Multi-target silencing, genome editing and epitope mapping of tomato allergens

拟议的研究项目旨在(I)提供原理证据，证明同时下调植物性食品中的几种过敏原是可能的，(Ii)开发和应用新的基因组编辑策略来调节番茄的致敏潜力，以及(Iii)改善对泛变应原轮廓蛋白的过敏原结构的分子理解。通过培育可用的RNAi介导的单基因、双基因和三基因敲除番茄品系，可以同时下调已知的番茄IgE结合蛋白。这些菌株将通过IgE免疫印迹和皮肤点刺试验来测试它们对常见病原体的敏感度和致敏潜力。为了避免RNAi介导的基因表达下调可能存在的缺陷，新开发的TAL-核酸酶和CRISP/Cas技术将被应用于基因组编辑，以特异性敲除番茄中的过敏原编码基因。虽然TAL-核酸酶已经在植物系统中成功地进行了测试，但由CRISP/CAS进行的RNA引导的基因组编辑在植物中还没有报道。因此，现有的系统将适用于植物，并将测试CRISP/CAS系统对植物基因组编辑的适用性。通过使用我们新开发的酵母选择系统无偏见地选择具有改变的IgE结合能力的Profilin变体，以及使用来自北欧、中欧和南欧国家的番茄过敏患者的血清对来自不同植物来源的Profilin进行表位扫描，将进行Profilin的结构阐明。拟议研究项目的预期结果是：(I)减少/取消过敏原表达的分子策略的评估，(Ii)针对植物基因的基因组编辑策略的适应，(Iii)关于选定过敏原潜在防御功能的知识，(Iv)对Profilin结构决定因素的分子洞察，以及(V)关于对表位偏好的潜在地理影响及其可能原因的信息。

The proposed research project aims at (i) providing proof-of-principle that simultaneous down regulation of several allergens in plant-derived food is possible, (ii) developing and applying novel genome editing strategies to modulate the allergenic potential of tomato and (iii) to improve the molecular understanding of allergenic structures of the panallergen profilin. Simultaneous down-regulation of known IgE-binding proteins of tomato will be achieved by breeding available RNAi-mediated mono-, double- and triple-knock-down tomato lines. These lines will be tested for their susceptibility towards common pathogens and for their allergenic potential by IgE immunoblots and skin prick tests. To avoid possible drawbacks of RNAi-mediated down-regulation of gene expression newly developed TAL-nuclease and CRISP/Cas technologies will be applied for genome editing to specifically knock-out allergen encoding genes in tomato. While TAL-nucleases have already successfully been tested in plant systems, RNA-guided genome editing by CRISP/Cas has not been reported for plants. Therefore, available systems will be adapted to plants and suitability of the CRISP/Cas system for genome editing in plants will be tested. The structural elucidation of profilin will be carried out by unbiased selection of profilin variants with altered IgE-binding capacities using our newly developed yeast selection system and by epitope scanning of profilins from different plant sources using sera from tomato allergic patients from north, central and south European countries. Expected outcomes of the proposed research project are: (i) the assessment of molecular strategies to reduce/abolish expression of allergens, (ii) adaptation of genome editing strategies to target plant genes, (iii) knowledge concerning the potential defense function of selected allergens, (iv) molecular insight into structural determinants of profilin and (v) information concerning the potential geographic influence on epitope preferences and their possible cause.