Multiplex CRISPR/Cas9-Mediated Editing of Seven Glycosyltransferase Homologs in Nicotiana benthamiana to Produce Stable, Cas9-Free, Glycoengineered Plants

Chetan Kaur¹Hayoung Song¹Myungjin Lee¹Seo-Young Kim¹Dong-Hoon Seo¹Hyangju Kang²Eun-Ju Sohn²Yidong Ran³Okjae Koo⁴Geung-Joo Lee^1*

• ¹Chungnam National University, Yuseong-gu, Republic of Korea
• ²BioApplications Inc., Pohang, Republic of Korea
• ³QI-Biodesign Technology Co Ltd, Beijing, China
• ⁴Kyungpook National University, Daegu, Republic of Korea

Producing therapeutic drugs in plants faces significant technical and regulatory challenges, mainly because of fundamental differences in the N-glycosylation pathways between plants and animals. 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 providing a robust genetic platform for future glycoengineering and recombinant protein production in plant-based systems. This study establishes the first fully Cas9-free, homozygous N. benthamiana lines edited at all seven glycosyltransferase loci, creating a stable foundation for future glycoengineering efforts.