Multiplex CRISPR/Cas9-mediated editing of seven glycosyltransferase homologs in Nicotiana benthamiana to produce stable, Cas9-free, glycoengineered plants

Introduction Plant-based systems hold great potential for producing therapeutic proteins, but differences in N-glycosylation pathways between plants and mammals present major technical and regulatory barriers. In particular, plant-specific α-1,3-fucosylation and β-1,2-xylosylation can generate immunogenic glycan structures, necessitating genome engineering to humanize plant glycosylation profiles. Methods We applied multiplex CRISPR/Cas9 genome editing in Nicotiana benthamiana to simultaneously target five α-1,3-fucosyltransferase genes and two β-1,2-xylosyltransferase genes. Resulting T 0 transformants were genotyped to assess mutagenesis, and subsequent T1 and T2 generations were screened to identify Cas9-free, homozygous plants. Growth and morphological characteristics were evaluated across germination, flowering, and seed production stages. Results Two T 0 lines (HL40 and HL64) exhibited successful edits in all seven target genes, with mutations consisting of single-base insertions and deletions up to 26 bp. In later generations, we identified stable Cas9-free homozygous lines containing mutations across all targeted loci. Three T 1 transformants with the highest number of homozygous alleles were selected to generate T 2 progeny. Heterozygous alleles segregated into homozygous genotypes in the T 2 generation, accompanied by confirmed loss of enzymatic activity. T 2 plants showed no detectable morphological or growth differences compared with wild-type plants, indicating no adverse phenotypic effects. Ultimately, we generated 12 independent Cas9-free, glycoengineered, homozygous lines. Discussion This work establishes the first N. benthamiana lines that are fully Cas9-free and homozygously edited at all seven key glycosyltransferase loci. These glycoengineered lines provide a stable and versatile genetic platform for future plant-based glycoengineering efforts and the production of recombinant therapeutic proteins.