CRISPR/Cas9-mediated knockout of six glycosyltransferase genes in Nicotiana benthamiana for the production of recombinant proteins lacking β-1,2-xylose and core α-1,3-fucose.

Plants offer fast, flexible and easily scalable alternative platforms for the production of pharmaceutical proteins, but differences between plant and mammalian N-linked glycans, including the presence of β-1,2-xylose and core α-1,3-fucose residues in plants, can affect the activity, potency and immunogenicity of plant-derived proteins. Nicotiana benthamiana is widely used for the transient expression of recombinant proteins so it is desirable to modify the endogenous N-glycosylation machinery to allow the synthesis of complex N-glycans lacking β-1,2-xylose and core α-1,3-fucose. Here, we used multiplex CRISPR/Cas9 genome editing to generate N. benthamiana production lines deficient in plant-specific α-1,3-fucosyltransferase and β-1,2-xylosyltransferase activity, reflecting the mutation of six different genes. We confirmed the functional gene knockouts by Sanger sequencing and mass spectrometry-based N-glycan analysis of endogenous proteins and the recombinant monoclonal antibody 2G12. Furthermore, we compared the CD64-binding affinity of 2G12 glycovariants produced in wild-type N. benthamiana, the newly generated FX-KO line, and Chinese hamster ovary (CHO) cells, confirming that the glyco-engineered antibody performed as well as its CHO-produced counterpart.