@article{0dbae97d1bbf463ea65b94990580e953,
title = "CRISPR/Cas9-mediated knockout of six glycosyltransferase genes in Nicotiana benthamiana for the production of recombinant proteins lacking β-1,2-xylose and core α-1,3-fucose",
abstract = "Plants offer fast, flexible and easily scalable alternative platforms for the production of pharmaceutical proteins, but differences between plant and mammalian N-linked glycans, including the presence of beta-1,2-xylose and core alpha-1,3-fucose residues in plants, can affect the activity, potency and immunogenicity of plant-derived proteins. Nicotiana benthamiana is widely used for the transient expression of recombinant proteins so it is desirable to modify the endogenous N-glycosylation machinery to allow the synthesis of complex N-glycans lacking beta-1,2-xylose and core alpha-1,3-fucose. Here, we used multiplex CRISPR/Cas9 genome editing to generate N. benthamiana production lines deficient in plant-specific alpha-1,3-fucosyltransferase and beta-1,2-xylosyltransferase activity, reflecting the mutation of six different genes. We confirmed the functional gene knockouts by Sanger sequencing and mass spectrometry-based N-glycan analysis of endogenous proteins and the recombinant monoclonal antibody 2G12. Furthermore, we compared the CD64-binding affinity of 2G12 glycovariants produced in wild-type N. benthamiana, the newly generated FX-KO line, and Chinese hamster ovary (CHO) cells, confirming that the glyco-engineered antibody performed as well as its CHO-produced counterpart.",
keywords = "CRISPR/Cas9, gene knockout, alpha-1,3-fucosyltransferase, beta-1,2-xylosyltransferase, glyco-engineering, molecular farming, HUMAN MONOCLONAL-ANTIBODY, N-GLYCANS, GUIDE RNA, GLYCOSYLATION, ARABIDOPSIS, PLANTS, ENDONUCLEASE, GLYCOPROTEIN, MUTAGENESIS, GENERATION",
author = "Julia Jansing and Markus Sack and Augustine, {Sruthy Marie} and Rainer Fischer and Luisa Bortesi",
note = "Funding Information: This work was partly supported by the ERC Advanced Grant Future-Pharma (269110) and by the Excellence Initiative of the German federal and state governments. The authors would like to acknowledge the help of the following colleagues: Flora Schuster (plant tissue culture), Ibrahim Al Amedi (greenhouse), Leonie Fritsch (NGS), Alexander Boes (protein purification), Claudia Hansen and Raphael Soeur (robot-assisted PCR clean-up, sequencing), Thomas Rademacher (2G12 construct). HBT-pcoCas9 was a gift from Jen Sheen (Addgene plasmid #52254). We thank Friedrich Altmann and Clemens Gruenwald-Gruber (BOKU Vienna) for the N-glycan analysis and Richard M. Twyman for editing the manuscript. The authors declare no conflict of interest. Funding Information: This work was partly supported by the ERC Advanced Grant Future-Pharma (269110) and by the Excellence Initiative of the German federal and state governments. The authors would like to acknowledge the help of the following colleagues: Flora Schuster (plant tissue culture), Ibrahim Al Amedi (greenhouse), Leonie Fritsch (NGS), Alexander Boes (protein purification), Claudia Hansen and Raphael Soeur (robot-assisted PCR clean-up, sequencing), Thomas Rademacher (2G12 construct). HBT-pco-Cas9 was a gift from Jen Sheen (Addgene plasmid #52254). We thank Friedrich Altmann and Clemens Gruenwald-Gruber (BOKU Vienna) for the N-glycan analysis and Richard M. Twyman for editing the manuscript. The authors declare no conflict of interest. Publisher Copyright: {\textcopyright} 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.",
year = "2019",
month = feb,
doi = "10.1111/pbi.12981",
language = "English",
volume = "17",
pages = "350--361",
journal = "Plant Biotechnology Journal",
issn = "1467-7644",
publisher = "Wiley-Blackwell",
number = "2",
}