There is a clear need, in plants, for an efficient and practical system for targeted mutagenesis. Planet Biotechnology's interest is in using the tobacco species Nicotiana benthamiana as a "factory" for the economical production of human therapeutic proteins, including a recombinant Fc fusion protein for treatment of inhalational anthrax. The CRISPR/Cas system could be used to modify numerous traits that interfere with this plant achieving its full potential as a therapeutic protein production system. This applicatio proposes the first attempt to use the CRISPR/Cas system for site-specific gene modification in plants. We intend to demonstrate a proof-of-concept by mutating fucosyltransferase (or fucosyltransferase and xylosyltransferase) in N. benthamiana, glycosyltransferases responsible for making plant-specific N-glycan residues. We will test two different CRISPR/Cas strategies. The first has been published and shown to work in human, zebrafish and Drosophila cells, and involves co-transfection of cells with Cas9 and a single synthetic guide RNA targeting the site to be mutated. The second strategy involves Cas9 plus two RNAs, the crRNA and tracrRNA, with the crRNA incorporating multiple target sites in a single gene. The Cas9 and RNA genes will be introduced into the plant binary plasmid vector pTRAkc. Cas9 and guide RNAs targeting the glycosyltransferases gene(s) will be delivered to the nuclei of N. benthamiana cells using an Agrobacterium tumefaciens binary vector system. The mutations will be initiated by infiltrating an Agrobacterium suspension carrying a CRISPR/Cas vector into N. benthamiana leaves and then regenerating plants. Leaf extracts from regenerated plants will be screened for the presence of ?1,3-fucose (or ?1,2-xylose) residues on glycoproteins using glycan-specific antibodies by ELISA. Any plants containing biallelic mutations of FucT will be identified by the loss of ?1,3- fucose (or ?1,2-xylose) on glycoproteins in primary (R0) regenerated plants. We will screen for monoallelic mutations by PCR followed by high resolution melting curve analysis. Plants identified as positive by this assay will be grown to maturity in the greenhouse and allowed to set seed. Second generation (R1) plants will again be screened by ELISA. DNA at and around the target sites will be amplified by PCR and the products will be sequenced to determine the exact nature of the mutations in FucT or XylT (insertions/deletions/other rearrangements). Mutant plants will be used for transient expression of a model glycoprotein, an Fc-fusion protein, which will be purified by Protein A chromatography and analyzed by immunoblotting for the presence of ?1,2-xylose and ?1,3-fucose. The absence of these two sugars will be confirmed by N-glycan analysis.
Planet Biotechnology and several other companies are using the plant Nicotiana benthamiana to produce human therapeutic proteins, such as antibodies. Several plant proteins, such as certain glycosyltransferases, proteases, proteins involved in phenolic biosynthesis and post-transcriptional gene silencing, limit the ability to produce functional, unmodified human proteins with normal human glycosylation in this plant species. We propose to develop a site-specific gene modification technology to allow direct mutation of undesired plant genes.