Inactivation of the β (1, 2)-xylosyltransferase and the α (1, 3)-fucosyltransferase gene in rice (Oryza sativa) by multiplex CRISPR/Cas9 strategy.

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    • Source:
      Publisher: Springer Country of Publication: Germany NLM ID: 9880970 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1432-203X (Electronic) Linking ISSN: 07217714 NLM ISO Abbreviation: Plant Cell Rep Subsets: MEDLINE
    • Publication Information:
      Original Publication: Berlin ; New York : Springer, 1981-
    • Subject Terms:
    • Abstract:
      Key Message: CRISPR/Cas9-mediated OsXylT and OsFucT mutation caused the elimination of plant-specific β1,2-xylose and α1,3-fucose residues on glycoproteins in rice, which is the first report of OsXylT/OsFucT double KO mutation in rice. N-glycosylation pathway is the one of post-translational mechanism and is known as highly conserved in eukaryotes. However, the process for complex-N-glycan modification is different between mammals and plants. In plant-specific manner, β1,2-xylose and α1,3-fucose residues are transferred to N-glycan core structure on glycoproteins by β1,2-xylosyltransferase (β1,2-XylT) and α1,3-fucosyltransferase (α1,3-FucT), respectively. As an effort to use plants as a platform to produce biopharmaceuticals, the plant-specific N-glycan genes of rice (Oryza sativa), β1,2-xylT (OsXylT) and α1,3-FucT (OsFucT), were knocked out using multiplex CRISPR/Cas9 technology. The double knock-out lines were found to have frameshift mutations by INDELs. Both β1,2-xylose and α1,3-fucose residues in the lines were not detected in Western blot analysis. Consistently, there was no peak corresponding to the N-glycans in MALDI-TOF/MS analysis. Although α1,3-fucose and β1,2-xylose residues were not detected in the line, other plant-specific residues of β1,3-galactose and α1,4-fucose were detected. Thus, we suggest that each enzymes working on the process for complex N-glycan biosynthesis might independently act in rice, hence the double knock-out of both OsXylT and OsFucT might be not enough to humanize N-glycan structure in rice.
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    • Grant Information:
      PJ013659 National Academy of Agricultural Science; 2020R1A2C1014133 National research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (KR); NRF-2018R1A6A3A01011033 the National Research Foundation of Korea (NRF) funded by the Ministry of Education; 714001-7 Ministry of Agriculture, Food and Rural Affairs
    • Contributed Indexing:
      Keywords: Lewis-a-epitope; N-glycosylation; Plant specific N-glycan; Rice; α1,3-fucosyltransferase; β1,2-xylosyltransferase
    • Accession Number:
      0 (Epitopes)
      0 (Plant Proteins)
      0 (Polysaccharides)
      EC 2.4.1.- (Fucosyltransferases)
      EC 2.4.1.152 (galactoside 3-fucosyltransferase)
      EC 2.4.2.- (Pentosyltransferases)
    • Publication Date:
      Date Created: 20210206 Date Completed: 20210625 Latest Revision: 20231213
    • Publication Date:
      20231215
    • Accession Number:
      10.1007/s00299-021-02667-8
    • Accession Number:
      33547931