ArticlePlant biotechnology journal2026
Highly efficient transgene-free ErCas12a RNP-protoplast genome editing and single-cell regeneration in Nicotiana benthamiana for glyco-engineering.
Article in Plant biotechnology journal, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 6 papers.
What it found
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The trial behind it
Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.
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Who cites it
6 citing papers in PubMed.
- Organ-on-a-Chip and Microfluidic Plant Cell Culture Systems: The Next Frontier for Controlled Secondary Metabolite Production and Real-Time Metabolomic Monitoring.Plants (Basel, Switzerland) · 2026Review
- Gene Editing of Nicotiana benthamiana Architecture for Space-Efficient Production of Recombinant Proteins in Closed Environments.Plant biotechnology journal · 2026Article
- Beyond GMOs: transgene-free gene-edited crops for global food security.Frontiers in plant science · 2026Review
- Genetic transformation and genome editing of flax (Frontiers in plant science · 2026Review
- Transgene-free genome editing in citrus and poplar trees using positive and negative selection markers.Plant cell reports · 2025Article
- Advances and challenges in plant N-glycoengineering: when fucosylation matters.Frontiers in plant science · 2025Review
Corrections and comments
PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.
Authors and funding
9 authors.
Funding
Abstract
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
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.