Engineering the N-glycosylation pathway of Nicotiana tabacum for molecular pharming using CRISPR/Cas9

Abstract

Molecular pharming in plants offers exciting possibilities to address global access to modern biologics. However, differences in the <i>N</i>-glycosylation pathway including the presence of β(1,2)-xylose and core α(1,3)-fucose can affect activity, potency and immunogenicity of plant-derived proteins. Successful glycoengineering approaches toward human-like structures with no changes in plant phenotype, growth, or recombinant protein expression levels have been reported for <i>Arabidopsis thaliana</i> and <i>Nicotiana benthamiana</i>. Such engineering of <i>N</i>-glycosylation would also be desirable for <i>Nicotiana tabacum</i>, which remains the crop of choice for recombinant protein pharmaceuticals required at massive scale and for manufacturing technology transfer to less developed countries. Here, we generated <i>N. tabacum</i> cv. SR-1 β(1,2)-xylosyltransferase (<i>XylT</i>) and α(1,3)-fucosyltransferase (<i>FucT</i>) knockout lines using CRISPR/Cas9 multiplex genome editing, targeting three conserved regions of the four <i>FucT</i> and two <i>XylT</i> genes. These two enzymes are responsible for generating non-human <i>N</i>-glycan structures. We confirmed full functional knockout of transformants by immunoblotting of total soluble protein by antibodies recognizing β(1,2)-xylose and core α(1,3)-fucose, mass spectrometry analysis of recombinantly produced VRC01, a broadly neutralizing anti-HIV-1 hIgG1 antibody, and Sanger sequencing of targeted regions of the putative transformants. These data represent an important step toward establishing <i>Nicotiana tabacum</i> as a biologics platform for Global Health.