajagopal J, Lonosky PM, Hall BD, Jondle MD, Voytas DF (2005) High-frequency homologous recombination in plants mediated by zinc-finger nucleases. Plant J 44(4):693–705CrossRefGoogle Scholar
  33. 33.
    Cai CQ, Doyon Y, Ainley WM, Miller JC, DeKelver RC, Moehle EA, Rock JM, Lee YL, Garrison R, Schulenberg L et al (2009) Targeted transgene integration in plant cells using designed zinc finger nucleases. Plant Mol Biol 69(6):699–709CrossRefGoogle Scholar
  34. 34.
    Ainley WM, Sastry-Dent L, Welter ME, Murray MG, Zeitler B, Amora R, Corbin DR, Miles RR, Arnold NL, Strange TL et al (2013) Trait stacking via targeted genome editing. Plant Biotechnol J 11(9):1126–1134CrossRefGoogle Scholar
  35. 35.
    Kumar S, AlAbed D, Worden A, Novak S, Wu H, Ausmus C, Beck M, Robinson H, Minnicks T, Hemingway D et al (2015) A modular gene targeting system for sequential transgene stacking in plants. J Biotechnol 207:12–20CrossRefGoogle Scholar
  36. 36.
    Schneider K, Schiermeyer A, Dolls A, Koch N, Herwartz D, Kirchhoff J, Fischer R, Russell SM, Cao ZH, Corbin DR et al (2016) Targeted gene exchange in plant cells mediated by a zinc finger nuclease double cut. Plant Biotechnol J 14(4):1151–1160CrossRefGoogle Scholar
  37. 37.
    Kumar S, Worden A, Novak S, Lee R, Petolino JF (2016) A trait stacking system via intra-genomic homologous recombination. Planta 244(5):1157–1166CrossRefGoogle Scholar
  38. 38.
    Butler NM, Baltes NJ, Voytas DF, Douches DS (2016) Geminivirus-mediated genome editing in potato (Solanum tuberosum L.) using sequence-specific nucleases. Front Plant Sci 7:1045CrossRefGoogle Scholar
  39. 39.
    Weinthal DM, Taylor RA, Tzfira T (2013) Nonhomologous end joining-mediated gene replacement in plant cells(1 C). Plant Physiol 162(1):390–400CrossRefGoogle Scholar
  40. 40.
    Mumm RH, Walters DS (2001) Quality control in the development of transgenic crop seed products. Crop Sci 41(5):1381–1389CrossRefGoogle Scholar
  41. 41.
    Cantos C, Francisco P, Trijatmiko KR, Slamet-Loedin I, Chadha-Mohanty PK (2014) Identification of “safe harbor” loci in indica rice genome by harnessing the property of zinc-finger nucleases to induce DNA damage and repair. Front Plant Sci 5:302CrossRefGoogle Scholar
  42. 42.
    Petolino JF, Worden A, Curlee K, Connell J, Moynahan TLS, Larsen C, Russell S (2010) Zinc finger nuclease-mediated transgene deletion. Plant Mol Biol 73(6):617–628CrossRefGoogle Scholar
  43. 43.
    Qi Y, Li X, Zhang Y, Starker CG, Baltes NJ, Zhang F, Sander JD, Reyon D, Joung JK, Voytas DF (2013) Targeted deletion and inversion of tandemly arrayed genes in Arabidopsis thaliana using zinc finger nucleases. G3 (Bethesda) 3(10):1707–1715CrossRefGoogle Scholar
  44. 44.
    Pavletich NP, Pabo CO (1991) Zinc finger DNA recognition – crystal-structure of a Zif268-DNA complex at 2.1-A. Science 252(5007):809–817CrossRefGoogle Scholar
  45. 45.
    Sainz MB, Goff SA, Chandler VL (1997) Extensive mutagenesis of a transcriptional activation domain identifies single hydrophobic and acidic amino acids important for activation in vivo. Mol Cell Biol 17(1):115–122CrossRefGoogle Scholar
  46. 46.
