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.
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.
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DOI：
10.3389/fpls.2017.00403
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发表时间：
2017
影响因子：
5.6
通讯作者：
Navarre C
中科院分区：
文献类型：
--
作者：
Mercx S;Smargiasso N;Chaumont F;De Pauw E;Boutry M;Navarre C
植物或植物细胞可以用来生产抗体或疫苗等药理糖蛋白。然而，这些蛋白质携带带有植物典型残基的N-糖链[β(1，2)-木糖和核心α(1，3)-岩藻糖]，这会极大地影响蛋白质的免疫原性、过敏性或活性。植物特有的两种酶：β(1，2)-木糖转移酶(XylT)和α(1，3)-岩藻糖基转移酶(Fuct)。我们的目标是利用CRISPR/Cas9敲除烟草BY-2悬浮细胞中的两个XylT基因和四个FucT基因(共12个等位基因)。三个XylT和六个Fuct sgRNAs被设计成针对保守区。将编码sgRNAs、Cas9和一个可选择标记(BAR)的基因导入烟草白粉菌-2细胞，获得转基因株系，并用识别β(1，2)-木糖和α(1，3)-岩藻糖的抗体进行Western blotting分析其胞外和胞内蛋白补体。3个品系表现出β(1，2)-木糖和α(1，3)-岩藻糖的强烈还原，而2个品系完全不存在它们，表明基因完全失活。胞外蛋白质的质谱分析证实了这些碳水化合物的缺失。靶区域的PCR扩增和测序表明靶点之间有小的插入和/或缺失。KO系没有表现出任何特殊的形态，生长为野生型。用编码人IgG2抗体的基因转化一株KO细胞。在未进行糖工程的对照转化子中，IgG2的表达水平与对照转化子一样高。结果表明，糖基化产物中不存在β(1，2)-木糖或α(1，3)-岩藻糖，主要糖型为GnGn结构。这些数据代表了由N.tabacum-2细胞表达的药理蛋白糖基化人源化的重要一步。
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.