Assessment of the CRISPR technology in tobacco for therapeutic protein production

PlantForm公司已经开发出烟草作为产生治疗性抗体的平台。植物有 在制造重组抗体方面比微生物更有优势，因为植物可以催化 N-糖基化，在蛋白质上添加糖，就像哺乳动物细胞一样。然而，两种植物特有的糖， 即岩藻糖和木糖也被结合到重组抗体中。这两种糖是 这些抗体不受欢迎，因为它们会在人体内引起过敏反应。这些糖被加入到 植物中岩藻糖基和木糖基转移酶产生的抗体。为了克服这个问题，PlantForm已经 利用RNA干扰(RNAi)减少岩藻糖基和木糖基转移酶mRNAs，但比例很小 (&lt；1%)抗体仍然含有这些不受欢迎的糖基。因此，重要的是要完全 通过其他方法消除烟草中岩藻糖基和木糖基转移酶的活性。这项提案的重点是 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 发展了一种在真核基因组中产生靶基因座缺失突变的分子方法。 烟草基因组学分析鉴定出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删除。具体地说，我们将在 并在烟草叶片中瞬时表达这些基因。基因组DNA将从叶片中分离出来，并且 通过聚合酶链式反应扩增靶基因座。使用测序和生物信息学，存在和频率 将为每个CRISPR构建确定扩增基因座中的缺失突变。这些结果将 向植物提供关于编码岩藻糖基和木糖基转移酶的基因是否可以 CRISPR永久删除的内容和CRISPR构造的内容最有效地运行。

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.