Targeted mutagenesis of plant genes by the CRISPR/Cas system

描述（由申请人提供）：在植物中，明显需要一种高效实用的靶向诱变系统。Planet Biotechnology的兴趣是利用烟草物种Nicotiana benthamiana作为经济生产人类治疗性蛋白质的“工厂”，包括用于治疗吸入性炭疽的重组Fc融合蛋白。CRISPR/Cas系统可用于修改许多干扰这种植物实现其作为治疗性蛋白质生产系统的全部潜力的性状。本申请首次尝试使用CRISPR/Cas系统在植物中进行位点特异性基因修饰。我们打算通过突变N. benthamiana中的focusyltransferase（或focusyltransferase和xylosyltransferase）来证明概念验证，这些糖基转移酶负责制造植物特异性n -聚糖残基。我们将测试两种不同的CRISPR/Cas策略。第一种方法已经发表，并显示在人类、斑马鱼和果蝇细胞中起作用，它涉及到用Cas9和单一合成的靶向突变位点的向导RNA共同转染细胞。第二种策略涉及Cas9加上两种rna， crRNA和tracrRNA，其中crRNA在单个基因中包含多个目标位点。Cas9和RNA基因将被导入植物双质粒载体pTRAkc中。利用农杆菌二元载体系统将Cas9和靶向糖基转移酶基因的引导rna传递到N. benthamiana细胞的细胞核中。突变将通过将携带CRISPR/Cas载体的农杆菌悬浮液渗透到N. benthamiana叶片中，然后再生植株而启动。再生植物的叶提取物将使用糖聚糖特异性抗体通过ELISA筛选糖蛋白上存在的¿1,3-焦点（或¿1,2-木糖）残基。任何含有FucT双等位基因突变的植物都可以通过在初代（R0）再生植物中糖蛋白上丢失¿1,3-焦点（或¿1,2-木糖）来识别。我们将通过PCR筛选单等位基因突变，然后进行高分辨率熔化曲线分析。通过该试验鉴定为阳性的植物将在温室中生长到成熟并允许种子。第二代（R1）植株将再次通过ELISA筛选。靶位点及其周围的DNA将通过PCR扩增，产物将被测序以确定FucT或XylT突变的确切性质（插入/缺失/其他重排）。突变植株将用于模型糖蛋白的瞬时表达，该糖蛋白是一种fc融合蛋白，将通过蛋白a层析纯化，并通过免疫印迹分析是否存在¿1,2-木糖和¿1,3-焦。这两种糖的缺失将通过n -聚糖分析得到证实。

DESCRIPTION (provided by applicant): 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.