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      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

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          Summary

          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 β‐1,2‐xylose and core α‐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 β‐1,2‐xylose and core α‐1,3‐fucose. Here, we used multiplex CRISPR/Cas9 genome editing to generate N. benthamiana production lines deficient in plant‐specific α‐1,3‐fucosyltransferase and β‐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 FXKO line, and Chinese hamster ovary ( CHO) cells, confirming that the glyco‐engineered antibody performed as well as its CHO‐produced counterpart.

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          Most cited references47

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          Genome-wide insertional mutagenesis of Arabidopsis thaliana.

          J Alonso (2003)
          Over 225,000 independent Agrobacterium transferred DNA (T-DNA) insertion events in the genome of the reference plant Arabidopsis thaliana have been created that represent near saturation of the gene space. The precise locations were determined for more than 88,000 T-DNA insertions, which resulted in the identification of mutations in more than 21,700 of the approximately 29,454 predicted Arabidopsis genes. Genome-wide analysis of the distribution of integration events revealed the existence of a large integration site bias at both the chromosome and gene levels. Insertion mutations were identified in genes that are regulated in response to the plant hormone ethylene.
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            Targeting DNA double-strand breaks with TAL effector nucleases.

            Engineered nucleases that cleave specific DNA sequences in vivo are valuable reagents for targeted mutagenesis. Here we report a new class of sequence-specific nucleases created by fusing transcription activator-like effectors (TALEs) to the catalytic domain of the FokI endonuclease. Both native and custom TALE-nuclease fusions direct DNA double-strand breaks to specific, targeted sites.
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              Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9.

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                Author and article information

                Contributors
                luisa.bortesi@maastrichtuniversity.nl
                Journal
                Plant Biotechnol J
                Plant Biotechnol. J
                10.1111/(ISSN)1467-7652
                PBI
                Plant Biotechnology Journal
                John Wiley and Sons Inc. (Hoboken )
                1467-7644
                1467-7652
                11 August 2018
                February 2019
                : 17
                : 2 ( doiID: 10.1111/pbi.2019.17.issue-2 )
                : 350-361
                Affiliations
                [ 1 ] Department for Molecular Biotechnology RWTH Aachen University Aachen Germany
                [ 2 ]Present address: Indiana Biosciences Research Institute Indianapolis IN USA
                [ 3 ]Present address: Aachen‐Maastricht Institute for Biobased Materials Maastricht University Geleen The Netherlands
                Author notes
                [*] [* ] Correspondence (Tel +31 (0) 433882568; email luisa.bortesi@ 123456maastrichtuniversity.nl )
                Author information
                http://orcid.org/0000-0003-2339-0083
                http://orcid.org/0000-0002-7380-9228
                Article
                PBI12981
                10.1111/pbi.12981
                6335070
                29969180
                72a38df6-834f-4db5-a907-f5c2975547bd
                © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

                This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 27 March 2018
                : 11 June 2018
                : 25 June 2018
                Page count
                Figures: 3, Tables: 3, Pages: 12, Words: 11311
                Funding
                Funded by: ERC
                Award ID: 269110
                Funded by: German federal and state governments
                Categories
                Research Article
                Research Articles
                Custom metadata
                2.0
                pbi12981
                February 2019
                Converter:WILEY_ML3GV2_TO_NLMPMC version:version=5.5.4 mode:remove_FC converted:16.01.2019

                Biotechnology
                crispr/cas9,gene knockout,α‐1,3‐fucosyltransferase,β‐1,2‐xylosyltransferase,glyco‐engineering,molecular farming

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