We report a comprehensive toolkit that enables targeted, specific modification of monocot and dicot genomes using a variety of genome engineering approaches Our reagents, based on transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, are systematized for fast, modular cloning and accommodate diverse regulatory sequences to drive reagent expression Vectors are optimized to create either single or multiple gene knockouts and large chromosomal deletions Moreover, integration of geminivirus-based vectors enables precise gene editing through homologous recombination Regulation of transcription is also possible A Web-based tool streamlines vector selection and construction One advantage of our platform is the use of the Csy-type (CRISPR system yersinia) ribonuclease 4 (Csy4) and tRNA processing enzymes to simultaneously express multiple guide RNAs (gRNAs) For example, we demonstrate targeted deletions in up to six genes by expressing 12 gRNAs from a single transcript Csy4 and tRNA expression systems are almost twice as effective in inducing mutations as gRNAs expressed from individual RNA polymerase III promoters Mutagenesis can be further enhanced 25-fold by incorporating the Trex2 exonuclease Finally, we demonstrate that Cas9 nickases induce gene targeting at frequencies comparable to native Cas9 when they are delivered on geminivirus replicons The reagents have been successfully validated in tomato (Solanum lycopersicum), tobacco (Nicotiana tabacum), Medicago truncatula, wheat (Triticum aestivum), and barley (Hordeum vulgare)

A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants

While N-glycan synthesis in the endoplasmic reticulum (ER) is relatively well conserved in eukaryotes, N-glycan processing and O-glycan biosynthesis in the Golgi apparatus are kingdom specific and result in different oligosaccharide structures attached to glycoproteins in plants and mammals. With the prospect of using plants as alternative hosts to mammalian cell lines for the production of therapeutic glycoproteins, significant progress has been made towards the humanization of protein N-glycosylation in plant cells. To date, successful efforts in this direction have mainly focused on the targeted expression of therapeutic proteins, the knockout of plant-specific N-glycan-processing genes, and/or the introduction of the enzymatic machinery catalyzing the synthesis, transport and addition of human sugars. By contrast, very little attention has been paid until now to the O-glycosylation status of plant-made therapeutic proteins, which is surprising considering that hundreds of human proteins represent good candidates for Hyp-O glycosylation when produced in a plant expression system. This review describes protein N- and O-linked glycosylation in plants and highlights the limitations and advantages of plant-specific glycosylation on plant-made biopharmaceuticals.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1467-7652.2009.00497.x

Plant-specific glycosylation patterns in the context of therapeutic protein production.

Expression systems for therapeutic glycoprotein production.

http://eprints.covenantuniversity.edu.ng/2284/1/Obembe_JBA2011%20Copy.pdf

Advances in plant molecular farming.

Therapeutic glycoprotein production in mammalian cells

Summary
Compared with other plant expression systems used for pharmaceutical protein production, alfalfa offers the advantage of very homogeneous N-glycosylation. Therefore, this plant was selected for further attempts at glycoengineering. Two main approaches were developed in order to humanize N-glycosylation in alfalfa. The first was a knock-down of two plant-specific N-glycan maturation enzymes, β1,2-xylosyltransferase and α1,3-fucosyltransferases, using sense, antisense and RNA interference strategies. In a second approach, with the ultimate goal of rebuilding the whole human sialylation pathway, human β1,4-galactosyltransferase was expressed in alfalfa in a native form or in fusion with a targeting domain from N-acetylglucosaminyltransferase I, a glycosyltransferase located in the early Golgi apparatus in Nicotiana tabacum. Both knock-down and knock-in strategies strongly, but not completely, inhibited the biosynthesis of α1,3-fucose- and β1,2-xylose-containing glycoepitopes in transgenic alfalfa. However, recombinant human β1,4-galactosyltransferase activity in transgenic alfalfa completely prevented the accumulation of the Lewis a glycoepitope on complex N-glycans.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1467-7652.2008.00353.x

Down-regulated expression of plant-specific glycoepitopes in alfalfa

Summary
Biopharmaceutical glycoproteins produced in plants carry N-glycans with plant-specific residues core α(1,3)-fucose and β(1,2)-xylose, which can significantly impact the activity, stability and immunogenicity of biopharmaceuticals. In this study, we have employed sequence-specific transcription activator-like effector nucleases (TALENs) to knock out two α(1,3)-fucosyltransferase (FucT) and the two β(1,2)-xylosyltransferase (XylT) genes within Nicotiana benthamiana to generate plants with improved capacity to produce glycoproteins devoid of plant-specific residues. Among plants regenerated from N. benthamiana protoplasts transformed with TALENs targeting either the FucT or XylT genes, 50% (80 of 160) and 73% (94 of 129) had mutations in at least one FucT or XylT allele, respectively. Among plants regenerated from protoplasts transformed with both TALEN pairs, 17% (18 of 105) had mutations in all four gene targets, and 3% (3 of 105) plants had mutations in all eight alleles comprising both gene families; these mutations were transmitted to the next generation. Endogenous proteins expressed in the complete knockout line had N-glycans that lacked β(1,2)-xylose and had a significant reduction in core α(1,3)-fucose levels (40% of wild type). A similar phenotype was observed in the N-glycans of a recombinant rituximab antibody transiently expressed in the homozygous mutant plants. More importantly, the most desirable glycoform, one lacking both core α(1,3)-fucose and β(1,2)-xylose residues, increased in the antibody from 2% when produced in the wild-type line to 55% in the mutant line. These results demonstrate the power of TALENs for multiplexed gene editing. Furthermore, the mutant N. benthamiana lines provide a valuable platform for producing highly potent biopharmaceutical products.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1111/pbi.12403

Marc André D'Aoust

Papers

Down-regulated expression of plant-specific glycoepitopes in alfalfa

Multiplexed, targeted gene editing in Nicotiana benthamiana for glyco-engineering and monoclonal antibody production.