Work

Highly efficient transgene‐free ErCas12a RNP‐protoplast genome editing and single‐cell regeneration in Nicotiana benthamiana for glyco‐engineering

Year: 2025

Type: article

Abstract: Summary Nicotiana benthamiana serves as a unique platform for biopharmaceutical production, offering advantages such as efficient and scalable protein synthesis. In addition, custom N‐glycans can be engineered on biopharmaceutical glycoproteins. Yet, plant‐native glycosyltransferases and glycoside hydrolases need to be removed to prevent undesired modifications of tailored N‐glycans. CRISPR‐based systems offer tremendous potential; however, the ploidy of the allotetraploid N. benthamiana can make genome editing challenging when attempting to knock out multiple undesired enzymes using transgenes. Here, we report a highly efficient CRISPR ribonucleoprotein (RNP)‐protoplast genome editing strategy for rapid, single‐generation platform engineering. We delineate the editing characteristics of ErCas12a RNPs and apply hydrogel protoplast immobilization to characterize true single‐cell regeneration. We target three β‐hexosaminidases responsible for removing terminal GlcNAc and/or GalNAc residues from N‐glycans and verify their inactivity via MALDI‐TOF‐MS N‐glycan analysis. We achieve up to 89.6%, 95.3% and 86.5% on‐target editing in the absence of off‐target editing. We demonstrate the feasibility of low cell density (10 4 ml −1 ) regeneration of individual CRISPR‐edited protoplasts in 12–14 weeks, carrying intended tetra‐allelic and/or deca‐allelic mutations while maintaining monoclonality. Despite the occurrence of genome duplications during the single‐cell regeneration of N. benthamiana protoplasts, high‐efficiency genome editing paired with shoot induction frequencies exceeding 89% facilitated the ubiquitous identification of desired β‐hexosaminidase mutants. We anticipate that this genome‐editing method will rapidly advance glyco‐engineering in polyploids such as N. benthamiana .

Source: Plant Biotechnology Journal

DOI: 10.1111/pbi.70141