CRISPR/Cas9‐Mediated Knockouts of the ALG3 and GNTI in N. benthamiana 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.