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.

植物场公司已开发烟草作为生产治疗抗体的平台。植物有 比微生物在制造重组抗体方面具有优势，因为植物可以催化 正如哺乳动物细胞一样，N-糖基化可以在蛋白质上添加糖。但是，有两个植物唯一的糖， 即叶谷糖和木糖也被掺入重组抗体。这两个糖是 在抗体中不良，因为它们会引起人类过敏反应。这些糖被添加到 植物中的岩藻糖基和木糖基转移酶的抗体。为了克服这个问题，植物形式有 利用RNA干扰（RNAI）减少岩藻糖基和木糖基转移酶mRNA，但很小的百分比 （<1％）抗体仍然具有这些不良糖基。因此，完全重要的是 通过其他方式消除烟草中烟素和木糖基转移酶的活性。该提议重点 评估产生突变烟草的可能性，缺乏岩藻糖基和木糖基转移酶活性的可能性， 使用CRISPR（群集定期间隔间隔短的回文重复序列）技术。 CRISPR是一个新的 开发的分子方法可以在真核基因组中的靶向基因座上产生缺失杂交。 烟草植物的基因组学分析鉴定 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 由CRISPR删除目标。具体而言，我们将在 质粒并在烟草叶中瞬时表达它们。基因组DNA将从叶子中分离出来，并 靶基因座将通过PCR扩增。使用测序和生物信息学，存在和频率 将确定每个CRISPR结构的放大基因座中的删除突变。这些结果将会 向植物形式提供知识，以了解编码岩藻糖基和木糖基转移酶是否可以是 由CRISPR永久删除以及CRISPR构造最有效地运作的内容。