CRISPR/Cas9‐Mediated Knockouts of the <i>ALG3</i> and <i>GNTI</i> in <i>N. benthamiana</i> and Their Application to Pharmaceutical Production

N-Glycosylation critically influences the efficacy, safety and pharmacokinetic properties of biopharmaceuticals. Plant expression platforms offer multiple advantages for the production of N-glycosylated proteins, but their use is impeded by the presence of plant-specific N-glycan epitopes, which raise concerns of possible immunogenicity to humans. In this study, N-glycoengineered Nicotiana benthamiana plants that produce more homogeneous N-glycans without plant-specific epitopes were generated using multiplex CRISPR/Cas9 genome editing. To achieve this N-glycosylation modification, ALG3 and GNTI, which function in N-glycosylation processes in the ER and Golgi, respectively, were characterised, and single- and double-knockout mutant plants were generated. Comprehensive N-glycan profiling revealed that while the ALG3-knockout plant line, alg3, maintained predominantly plant-specific N-glycans with altered mannose content, the GNTI-knockout line, gntI, produced exclusively high-mannose-type N-glycans. Notably, the alg3gntI double-knockout mutants yielded highly uniform trimannosidic N-glycans. To validate our N-glycoengineering approach, we expressed two model biopharmaceuticals, Varlilumab (anti-CD27 antibody) and β-glucocerebrosidase (GCase), in wild-type and mutant plants. While the antibodies expressed in alg3 and alg3gntI showed a certain level of glucosylated endoplasmic reticulum-type N-glycan, with increased non-N-glycosylated heavy chains, GCase exhibited a more consistent N-glycosylation profile, reflecting the engineered N-glycosylation pathway. Our findings provide valuable insights into N-glycan biosynthesis in N. benthamiana and demonstrate the potential of targeted N-glycoengineering for producing biopharmaceuticals with more homogeneous mannose-type N-glycan profiles.