Assessment of the CRISPR technology in tobacco for therapeutic protein production

评估烟草中用于治疗性蛋白质生产的 CRISPR 技术

• 批准号: 478461-2015
• 负责人: Ro, DaeKyun
• 金额: $ 1.82万
• 依托单位: University of Calgary
• 依托单位国家: 加拿大
• 项目类别: Engage Grants Program
• 财政年份: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=579527
• 关键词: Assessment; CRISPR; technology; tobacco; therapeutic

The PlantForm Corporation has developed tobacco as a platform to produce therapeutic antibodies. Plant has an advantage over microbes in manufacturing recombinant antibodies because plant can catalyze N-glycosylations to add sugars on the proteins as mammalian cells do. However, two plant-unique sugars, namely fucose and xylose, are also incorporated to the recombinant antibodies. These two sugars are undesirable in the antibodies as they can cause allergic reactions in human. These sugars are added to the antibodies by fucosyl and xylosyl transferase enzymes in plant. To overcome this problem, the PlantForm has utilized the RNA interference (RNAi) to reduce fucosyl and xylosyl transferase mRNAs, but a small percentage (<1%) of the antibodies still possess these undesirable sugar groups. It is therefore important to entirely eliminate the fucosyl and xylosyl transferase activities in tobacco by other means. This proposal focuses on assessing the possibility of generating a mutant tobacco, deficient of fucosyl and xylosyl transferase activities, using the CRISPR (clustered regularly interspaced short palindromic repeats) technology. CRISPR is a newly developed molecular method which can generate deletion-mutations on targeted loci in eukaryotic genomes. Genomics analysis of the tobacco plant identified three pairs of highly homologous fucosyl and xylosyl transferases (one pair of xylosyl transferase and two pairs of fucosyl transferases). These three gene pairs will be targeted for deletions by CRISPR. Specifically, we will constructs multiple CRISPR components in plasmids and transiently express these in tobacco leaves. Genomic DNAs will be isolated from the leaves, and targeted loci will be amplified by PCR. Using sequencing and bioinformatics, presence and frequency of the deletion mutations in the amplified loci will be determined for each CRISPR construct. These results will provide knowledge to the PlantForm about whether the genes encoding fucosyl and xylosyl transferase can be permanently deleted by CRISPR and what CRISPR constructs operate most efficiently.

PlantForm 公司开发了烟草作为生产治疗性抗体的平台。工厂有 在生产重组抗体方面比微生物有优势，因为植物可以催化 像哺乳动物细胞一样，通过 N-糖基化在蛋白质上添加糖。然而，两种植物特有的糖， 即岩藻糖和木糖，也被掺入重组抗体中。这两种糖是 抗体中不受欢迎，因为它们会引起人类过敏反应。这些糖被添加到 植物中岩藻糖基和木糖基转移酶产生的抗体。为了解决这个问题，PlantForm 利用 RNA 干扰 (RNAi) 来减少岩藻糖基和木糖基转移酶 mRNA，但比例很小 (<1%) 的抗体仍然具有这些不需要的糖基团。因此，重要的是要完全 通过其他方式消除烟草中的岩藻糖基和木糖基转移酶活性。该提案的重点是 评估产生缺乏岩藻糖基和木糖基转移酶活性的突变烟草的可能性， 使用 CRISPR（成簇规则间隔短回文重复）技术。 CRISPR是一种新的 开发了可以在真核基因组中的目标位点上产生缺失突变的分子方法。 烟草植物的基因组分析确定了三对高度同源的岩藻糖基和木糖基 转移酶（一对木糖基转移酶和两对岩藻糖基转移酶）。这三个基因对将 成为 CRISPR 删除的目标。具体来说，我们将构建多个 CRISPR 组件 质粒并在烟叶中瞬时表达它们。基因组 DNA 将从叶子中分离出来，并且 目标位点将通过 PCR 进行扩增。利用测序和生物信息学，确定 将为每个 CRISPR 构建体确定扩增基因座中的缺失突变。这些结果将 向 PlantForm 提供关于编码岩藻糖基和木糖基转移酶的基因是否可以被 被 CRISPR 永久删除以及 CRISPR 构建体运行最有效。