TY - JOUR
T1 - Knockout of Glycosyltransferases in Nicotiana benthamiana by Genome Editing to Improve Glycosylation of Plant-Produced Proteins
AU - Jansing, Julia
AU - Bortesi, Luisa
N1 - © 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
PY - 2022
Y1 - 2022
N2 - 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.
AB - 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.
U2 - 10.1007/978-1-0716-2241-4_14
DO - 10.1007/978-1-0716-2241-4_14
M3 - Article
C2 - 35616867
SN - 1064-3745
VL - 2480
SP - 241
EP - 284
JO - Methods in Molecular Biology
JF - Methods in Molecular Biology
ER -