Overcoming N-glycosylation challenges through multiplex CRISPR/Cas9-mediated editing of seven glycosyltransferase homologs in Nicotiana benthamiana

Abstract Utilizing plants for the manufacturing of therapeutic drugs for human and animal disease treatment faces technical and regulatory challenges because of disparities in the N-glycosylation pathway between animals and plants. The key challenge involves differences in the post-translational modification machinery in the N-glycosylation pathway. We used multiplex CRISPR/Cas9 genome editing to target five α-1,3-fucosyltransferase and two β-1,2-xylosyltransferase genes to modify N-glycosylation in Nicotiana benthamiana. We obtained two T₀ transformants, HL40 and HL64, which exhibited successful mutagenesis in all seven target genes. Mutations in these genes resulted from deletions ranging from a single base to up to 26 bases, and single-base insertions. In subsequent generations, stable Cas9-free homozygous lines exhibiting mutations in all seven genes were identified. Three Cas9-free T₁ transformants with the highest number of homozygous mutations were selected to generate T₂ transformants. Heterozygous alleles in the T₁ transformants segregated into homozygous genotypes in the T₂ generation with a confirmed loss of enzyme activity. The morphology and growth rate of the T₂ transformants showed no notable variations compared to those of the wild type throughout germination, flowering, and seed production, indicating the absence of discernible side effects from the mutations. Our experiment yielded 12 Cas9-free, glycoengineered, homozygous plants suitable for plant-based recombinant protein production in molecular farming systems, eliminating regulatory and immunogenic concerns.