Targeted mutagenesis of plant genes by the CRISPR/Cas system

CRISPR/Cas系统对植物基因进行定点突变

• 批准号: 8713873
• 负责人: KEITH WYCOFF
• 金额: $ 22.41万
• 依托单位: PLANET BIOTECHNOLOGY, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8713873
• 关键词: Agrobacterium; Amino Acid Sequence; Anabolism; Animals; Anthrax disease; Antibodies; Biological Assay; Biological Products; Biotechnology; Cell Nucleus; Cells; Chimeric Proteins; Chromatography; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; DNA Polymerase II; DNA Polymerase III; DNA Sequence Rearrangement; Digestion; Drosophila genus; Enzyme-Linked Immunosorbent Assay; Enzymes; Eukaryota; Fucose; Fucosyltransferase; Gaucher Disease; Gene Cluster; Gene-Modified; Generations; Genes; Glycoproteins; Glycosyltransferase Gene; Guide RNA; High Pressure Liquid Chromatography; Human; Immunoblotting; Kentucky; Larix; Medicago; Methods; Modeling; Mus; Mutagenesis; Mutate; Mutation; Natural regeneration; Nature; Nicotiana; Oligosaccharides; Peptide Hydrolases; Peptide Sequence Determination; Phase; Phase I Clinical Trials; Planets; Plant Genes; Plant Leaves; Plant Proteins; Plants; Plasmid Cloning Vector; Plasmids; Polysaccharides; Production; Prokaryotic Cells; Proteins; Publishing; RNA; RNA Interference; Reagent; Recombinants; Resolution; Rhizobium radiobacter; Seeds; Site; Structure; Suspension substance; Suspensions; System; T-DNA; Technology; Testing; Therapeutic; Tobacco use; Transfection; Virus; Work; Xylose; Zebrafish; adaptive immunity; anthranilamide; bioprocess; glucosylceramidase; glycosylation; glycosyltransferase; influenza virus vaccine; insertion/deletion mutation; interest; melting; mutant; promoter; public health relevance; sugar; therapeutic protein; trait; vector; xylosyltransferase

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.

描述（由申请人提供）：在植物中，显然需要一种用于靶向诱变的有效和实用的系统。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系统可用于修饰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系统进行植物中的位点特异性基因修饰。我们打算通过突变N. benthamiana，负责制造植物特异性N-聚糖残基的糖基转移酶。 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，并且涉及用Cas9和靶向待突变位点的单一合成指导RNA共转染细胞。第二种策略涉及Cas9加上两种RNA，crRNA和tracrRNA，其中crRNA在单个基因中掺入多个靶位点。将Cas9和RNA基因引入植物双元质粒载体pTRAkc中。靶向糖基转移酶基因的Cas9和向导RNA将被递送到N.本塞姆氏菌细胞，使用根癌农杆菌双元载体系统。 通过将携带CRISPR/Cas载体的农杆菌悬浮液渗入N. benthamiana叶片，然后再生植株。将通过ELISA使用聚糖特异性抗体筛选再生植物的叶提取物中糖蛋白上是否存在<$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（RO）再生植物中糖蛋白上的1，3-岩藻糖（或1，2-木糖）的损失来鉴定。 我们将通过PCR和高分辨率熔解曲线分析筛选单等位基因突变。通过该测定鉴定为阳性的植物将在温室中生长至成熟并允许结籽。将再次通过ELISA筛选第二代（R1）植物。将通过PCR扩增靶位点处及其周围的DNA，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）。突变体植物将用于瞬时表达模型糖蛋白，一种Fc融合蛋白，其将通过蛋白A色谱法纯化并通过免疫印迹分析1，2-木糖和1，3-岩藻糖的存在。将通过N-聚糖分析确认不存在这两种糖。