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

N-glycosylation pathway is the one of post-translational mechanism and is known as highlconserved in eukaryotes. However, the process for complex-N-glycan modification is different between mammals and plants. In plant-specific manner, beta 1,2-xylose and alpha 1,3-fucose residues are transferred to N-glycan core structure on glycoproteins by beta 1,2-xylosyltransferase (beta 1,2-XylT) and alpha 1,3-fucosyltransferase (alpha 1,3-FucT), respectively. As an effort to use plants as a platform to produce biopharmaceuticals, the plant-specific N-glycan genes of rice (Oryza sativa), beta 1,2-xylT (OsXylT) and alpha 1,3-FucT (OsFucT), were knocked out using multiplex CRISPR/Cas9 technology. The double knock-out lines were found to have frameshift mutations by INDELs. Both beta 1,2-xylose and alpha 1,3-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 alpha 1,3-fucose and beta 1,2-xylose residues were not detected in the line, other plant-specific residues of beta 1,3-galactose and alpha 1,4-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.

Automated summary

The N-glycosylation pathway is highly conserved in eukaryotes as a post-translational mechanism, but the process for complex N-glycan modification differs between mammals and plants. Knocking out plant-specific N-glycan genes in rice using CRISPR/Cas9 technology revealed that double knockout of OsXylT and OsFucT was not sufficient to humanize N-glycan structure in rice.