An efficient DNA- and selectable-marker-free genome-editing system using zygotes in rice View Full Text


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Article Info

DATE

2019-04

AUTHORS

Erika Toda, Narumi Koiso, Arika Takebayashi, Masako Ichikawa, Takatoshi Kiba, Keishi Osakabe, Yuriko Osakabe, Hitoshi Sakakibara, Norio Kato, Takashi Okamoto

ABSTRACT

Technology involving the targeted mutagenesis of plants using programmable nucleases has been developing rapidly and has enormous potential in next-generation plant breeding. Notably, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) (CRISPR-Cas9) system has paved the way for the development of rapid and cost-effective procedures to create new mutant populations in plants1,2. Although genome-edited plants from multiple species have been produced successfully using a method in which a Cas9-guide RNA (gRNA) expression cassette and selectable marker are integrated into the genomic DNA by Agrobacterium tumefaciens-mediated transformation or particle bombardment3, CRISPR-Cas9 integration increases the chance of off-target modifications4, and foreign DNA sequences cause legislative concerns about genetically modified organisms5. Therefore, DNA-free genome editing has been developed, involving the delivery of preassembled Cas9-gRNA ribonucleoproteins (RNPs) into protoplasts derived from somatic tissues by polyethylene glycol-calcium (PEG-Ca2+)-mediated transfection in tobacco, Arabidopsis, lettuce, rice6, Petunia7, grapevine, apple8 and potato9, or into embryo cells by biolistic bombardment in maize10 and wheat11. However, the isolation and culture of protoplasts is not feasible in most plant species and the frequency of obtaining genome-edited plants through biolistic bombardment is relatively low. Here, we report a genome-editing system via direct delivery of Cas9-gRNA RNPs into plant zygotes. Cas9-gRNA RNPs were transfected into rice zygotes produced by in vitro fertilization of isolated gametes12 and the zygotes were cultured into mature plants in the absence of selection agents, resulting in the regeneration of rice plants with targeted mutations in around 14-64% of plants. This efficient plant-genome-editing system has enormous potential for the improvement of rice as well as other important crop species. More... »

PAGES

1-6

References to SciGraph publications

  • 2006-03. Isolation of gametes and central cells from Oryza sativa L. in PLANT REPRODUCTION
  • 2013-12. Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system in PLANT METHODS
  • 2007-08. Establishment of an in vitro fertilization system in rice (Oryza sativa L.) in PLANTA
  • 2016-07. Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins in PLANT CELL REPORTS
  • 1985-12. High Efficiency Direct Gene Transfer to Plants in NATURE BIOTECHNOLOGY
  • 2013-03. Efficient genome editing in zebrafish using a CRISPR-Cas system in NATURE BIOTECHNOLOGY
  • 2015-12. Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease in GENOME BIOLOGY
  • 2015-11. DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins in NATURE BIOTECHNOLOGY
  • 1995-12. Electro-fused isolated wheat (Triticum aestivum L.) gametes develop into multicellular structures in PLANT CELL REPORTS
  • 2017-01-18. Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes in NATURE COMMUNICATIONS
  • 2015-08. Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice in PLANT MOLECULAR BIOLOGY
  • 2015-01-08. Regulatory uncertainty over genome editing in NATURE PLANTS
  • 2016-07. Optimization of CRISPR/Cas9 genome editing to modify abiotic stress responses in plants in SCIENTIFIC REPORTS
  • 1998-05. Differentiation of isolated wheat zygotes into embryos and normal plants in PLANTA
  • 2016-11-16. Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes in NATURE COMMUNICATIONS
  • Identifiers

    URI

    http://scigraph.springernature.com/pub.10.1038/s41477-019-0386-z

    DOI

    http://dx.doi.org/10.1038/s41477-019-0386-z

    DIMENSIONS

    https://app.dimensions.ai/details/publication/pub.1112971390

    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/30911123


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