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（由适用提供）：在植物中，明确需要有针对性诱变的有效且实用的系统。 Planet Biotechnology的兴趣在于使用烟草种类烟草烟草本植物作为人类治疗蛋白的经济生产的“工厂”，包括重组FC融合蛋白来治疗衰竭。 CRISPR/CAS系统可用于修改许多干扰该植物的特征，从而充分发挥其作为治疗性蛋白质生产系统的潜力。该应用程序提出，首次尝试使用CRISPR/CAS系统进行植物中特定地点基因修饰的尝试。我们打算通过突变构成岩藻糖基转移酶（或岩糖基转移酶和木糖基转移酶）的概念证明，这是糖基转移酶，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。第一个已发表并证明在人，斑马鱼和果蝇细胞中起作用，涉及与CAS9的细胞共转染和一个靶向要突变位点的单个合成指南RNA。第二种策略涉及CAS9加两个RNA，CRRNA和TRACRRNA，CRRNA与单个基因中的多个靶位点结合。 CAS9和RNA基因将被引入植物二元质粒载体PTRAKC中。 CAS9和引导RNA靶向糖基转移酶基因（S）将使用农业菌群Tumefaciens二进制载体系统传递到本氏乳杆菌细胞的核。该突变将通过渗入携带CRISPR/CAS载体的农杆菌悬浮液进入本氏乳杆菌，然后再生植物。将使用ELISA的聚糖特异性抗体筛选从再生植物中的叶提取物，以筛选糖蛋白上的1,3-羟基糖（或1,2-木糖）残基。任何含有双质突变的植物都将通过在原发性（R0）再生植物中糖蛋白上的1,3-富谷糖（或1,2-木糖）损失来鉴定。我们将通过PCR筛选单性突变，然后进行高分辨率熔解曲线分析。通过这种测定鉴定为阳性的植物将在温室中成熟到成熟，并允许固定种子。第二代（R1）植物将再次由Elisa筛选。 PCR将扩大目标位点及其周围周围的DNA，并将测序确定FUCT或XYLT中突变的确切性质（插入/缺失/其他重排）。突变植物将用于模型糖蛋白（FC融合蛋白）的瞬时表达，该蛋白将通过蛋白质A色谱法纯化，并通过免疫印迹分析，以在1，2-木糖和1,3-浓度糖中存在。这两种糖的缺乏将通过N-聚糖分析确认。