Inactivation of the β(1,2)-xylosyltransferase and the α(1,3)-fucosyltransferase genes in Nicotiana tabacum BY-2 Cells by a Multiplex CRISPR/Cas9 Strategy Results in Glycoproteins without Plant-Specific Glycans

Abstract

Plants or plant cells can be used to produce pharmacological glycoproteins such as antibodies or vaccines. However these proteins carry N-glycans with plant-typical residues [β(1,2)-xylose and core α(1,3)-fucose], which can greatly impact the immunogenicity, allergenicity, or activity of the protein. Two enzymes are responsible for the addition of plant-specific glycans: β(1,2)-xylosyltransferase (XylT) and α(1,3)-fucosyltransferase (FucT). Our aim consisted of knocking-out two XylT genes and four FucT genes (12 alleles altogether) in Nicotiana tabacum BY-2 suspension cells using CRISPR/Cas9. Three XylT and six FucT sgRNAs were designed to target conserved regions. After transformation of N. tabacum BY-2 cells with genes coding for sgRNAs, Cas9, and a selectable marker (bar), transgenic lines were obtained and their extracellular as well as intracellular protein complements were analyzed by Western blotting using antibodies recognizing β(1,2)-xylose and α(1,3)-fucose. Three lines showed a strong reduction of β(1,2)-xylose and α(1,3)-fucose, while two lines were completely devoid of them, indicating complete gene inactivation. The absence of these carbohydrates was confirmed by mass spectrometry analysis of the extracellular proteins. PCR amplification and sequencing of the targeted region indicated small INDEL and/or deletions between the target sites. The KO lines did not show any particular morphology and grew as the wild-type. One KO line was transformed with genes encoding a human IgG2 antibody. The IgG2 expression level was as high as in a control transformant which had not been glycoengineered. The IgG glycosylation profile determined by mass spectrometry confirmed that no β(1,2)-xylose or α(1,3)-fucose were present on the glycosylation moiety and that the dominant glycoform was the GnGn structure. These data represent an important step toward humanizing the glycosylation of pharmacological proteins expressed in N. tabacum BY-2 cells.

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

“…Ever since scientists became interested in using plants as a production platform for recombinant proteins, there was an awareness that the N-glycans produced in plants could influence the behaviour of pharmaceutical glycoproteins in vivo (Parekh et al, 1989). Consequently, there has a been a lot of effort to modify the N-glycosylation pathway in a variety of plant species, using T-DNA insertion mutants (Strasser et al, 2004), RNAi (Cox et al, 2006;Sourrouille et al, 2008;Strasser et al, 2008), chemical mutagenesis (Weterings and Van Eldik, 2013) and, more recently, targeted nucleases (Hanania et al, 2017;Li et al, 2016;Mercx et al, 2017). N. benthamiana is of particular interest for molecular farming because the transient expression of proteins is fast and yields of more than 2 mg/g fresh leaf weight have been reported for antibodies (Zischewski et al, 2015).…”

“…In N. benthamiana , the two XylT genes and two of the five FucT genes were knocked out with TALENs to completely eliminate the β‐1,2‐xylosyltransferase activity and reduce core α‐1,3‐fucosyltransferase activity by 60%, the latter confirming that the other FucT genes would need to be targeted to eliminate FucT activity completely (Li et al ., ). Two recent publications describe the successful multiplex CRISPR‐mediated knockout of four or five FucT genes and two XylT genes in Nicotiana tabacum BY‐2 suspension cells (Hanania et al ., ; Mercx et al ., ), resulting in the absence of α‐1,3‐fucose and β‐1,2‐xylose in endogenous and recombinant proteins, and no differences in cell morphology or growth between the knockout and wild‐type lines. However, to the best of our knowledge, the comprehensive knockout of all active XylT and FucT genes in intact N. benthamiana plants has not been achieved thus far.…”

Transgene Biocontainment Strategies for Molecular Farming

“…Plant production is also free from human pathogensa major concern in mammalian cell culture production systems -and free from endotoxins, which are a risk in bacterial systems (Commandeur et al, 2002). Protein glycosylation patterns can be manipulated in plants, including to produce 'humanized' glycosylation patterns (Hanania et al, 2017;Mercx et al, 2017). This is important for complex glycoproteins such as monoclonal antibodies or membrane proteins as glycosylation can affect protein stability, subcellular targeting, biological activity, and immunogenicity (He et al, 2014).…”

Sialoglycans and genetically engineered plants

“…Different approaches have been used to modify the N-glycosylation pathway in different plant species, using T-DNA insertion mutants [11], RNA interference (RNAi) [12][13][14], chemical mutagenesis [15], and targeted nuclease [16][17][18] approaches. N. benthamiana finds importance in molecular farming as the transient expression of proteins is fast and yields antibodies [19] by different transient expression systems, including the MagnICON system [20], the pEAQ vector [21], and the pTRA vector [22].…”