Precise Engineering of Plant Genomes using Zinc Finger Nucleases

PI：丹尼尔F. Voytas（明尼苏达大学）CoPIs：Drena L.多布斯（爱荷华州州立大学）、J.基思·贾斯珀（马萨诸塞州总医院）、詹妮弗·库兹马（明尼苏达大学）和王侃（爱荷华州州立大学）植物具有非凡的生物合成能力，可以利用这些能力生产对食品、燃料、医药和工业有价值的化合物。然而，充分实现植物的生物合成潜力需要复杂的工具来操纵植物基因组。 具体地，期望对植物遗传密码进行精确改变，包括DNA插入、缺失和取代。 这种精确的修饰可以通过称为基因靶向或同源重组的过程进行。 从根本上讲，基因打靶是一种DNA交换反应：将携带所需序列的DNA片段引入植物细胞，并取代基因的天然拷贝。 为了提高基因靶向的效率，在修饰位点（靶标）处产生染色体断裂。 一种叫做锌指核酸酶（ZFN）的酶被用来产生染色体断裂。 ZFN有两个组成部分：DNA识别结构域（锌指阵列）和切割染色体的核酸酶。 锌指阵列可以被设计为识别不同的DNA序列，从而使得修饰任何染色体靶成为可能。 目前的研究是针对开发锌指核酸酶辅助基因打靶在植物中的广泛使用，包括建立高频基因修饰的关键参数和锌指阵列设计的鲁棒方法。 该项目的重点是在水稻中实施基因靶向，水稻可以说是世界上最重要的粮食作物。 这项研究的成果将是一个非常容易的基因靶向系统，可用于各种植物物种。 由于基因靶向以高度特异性和可控的方式引入植物基因组的变化，通过基因靶向产生的作物可能比传统的转基因作物更容易被公众接受。 一种对植物基因组进行精确修饰的有效方法（基因打靶）对于基因和遗传途径的详细功能分析至关重要。 基因靶向还将使新作物品种的开发成为可能，包括那些更好地抵抗害虫、提高食品价值和生产具有工业重要性的化合物的品种。 基因靶向与转基因的根本区别在于，所产生的植物材料可能仅具有将其与亲本区分开的单个或几个核苷酸变化。 这种精确性表明，基因靶向可能会减轻对使用转基因作物的一些担忧，这种担忧限制了基因工程在植物农业中的应用。 该项目专门探讨了基因靶向的潜在社会影响。 此外，该项目还将培训本科生和研究生从事植物分子生物学、计算生物学和公共政策方面的工作。 研究课题的多样性为所有项目参与者提供了丰富的跨学科培训环境和了解科学对社会影响的独特机会。 该项目产生的软件和数据可在www.zincfinger.org上查阅，DNA试剂可在www.addgene.org上查阅。

PI: Daniel F. Voytas (University of Minnesota)CoPIs: Drena L. Dobbs (Iowa State University), J. Keith Joung (Massachusetts General Hospital), Jennifer Kuzma (University of Minnesota) and Kan Wang (Iowa State University) Plants have remarkable biosynthetic capacities that can be harnessed to produce compounds of value for food, fuel, medicine and industry. Fully realizing the biosynthetic potential of plants, however, requires sophisticated tools to manipulate plant genomes. Specifically, it is desirable to make precise alterations to the plant genetic code, including DNA insertions, deletions and substitutions. Such precise modifications can be made through a process known as gene targeting or homologous recombination. Fundamentally, gene targeting is a DNA swapping reaction: a DNA fragment carrying a desired sequence is introduced into a plant cell, and it replaces the native copy of the gene. To enhance the efficiency of gene targeting, a chromosome break is created at the site of modification (the target). An enzyme called a zinc finger nuclease (ZFN) is used to generate the chromosome break. ZFNs have two components: a DNA recognition domain (a zinc finger array) and a nuclease that cleaves the chromosome. Zinc finger arrays can be designed to recognize diverse DNA sequences, thereby making it possible to modify any chromosomal target. Current research is directed at developing zinc finger nuclease-assisted gene targeting for widespread use in plants, including establishing key parameters for high frequency gene modification and robust methods for the design of zinc finger arrays. The project focuses on implementing gene targeting in rice, arguably the world's most important food crop. The outcome of the research will be a highly facile gene targeting system that can be employed in a variety of plant species. Because gene targeting introduces changes in plant genomes in a highly specific and controlled manner, crops generated through gene targeting may be met with greater public acceptance than traditional genetically modified crops. An efficient method for making precise modifications to plant genomes (gene targeting) is critical for detailed functional analysis of genes and genetic pathways. Gene targeting will also enable the development of new crop varieties, including those that better withstand pests, have enhanced food value, and produce compounds of industrial importance. Gene targeting differs fundamentally from transgenesis in that the resulting plant material may only have a single or few nucleotide changes that distinguish it from the parent. This precision suggests that gene targeting may mitigate some concerns about the use of genetically modified crops, which has limited the application of genetic engineering to plant agriculture. The project specifically explores the potential societal impacts of gene targeting. In addition, the project will train undergraduate and graduate students for work in plant molecular biology, computational biology and public policy. This diversity of research topics provides a rich interdisciplinary training environment and a unique opportunity for all project participants to learn about the impact of science on society. Access to software and data generated from this project can be obtained at www.zincfingers.org. DNA reagents are available at www.addgene.org.