Assessment of the CRISPR technology in tobacco for therapeutic protein production

PlantForm公司开发了烟草作为生产治疗性抗体的平台。厂 在制造重组抗体方面优于微生物， N-糖基化，像哺乳动物细胞一样在蛋白质上添加糖。然而，两种植物特有的糖， 即岩藻糖和木糖也掺入重组抗体中。这两种糖是 抗体中不期望的，因为它们可在人体中引起过敏反应。这些糖被添加到 抗体通过岩藻糖基和木糖基转移酶在植物中。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。因此，重要的是， 通过其它手段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 评估产生岩藻糖基和木糖基转移酶活性缺陷的突变烟草的可能性， 使用CRISPR（成簇的规则间隔短回文重复序列）技术。CRISPR是一种新的 发展了一种在真核生物基因组靶位点产生缺失突变的分子方法。 烟草植物的基因组学分析鉴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）。这三对基因将 被CRISPR作为删除的目标。具体来说，我们将构建多个CRISPR组件， 质粒并在烟草叶片中瞬时表达。基因组DNA将从叶子中分离出来， 通过PCR扩增靶基因座。使用测序和生物信息学， 将针对每个CRISPR构建体确定扩增基因座中的缺失突变。这些结果将 为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CRISPR永久删除，以及什么CRISPR构建体最有效地运作。

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.