CRISPR/Cas9‐mediated knockout of a prolyl‐4‐hydroxylase subfamily in Nicotiana benthamiana using DsRed2 for plant selection

Abstract

The properties of host plants used for molecular farming can be modified by CRISPR/Cas9 genome editing to improve the quality and yield of recombinant proteins. However, it is often necessary to target multiple genes simultaneously, particularly when using host plants with large and complex genomes. This is the case for Nicotiana benthamiana, an allotetraploid relative of tobacco frequently used for transient protein expression. A multiplex genome editing system incorporating the DsRed2 fluorescent marker for the identification and selection of transgenic plants was established. As proof of principle, NbP4H4 was targeted encoding a prolyl-4-hydroxylase involved in protein O-linked glycosylation. Using preselected gRNAs with efficiencies confirmed by transient expression, transgenic plant lines with knockout mutations in all four NbP4H4 genes were obtained. Leaf fluorescence was then used to screen for the absence of the SpCas9 transgene in T1 plants, and transgene-free lines with homozygous or biallelic mutations were identified. The analysis of plant-produced recombinant IgA1 as a reporter protein revealed changes in the number of peptides containing hydroxyproline residues and pentoses in the knockout plants. The selection of efficient gRNAs combined with the DsRed2 marker reduces the effort needed to generate N. benthamiana mutants and simplifies the screening processes to obtain transgene-free progeny.

Differential prolyl hydroxylation by six Physcomitrella prolyl-4 hydroxylases

“…However, functional compensation apart from transcriptional upregulation is also considerable since all p4h genes are expressed in protonema under standard growth conditions. A possible rebalancing effect by the remaining P4Hs was also reported after a quadruple KO of the Nicotiana P4H4 subset, where the KO led to a reduced abundance of the unmodified version of the hinge region from a recombinant IgA1 antibody and to an increased fraction of peptides O -glycosylated with pentoses (Uetz et al, 2022). In our data, the abundance of the prolyl-hydroxylated form of the peptide GANYAITFCPTVTPVAK was strongly reduced in the P4H5 KO line, indicating that in this case loss of P4H5 is hardly compensated.…”

Protein Glycosylation in Bryophytes Differs Subtly from That in Vascular Plants

“…The remarkable fact now is that apparently, the sites of mucin-type O-glycosylation of mammalian proteins are also the sites prone to be modified by prolyl-4-hydroxylase and then by arabinosyl-transferase as exemplified by human erythropoietin expressed in the moss P. patens [38] or human IgA1 [39]. Hardly surprising, the same holds true for N. benthamiana with the only, albeit technologically relevant difference, that several prolyl-4-hydroxylase are redundantly at work in vascular plants [39,40], whereas knock-out of just one paralogous gene sufficed to suppress erythropoietin oxidation in P. patens [41].…”

Implications of O-glycan modifications in the hinge region of a plant-produced SARS-CoV-2-IgA antibody on functionality

“…Their endogenous N- and O-glycosylation patterns differ from humans, which has hampered the broad application of the expression system to produce human therapeutics ( Strasser et al, 2021 ). While there has been considerable effort in glycoengineering popular expression hosts such as Nicotiana benthamiana , N. tabacum , tobacco BY2 cells and the moss Physcomitrella patens towards humanized- and customized N-glycosylation ( Bakker et al, 2001 ; Koprivova et al, 2004 ; Strasser et al, 2008 ; Kallolimath et al, 2016 ; Limkul et al, 2016 ; Mercx et al, 2017 ; Jansing et al, 2019 ; Bohlender et al, 2020 ; Herman et al, 2021 ; Göritzer et al, 2022 ; Kogelmann et al, 2023 ) limited attention was cast towards modulating the plant endogenous O-glycosylation pathway ( Castilho et al, 2012 ; Yang et al, 2012 ; Parsons et al, 2013 ; Dicker et al, 2016 ; Ramírez-Alanis et al, 2018 ; Mócsai et al, 2021 ; Uetz et al, 2022 ). Plant-endogenous HyP and further modifications with pentoses (arabinoses) were found in several recombinantly produced proteins such as IgA1, MUC1, EPO-Fc, and Ara h 2 ( Karnoup et al, 2005 ; Weise et al, 2007 ; Pinkhasov et al, 2011 ; Castilho et al, 2012 ; Yang et al, 2012 ; Üzülmez et al, 2021 ).…”

“…Several P4H members from Arabidopsis thaliana , tobacco, and Solanum lycopersicum have been identified and characterized ( Yuasa et al, 2005 ; Kalaitzis et al, 2010 ; Velasquez et al, 2011 ). This paved the way for the discovery of similar enzymes in plant species utilized in plant molecular farming, such as N. benthamiana or P. patens ( Parsons et al, 2013 ; Mócsai et al, 2021 ; Uetz et al, 2022 ). While CRISPR/Cas9-mediated knockout of the P4H4 subfamily in N. benthamiana resulted in a noticeable change of the O-glycosylation pattern in the hinge region of a recombinant IgA1 antibody, the presence of considerable amounts of HyP residues suggested that other P4H enzymes are mainly responsible for proline oxidation in the IgA1 hinge region ( Uetz et al, 2022 ).…”