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

First author: Julia Jansing; Affiliations: RWTH Aachen University (亚琛工业大学): Aachen, Germany

Corresponding author: Luisa Bortesi

Plants offer fast, flexible and easily scalable alternative platforms for the production of pharmaceutical proteins (药用蛋白质), but differences between plant and mammalian N‐linked glycans (葡聚糖), including the presence of β‐1,2‐xylose (β‐1,2‐木糖) and core α‐1,3‐fucose (α‐1,3‐海藻糖) residues in plants, can affect the activity, potency and immunogenicity (免疫原性) of plant‐derived proteins. Nicotiana benthamianais (本氏烟草) widely used for the transient expression of recombinant proteins (重组蛋白) so it is desirable to modify the endogenous N‐glycosylation (N-糖基化) 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 (α‐1,3‐海藻糖基转移酶) and β‐1,2‐xylosyltransferase (β‐1,2‐木糖基转移酶) activity, reflecting the mutation of six different genes. We confirmed the functional gene knockouts by Sanger sequencing and mass spectrometry‐ased N‐glycan analysis of endogenous proteins and the recombinant monoclonal antibody (单克隆抗体) 2G12. Furthermore, we compared the CD64‐inding affinity of 2G12 glycovariants (糖原变异体) produced in wild‐ ype 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.

植物提供药用蛋白制备的快速、灵活和易改良的平台，但植物与哺乳动物之间N-葡聚糖，包括植物特有的β‐1,2‐木糖和核心α‐1,3‐海藻糖残基严重影响了植物源蛋白的活性，应用潜力和免疫原性。本氏烟草广泛用于重组蛋白的转基因试验，比较适合用于修改内源N-糖基化以合成缺失β‐1,2‐木糖和核心α‐1,3‐海藻糖的复合N-葡聚糖。本文通过多重的CRISPR/Cas9基因组编辑技术构建了缺失植物特异性α‐1,3‐海藻糖基转移酶和β‐1,2‐木糖基转移酶活性的本氏烟生产株系，共突变了6个不同的基因。作者通过Sanger测序，基于内源蛋白的N-葡聚糖质谱鉴定及重组单克隆抗体2G12进一步确认了基因敲除的功能性。此外，作者还比较了野生本氏烟、新FX‐KO株系及中国仓鼠卵巢CHO细胞系中2G12糖原变异体的CD64结合亲和力，结果显示基因工程糖抗体与CHO细胞系产生的同类物功能类似。

通讯：Luisa Bortesi (https://www.maastrichtuniversity.nl/luisa.bortesi)

研究方向：利用CRISPR/Cas9基因组编辑技术改良植物作为生产平台。

doi: https://doi.org/10.1111/pbi.12981

Journal: Plant Biotechnology Journal

First Published: 03 July, 2018

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