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

CRISPR/Cas9-mediated OsXylT and OsFucT mutation caused the elimination of plant-specific ß1,2-xylose and a1,3-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, ß1,2-xylose and a1,3-fucose residues are transferred to N-glycan core structure on glycoproteins by ß1,2-xylosyltransferase (ß1,2-XylT) and a1,3-fucosyltransferase (a1,3-FucT), respectively. As an effort to use plants as a platform to produce biopharmaceuticals, the plant-specific N-glycan genes of rice (Oryza sativa), ß1,2-xylT (OsXylT) and a1,3-FucT (OsFucT), were knocked out using multiplex CRISPR/Cas9 technology. The double knock-out lines were found to have frameshift mutations by INDELs. Both ß1,2-xylose and a1,3-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 a1,3-fucose and ß1,2-xylose residues were not detected in the line, other plant-specific residues of ß1,3-galactose and a1,4-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.