Highly efficient DNA-free MAD7 gene editing in N. benthamiana protoplasts with subsequent monoclonal single-cell regeneration for helminth glycan engineering

N. benthamiana serves as a unique platform to produce helminth glycoproteins with uncommon glycan motifs. Helminth glycoproteins with tailormade N-glycans can be used as vaccines against helminths and additionally have the potential to treat inflammatory disorders. To facilitate this, plant-native glycosyltransferases and glycoside hydrolases should be knocked-out to prevent undesired N-glycan modifications and achieve more homogenous N-glycans. CRISPR-based editing systems offer tremendous potential in knocking out undesired enzymes. However, the high ploidy levels of the allotetraploid N. benthamiana make genome editing challenging when attempting to remove (multiple) undesired enzymes using DNA-encoded delivery of CRISPR cargoes. Here, we report the development of a highly efficient CRISPR ribonucleoprotein (RNP)-protoplast gene editing strategy for rapid one-generation platform engineering. Using MAD7 RNPs and PEG-mediated transfections of protoplast populations, we target three glycoside hydrolase (β-hexosaminidases) genes responsible for the removal of terminal GlcNAc and/or GalNAc residues from N-glycans. We achieved up to 87.5%, 95.6%, and 83.8% on-target editing of Nbhexo1, Nbhexo2, and Nbhexo3, respectively. Using protoplast immobilization, we demonstrate the feasibility of low cell density (104/ml) regeneration of individual CRISPR-edited protoplasts carrying tetra-allelic mutations at the target sites whilst maintaining monoclonality. Deep sequencing of predicted off-targets in protoplast populations revealed the absence of off-target editing. Through MALDI-TOF-MS N-glycan analysis, we demonstrate that all generated Nbhexo lines are functional knock-outs. We anticipate this gene-editing method to rapidly advance glycoengineering in polyploids such as N. benthamiana, while paving the way for trait engineering in other economically relevant polyploids.