水稻靶标基因单碱基定向替换技术的优化及抗病相关新材料的创制

Targeted genome editing technologies, such as zinc finger endonuclease, TALEN, CRISPR/Cas9 etc. have been well developed and widely adopted in gene functional study by the plant research community, it is easy than ever to characterize a specific gene in plants by generating the loss-of-function mutants through site-specific endonucleases. However, introducing specific DNA sequence changes into genome at the desired site, which leads to gain-of-function mutant, remains challenging. In this proposal, we present a simple base editing method (rBE) for this purpose in rice. The base editors include a Cas9n nickase (Cas9n and Cas9n(VQR)), cytosine deaminase (human AID and rat APOBEC1) and Uracil DNA glycosylase inhibitor (UGI). The sgRNA directs the Cas9n nickase fusion protein to the target site in the genome, cytosine deaminase converts any cytosine within reach into uracil, which is then repaired by error-prone mechanisms and leads to various point mutation, notably thymines when UGI exists. Four Gateway-based binary vector systems will be designed and constructed, genes involved in rice defense response, such as OsSERK1, OsFLS2, Pi-ta, Pi-d2 etc. will be targeted for base editing. The editing efficiency, base substitution pattern and inheritance of modified loci in progenies will be evaluated. In a word, the tool kit is valuable for both basic researches and agricultural applications in rice in the future.

近年来，基因组定点编辑技术作为一种重要技术手段在植物学研究中得到了广泛应用。然而，相对于常见的基因功能缺失突变体创制，功能获得性突变体的创制还比较困难。本项目前期工作中，借助CRISPR/Cas9系统引导，利用胞嘧啶脱氨酶定点修饰水稻基因组中靶位点处胞嘧啶碱基，成功实现了靶基因的单碱基定向替换，人工创制基因功能获得性水稻材料。但初步结果表明，对于不同碱基组合模式的靶位点，其编辑效果不同。因此，本项目拟进行优化，最终建立一个简单、易操作的rBE载体工具盒，以完全满足TC、AC、GC和CC4种不同碱基组合模式的特定靶位点的需求。以水稻抗性相关基因OsSERK1、OsFLS2、 Pi-d2、Pi-ta等为靶标，创制其新功能材料，明确新材料后代的修饰位点遗传稳定性和相关病原菌抗性。上述工作的开展，将为科研人员提供一个重要的基因功能研究工具，并在生产上为培育水稻抗病新品种提供新思路和新策略。

该项目以hAID基因为基础优化碱基编辑工具rBE3，形成rBE5和rBE9，并利用其成功改造水稻抗性相关基因OsSERK1、OsFLS2、 Pi-d2、Pi-ta，创制其新功能材料。以水稻内源基因OsAOS1、OsJAR1、OsJAR2和OsCOI2为作用靶标，分析优化后rBE9的优势。结果发现rBE9的碱基编辑效率高于rBE3，且无碱基偏好性，即hAID*Δ介导的rBE5/9能高效进行碱基编辑，能够完全满足TC、AC、GC和CC4种不同碱基组合模式的特定靶位点的需求，扩展了水稻碱基编辑技术的应用。另一方面本研究利用TadA*7.10和TadA*7.8构建了一系列腺嘌呤碱基编辑工具，结果发现以TadA*7.10为基础构建的rBE14实现了靶位点碱基的编辑，成功实现了目标碱基的编辑替换（A>G）。并利用rBE14对水稻OsSERK2和OsWRKY45中的病原响应磷酸化位点成功进行改造，获得预期的突变体材料，编辑效率分别为32.05%和62.26%。且在该系统中引入了全新的紫外激活GFP检测系统，可以通过手持式紫外灯直接照射水稻抗性愈伤筛选得到发生编辑事件的水稻愈伤，也使后代群体中的转基因分离检测变得十分便利。rBE5、rBE9及rBE14可在水稻基因组中实现对DNA四种碱基的编辑替换（A>G, T>C, C>T, G>A），这对于对水稻基因功能解析和现代分子育种研究具有重大推进作用，特别是对水稻品种的缺陷型基因进行修饰矫正，有利于加快水稻育种进程以及延长现有品种的商业周转期。