Targeted mutagenesis of plant genes by the CRISPR/Cas system

描述(由申请人提供)：在植物中，显然需要一种有效和实用的定向突变系统。Planet BioTechnology感兴趣的是将烟草品种本特米纳烟草用作经济地生产人类治疗性蛋白的“工厂”，包括用于治疗吸入性炭疽的重组Fc融合蛋白。CRISPR/Cas系统可以用来改变干扰这种植物发挥其作为治疗性蛋白质生产系统的全部潜力的许多性状。本申请首次尝试利用CRISPR/Cas系统对植物进行定点基因修饰。我们打算通过突变N.benthamiana中的岩藻糖基转移酶(或岩藻糖基转移酶和木糖转移酶)来展示一种概念验证，这种糖基转移酶负责制造植物特有的N-糖链残基。我们将测试两种不同的CRISPR/CAS策略。第一个已经发表，并被证明在人类、斑马鱼和果蝇细胞中起作用，涉及到与Cas9和针对要突变的位置的单一合成引导RNA共转染细胞。第二种策略涉及Cas9加上两个RNA，即crRNA和trrRNA，其中crRNA在单个基因中整合了多个靶点。将Cas9和RNA基因导入植物双质粒载体pTRAkc。针对糖基转移酶基因(S)的Cas9和GUIDE RNA将使用农杆菌双元载体系统传递到本底农杆菌细胞的核中。这些突变将通过将携带CRISPR/Cas载体的农杆菌悬浮液渗透到N.benthamiana叶片中，然后再生植株来启动。再生植物的叶提取物将通过酶联免疫吸附试验筛选糖蛋白上是否存在1，3-岩藻糖(或1，2-木糖)残基。任何含有Fuct双等位基因突变的植物将通过初级(R0)再生植株糖蛋白上1，3-岩藻糖(或1，2-木糖)的丢失来识别。我们将通过聚合酶链式反应和高分辨率熔化曲线分析来筛查单等位基因突变。经检测呈阳性的植物将在温室中生长到成熟，并允许结籽。第二代(R1)植株将再次通过酶联免疫吸附试验进行筛选。目标部位及其周围的DNA将通过聚合酶链式反应进行扩增，并对产物进行测序，以确定Fuct或XylT突变的确切性质(插入/缺失/其他重排)。突变植株将用于瞬时表达模型糖蛋白Fc融合蛋白，该蛋白将通过蛋白A层析纯化，并通过免疫印迹分析1，2-木糖和1，3-岩藻糖的存在。N-糖链分析将证实这两种糖的缺失。

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.