2010: Targeted Mutagenesis in Arabidopsis Using Zinc Finger Nucleases

植物生物学的下一个挑战是通过各种基因组测序项目来识别许多基因的功能。了解基因功能的一个重要方法是研究去除或敲除特定基因功能的后果。在模式植物拟南芥中，有有效的方法可以敲除许多感兴趣的基因。然而，功能分析往往会因遗传冗余或相同或密切相关基因的多个拷贝的存在而受挫。 在植物中特别普遍的是串联基因复制，并且多达15%的拟南芥基因以串联阵列的形式组织。 一个有针对性的诱变协议将开发，以增强现有的方法来了解拟南芥基因功能，特别是基因的功能分析已混淆遗传冗余。该方法使用锌指核酸酶（ZFN）-嵌合蛋白，其由与FokI内切核酸酶的DNA切割结构域融合的锌指阵列组成。 高亲和力和特异性的锌指阵列可以被设计成识别几乎任何靶基因。 当靶DNA被ZFN切割时，断裂的末端被无效地修复，导致基因座特异性突变。在初始实验中，将使用模型锌指核酸酶和含有同源靶位点的报告基因来优化用于有效回收可遗传的ZFN诱导的突变的方法。 接下来，ZFN将被工程化以识别天然拟南芥基因座，包括突变时产生可观察表型的那些基因座。ZFN还将针对突变不导致可观察表型的基因座进行工程改造。 为了恢复这样的基因座处的突变，将采用稳健的DNA扩增和基于序列的检测方法。 最后，将靶向重复基因的串联阵列。 阵列的缺失将通过工程化ZFN以在最外层基因内切割来完成。 将确定恢复缺失和单个突变的频率以评估该方法的相对效率。 除了拟南芥，这种诱变方法也将是有价值的植物物种，如水稻和玉米，其中基因组测序项目正在进行和串联基因丰富。此外，产生和调节特定染色体双链断裂的能力将促进植物染色体结构和DNA修复机制的研究。可以访问基因敲除研究的方案和结果的网站的URL是http://www.beckmancenter.umn.edu/html/2010.html。 如果要充分利用植物来满足世界对燃料、饲料和工业原材料日益增长的需求，了解植物基因的功能至关重要。 虽然在辨别植物基因功能方面已经取得了重大进展，但现有方法具有固有的局限性，特别是在将功能归于冗余或重复基因方面。 本项目的目标是实施一种有效的靶向诱变方法来克服这些限制。 本计画将训练大学部实习生及研究生从事植物分子生物学之工作。 来自代表性不足群体的学生将通过旨在提高少数民族参与科学的计划被招募到该项目中。

The next challenge in plant biology is to discern the function of the many genes revealed through the various genome sequencing projects. One important approach for understanding gene function is to study the consequence of removing or knocking out the function of a specific gene. In the model plant Arabidopsis, efficient methods are available to knock out many genes of interest. Functional analysis, however, is often frustrated by genetic redundancy or the presence of multiple copies of the same or closely related genes. Particularly prevalent in plants are tandem gene duplications, and as many as 15% of Arabidopsis genes are organized in tandem arrays. A targeted mutagenesis protocol will be developed to augment existing approaches for understanding Arabidopsis gene function, particularly for genes for which functional analysis has been confounded by genetic redundancy. The approach uses zinc finger nucleases (ZFNs) - chimeric proteins made up of a zinc finger array fused to the DNA cleavage domain of Fokl endonuclease. Zinc finger arrays of high affinity and specificity can be engineered to recognize virtually any target gene. Upon cleavage of the target DNA by the ZFN, the broken ends are repaired inefficiently, resulting in locus-specific mutations. In initial experiments, a model zinc finger nuclease and a reporter gene containing the cognate target site will be used to optimize methods for efficient recovery of heritable ZFN-induced mutations. Next, ZFNs will be engineered to recognized native Arabidopsis loci, including those that give observable phenotypes when mutated. ZFNs will also be engineered for a locus where mutatations do not result in an observable phenotype. To recover mutations at such a locus, robust DNA amplification and sequence-based detection methods will be employed. Lastly, a tandem array of duplicate genes will be targeted. Deletion of the array will be accomplished by engineering ZFNs to cleave within the outermost genes. The frequency of recovering deletions and individual mutations will be determined to assess the relative efficiency of this approach. In addition to Arabidopsis, this mutagenesis approach will also be valuable for plant species such as rice and maize, where genome sequencing projects are ongoing and tandem genes are abundant. Moreover, the ability to generate and regulate specific chromosomal double-strand breaks will facilitate studies of chromosome structure and DNA repair mechanisms in plants. The URL for the web site where protocols as well as results of gene knock-out studies can be accessed is http://www.beckmancenter.umn.edu/html/2010.html. Understanding the function of plant genes is critical if plants are to be fully harnessed to meet the world's burgeoning need for fuel, feed and industrial raw materials. Although significant progress has been made in discerning plant gene function, existing approaches have inherent limitations, particularly with respect to ascribing function to redundant or duplicated genes. The goal of this project is to implement an efficient targeted mutagenesis approach to overcome these limitations. This project will train undergraduate interns and graduate students for work in plant molecular biology. Students from underrepresented groups will be recruited to the project through programs designed to enhance minority participation in science.