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

• Published in: Plant cell reports Vol. 40; no. 6; pp. 1025 - 1035
• Main Authors: Jung, Jae-Wan, Shin, Jun-Hye, Lee, Won-Kyung, Begum, Hilal, Min, Chan-Hong, Jang, Mi-Hwa, Oh, Han-Bin, Yang, Moon-Sik, Kim, Seong-Ryong
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.06.2021; Springer Nature B.V
• Subjects: Biomedical and Life Sciences; Biosynthesis; Biotechnology; Cell Biology; CRISPR; CRISPR-Cas Systems; Epitopes - genetics; Eukaryotes; Frameshift mutation; Fucose; Fucosyltransferases - genetics; Galactose; Gene Editing - methods; Gene Silencing; Glycan; Glycoproteins; Glycosylation; Green Genome Editing Innovations: Precision and Safety; Inactivation; Life Sciences; Multiplexing; Mutation; N-glycans; Original Article; Oryza - genetics; Oryza sativa; Pentosyltransferases - genetics; Plant Biochemistry; Plant Proteins - genetics; Plant Sciences; Plants, Genetically Modified - genetics; Polysaccharides; Polysaccharides - genetics; Polysaccharides - immunology; Polysaccharides - metabolism; Post-translation; Residues; Rice; UDP Xylose-Protein Xylosyltransferase; Xylose; glycosylation; α1,3-fucosyltransferase; Plant specific N-glycan; β1,2-xylosyltransferase; Lewis-a-epitope; Rice; N-glycosylation
• ISSN: 0721-7714; 1432-203X
• DOI: 10.1007/s00299-021-02667-8

Key message CRISPR/Cas9-mediated OsXylT and OsFucT mutation caused the elimination of plant-specific β 1,2-xylose and α 1,3-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.