Seamless Genome Editing in Rice via Gene Targeting and Precise Marker Elimination

  • Ayako Nishizawa-Yokoi
  • Hiroaki Saika
  • Seiichi TokiEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1469)


Positive–negative selection using hygromycin phosphotransferase (hpt) and diphtheria toxin A-fragment (DT-A) as positive and negative selection markers, respectively, allows enrichment of cells harboring target genes modified via gene targeting (GT). We have developed a successful GT system employing positive–negative selection and subsequent precise marker excision via the piggyBac transposon derived from the cabbage looper moth to introduce desired modifications into target genes in the rice genome. This approach could be applied to the precision genome editing of almost all endogenous genes throughout the genome, at least in rice.

Key word

Gene targeting Positive–negative selection Marker excision piggyBac transposon Homologous recombination 



We acknowledge Dr. R. Terada (Meijo University) and Dr. S. Iida (Shizuoka Pref. University) for providing the DT-A gene, Dr. K. Uchino and Dr. H. Sezutsu (National Institute of Agrobiological Sciences) for providing hyPBase gene, and Dr. H. Rothnie for English editing. This research was supported by a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Genomics for Agricultural Innovation PGE1001), KAKENHI (23658012 and 23310142) and the Cross-ministerial Strategic Innovation Promotion Program (SIP).


  1. 1.
    Terada R, Urawa H, Inagaki Y, Tsugane K, Iida S (2002) Efficient gene targeting by homologous recombination in rice. Nat Biotechnol 20:1030–1034CrossRefPubMedGoogle Scholar
  2. 2.
    Shimatani Z, Nishizawa-Yokoi A, Endo M, Toki S, Terada R (2015) Positive-negative-selection-mediated gene targeting in rice. Front Plant Sci 5:748CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Osakabe K, Nishizawa-Yokoi A, Ohtsuki N, Osakabe Y, Toki S (2014) A mutated cytosine deaminase gene, codA (D314A), as an efficient negative selection marker for gene targeting in rice. Plant Cell Physiol 55:658–665CrossRefPubMedGoogle Scholar
  4. 4.
    Nishizawa-Yokoi A, Nonaka S, Osakabe K, Saika H, Toki S (2015) A universal positive-negative selection system for gene targeting in plants combining an antibiotic resistance gene and its antisense RNA. Plant Physiol 169:362–370CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Cary LC, Goebel M, Corsaro BG, Wang HG, Rosen E, Fraser MJ (1989) Transposon mutagenesis of baculoviruses: analysis of Trichoplusia ni transposon IFP2 insertions within the FP-locus of nuclear polyhedrosis viruses. Virology 172:156–169CrossRefPubMedGoogle Scholar
  6. 6.
    Ryder E, Russell S (2003) Transposable elements as tools for genomics and genetics in Drosophila. Brief Funct Genomic Proteomic 2:57–71CrossRefPubMedGoogle Scholar
  7. 7.
    Woodard LE, Wilson MH (2015) piggyBac-ing models and new therapeutic strategies. Trends Biotechnol 33:525–533CrossRefPubMedGoogle Scholar
  8. 8.
    Nishizawa-Yokoi A, Endo M, Osakabe K, Saika H, Toki S (2014) Precise marker excision system using an animal-derived piggyBac transposon in plants. Plant J 77:454–463CrossRefPubMedGoogle Scholar
  9. 9.
    Nishizawa-Yokoi A, Endo M, Ohtsuki N, Saika H, Toki S (2015) Precision genome editing in plants via gene targeting and piggyBac-mediated marker excision. Plant J 81:160–168CrossRefPubMedGoogle Scholar
  10. 10.
    Hood E, Gelvin S, Melchers L, Hoekema A (1993) New Agrobacterium helper plasmids for gene-transfer to plants. Transgenic Res 2:208–218CrossRefGoogle Scholar
  11. 11.
    Yusa K, Zhou L, Li M, Bradley A, Craig N (2011) A hyperactive piggyBac transposase for mammalian applications. Proc Natl Acad Sci U S A 108:1531–1536CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Saika H, Onodera H, Seiichi T (2012) Visual selection in rice: a strategy for the efficient identification of transgenic calli accumulating transgene products. In: Dunwell JM, Wetten AC (eds). Transgenic plants. Humana, Totawa. Methods Mol Biol 847:67–74Google Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Ayako Nishizawa-Yokoi
    • 1
  • Hiroaki Saika
    • 1
  • Seiichi Toki
    • 1
    • 2
    Email author
  1. 1.Plant Genome Engineering Research UnitNational Institute of Agrobiological SciencesTsukubaJapan
  2. 2.Kihara Institute for Biological ResearchYokohama City UniversityYokohamaJapan

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