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

Affiliation

Jung JW(#)(1)(2), Shin JH(#)(3)(4), Lee WK(3), Begum H(3), Min CH(5), Jang MH(3), Oh HB(5), Yang MS(1), Kim SR(6).
Author information:
(1)Department of Molecular Biology, Chonbuk National University, Jeonju, Jeollabuk-do, 561-756, Republic of Korea.
(2)Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK.
(3)Department of Life Science, Sogang University, Seoul, Republic of Korea.
(4)PhytoMab Co., Seoul, Republic of Korea.
(5)Department of Chemistry, Sogang University, Seoul, Republic of Korea.
(6)Department of Life Science, Sogang University, Seoul, Republic of Korea. [Email]
(#)Contributed equally

Abstract

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.