Assessment of the CRISPR technology in tobacco for therapeutic protein production

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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.