    Liu PQ, Rebar EJ, Zhang L, Liu Q, Jamieson AC, Liang YX, Qi H, Li PX, Chen BL, Mendel MC et al (2001) Regulation of an endogenous locus using a panel of designed zinc finger proteins targeted to accessible chromatin regions - Activation of vascular endothelial growth factor A. J Biol Chem 276(14):11323–11334CrossRefGoogle Scholar
  47. 47.
    Guan X, Stege J, Kim M, Dahmani Z, Fan N, Heifetz P, Barbas CF, Briggs SP (2002) Heritable endogenous gene regulation in plants with designed polydactyl zinc finger transcription factors. Proc Natl Acad Sci U S A 99(20):13296–13301CrossRefGoogle Scholar
  48. 48.
    Ordiz MI, Barbas CF, Beachy RN (2002) Regulation of transgene expression in plants with polydactyl zinc finger transcription factors. Proc Natl Acad Sci U S A 99(20):13290–13295CrossRefGoogle Scholar
  49. 49.
    Sanchez JP, Ullman C, Moore M, Choo Y, Chua NH (2002) Regulation of gene expression in Arabidopsis thaliana by artificial zinc finger chimeras. Plant Cell Physiol 43(12):1465–1472CrossRefGoogle Scholar
  50. 50.
    Stege JT, Guan X, Ho T, Beachy RN, Barbas CF (2002) Controlling gene expression in plants using synthetic zinc finger transcription factors. Plant J 32(6):1077–1086CrossRefGoogle Scholar
  51. 51.
    Li JQ, Blue R, Zeitler B, Strange TL, Pearl JR, Huizinga DH, Evans S, Gregory PD, Urnov FD, Petolino JF (2013) Activation domains for controlling plant gene expression using designed transcription factors. Plant Biotechnol J 11(6):671–680CrossRefGoogle Scholar
  52. 52.
    Van Eenennaam AL, Li GF, Venkatramesh M, Levering C, Gong XS, Jamieson AC, Rebar EJ, Shewmaker CK, Case CC (2004) Elevation of seed alpha-tocopherol levels using plant-based transcription factors targeted to an endogenous locus. Metab Eng 6(2):101–108CrossRefGoogle Scholar
  53. 53.
    Sanchez JP, Ullman C, Moore M, Choo Y, Chua NH (2006) Regulation of Arabidopsis thaliana 4-coumarate:coenzyme-A ligase-1 expression by artificial zinc finger chimeras. Plant Biotechnol J 4(1):103–114CrossRefGoogle Scholar
  54. 54.
    Gupta M, DeKelver RC, Palta A, Clifford C, Gopalan S, Miller JC, Novak S, Desloover D, Gachotte D, Connell J (2012) Transcriptional activation of Brassica napus β-ketoacyl-ACP synthase II with an engineered zinc finger protein transcription factor. Plant Biotechnol J 10(7):783–791CrossRefGoogle Scholar
  55. 55.
    DeKelver RG, Manju G, Miller Jeffrey C, Stephen N, Petolino Joseph F (2013) Engineered zinc finger proteins targeting plant genes involved in fatty acid biosynthesis. In: USPTO (ed) USPTO. Dow Agrosciences LLC, Sangamo Biosciences, Inc., Unites States of AmericaGoogle Scholar
  56. 56.
    Johnson LM, Du J, Hale CJ, Bischof S, Feng S, Chodavarapu RK, Zhong X, Marson G, Pellegrini M, Segal DJ et al (2014) SRA- and SET-domain-containing proteins link RNA polymerase V occupancy to DNA methylation. Nature 507(7490):124–128CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Stephen Novak
    • 1
  1. 1.Corteva Agriscience™Agriculture Division of DowDuPont™IndianapolisUSA

Personalised recommendations