Knockout of Glycosyltransferases in Nicotiana benthamiana by Genome Editing to Improve Glycosylation of Plant-Produced Proteins.
Allotetraploid genome
CRISPR/Cas9
Genome editing
Glycosyltransferase
Nicotiana benthamiana
Targeted gene knockout
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2022
2022
Historique:
entrez:
26
5
2022
pubmed:
27
5
2022
medline:
31
5
2022
Statut:
ppublish
Résumé
Plants are excellent production hosts for the in vivo synthesis of complex glycosylated proteins such as antibodies. The plant N-glycosylation machinery is largely similar to that found in humans and other mammalian organisms, which is an advantage in comparison to microbial production systems in particular. However, there are some differences in the identity and chemical linkage of the sugars that plants and mammals use to build their N-glycans. These differences can affect important properties of glycosylated proteins produced recombinantly in plants. Here we describe the complete procedure of multiplex targeted gene knockout with CRISPR/Cas9 in Nicotiana benthamiana in order to eliminate the undesirable sugars α-1,3-fucose and β-1,2-xylose from the plant N-glycans. The workflow includes target gene identification, guide RNA design and testing, plant transformation, and the analysis of the regenerated transgenic plants by Sanger sequencing, immunoblot, and mass-spectrometric analysis of recombinant and endogenous proteins.
Identifiants
pubmed: 35616867
doi: 10.1007/978-1-0716-2241-4_14
doi:
Substances chimiques
Plant Proteins
0
Polysaccharides
0
Recombinant Proteins
0
Xylose
A1TA934AKO
Glycosyltransferases
EC 2.4.-
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
241-284Informations de copyright
© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
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