CRISPR/Cas9-mediated knockout of six glycosyltransferase genes in Nicotiana benthamiana for the production of recombinant proteins lacking ß-1,2-xylose and core α-1,3-fucose.

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 ß-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 ß-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 ß-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.

Genome Editing in Agriculture: Technical and Practical Considerations.

The advent of precise genome-editing tools has revolutionized the way we create new plant varieties. Three groups of tools are now available, classified according to their mechanism of action: Programmable sequence-specific nucleases, base-editing enzymes, and oligonucleotides. The corresponding techniques not only lead to different outcomes, but also have implications for the public acceptance and regulatory approval of genome-edited plants. Despite the high efficiency and precision of the tools, there are still major bottlenecks in the generation of new and improved varieties, including the efficient delivery of the genome-editing reagents, the selection of desired events, and the regeneration of intact plants. In this review, we evaluate current delivery and regeneration methods, discuss their suitability for important crop species, and consider the practical aspects of applying the different genome-editing techniques in agriculture.

Knockout of Glycosyltransferases in Nicotiana benthamiana by Genome Editing to Improve Glycosylation of Plant-Produced Proteins.

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 ß-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.

The Correlation Between DsRed mRNA Levels and Transient DsRed Protein Expression in Plants Depends on Leaf Age and the 5' Untranslated Region.

The yield of recombinant proteins in plants determines their economic competitiveness as a production platform compared to microbes and mammalian cells. The promoter, untranslated regions (UTRs) and codon usage can all contribute to the yield, but potential interactions among these components have not been examined in detail. Here the effect of two promoters (35SS and nos) and four 5'UTRs on the spatiotemporal expression of DsRed mRNA and the accumulation of DsRed protein during transient expression in tobacco (Nicotiana tabacum) mediated by Agrobacterium tumefaciens is investigated. The authors found that the mRNA levels peaked 2-3 days post-infiltration (dpi), and rapidly declined thereafter, whereas DsRed protein was first detected after ≈3 days and concentrations continued to increase until at least 5 dpi. This temporal decoupling of mRNA and protein expression was strongest in the older leaves, which also produced the lowest DsRed yields. The accumulation of DsRed linearly correlated with mRNA levels in all but the youngest leaves, where more DsRed was synthesized per mRNA molecule. This was the case for both promoters, although the nos promoter had a higher protein/mRNA ratio than the 35SS promoter. Furthermore, the type of 5'UTR affected DsRed protein accumulation by 50% starting from similar levels of mRNA. The authors concluded that DsRed mRNA levels are not the limiting factor for DsRed protein expression in plants, but that translation-associated processes such as initiation, elongation, and release are bottlenecks that should be addressed in future studies.