Plant Genome Engineering using DNA Replicons

PI: Daniel F. Voytas（明尼苏达大学-双城分校）高级人员：Colby G. Starker（明尼苏达大学-双城分校）在植物领域，基因组工程通过将DNA序列与生物功能联系起来，有望推进基础研究。此外，基因组工程将使植物的生物合成能力能够被利用来生产不断增长的世界人口所需的许多农产品。如果成功，该研究项目的成果将是高效植物基因组工程的协议和试剂，这些协议和试剂将存放在公共存储库中，以提供不受限制的访问。在外联和培训方面，该项目将每年举办研讨会，教授科学家如何使用DNA复制子和CRISPR/Cas9试剂有效地创建靶向基因组修饰。本科生和研究生也将接受植物分子生物学方面的培训。研究生将有机会在世界各地的实验室学习，从而成为全球努力实施基因组工程以促进植物生物学进步的一部分。基因组工程的最新进展使精确改变生物体的遗传蓝图成为可能。该技术的应用是广泛的，从纠正人类的遗传缺陷（基因治疗）到了解模式生物的基因功能。在植物方面，基因组工程不仅有望推进基础植物研究，而且还有望利用植物的生物合成能力来生产不断增长的世界人口所需的许多农产品。在这个项目中，方法将开发有效的工程植物基因组。其中一个重点是克服实施这项技术的主要障碍，即将基因组工程试剂输送到植物细胞中的挑战。在这里，将开发双病毒——植物DNA病毒——作为试剂递送的载体。因为它们有DNA基因组，所以双病毒非常适合传递产生目标序列改变所需的分子，包括刺激目标基因组修饰的序列特异性核酸酶和将序列改变纳入基因组的DNA模板。基于双病毒的复制子将首先以烟草为模型进行优化，以进行多种基因组修饰，包括靶向诱变、基因替换和基因插入。然后，这些复制子将被用于在密切相关的物种，西红柿和土豆中进行类似的修改，目的是在这些物种中开发具有有价值特征的新作物品种。此外，一种新描述的序列特异性核酸酶——CRISPR/Cas9系统——将被评估其在植物基因组工程中的实用性。将优化CRISPR/Cas9试剂在植物中的表达和活性，并开发同时修饰单个和多个植物基因的方案。

PI: Daniel F. Voytas (University of Minnesota - Twin Cities)Senior personnel: Colby G. Starker (University of Minnesota - Twin Cities)In plants, genome engineering promises to advance basic research by linking DNA sequences to biological function. Further, genome engineering will enable plants' biosynthetic capacity to be harnessed to produce the many agricultural products required by an expanding world population. If successful, the outcome of this research project will be protocols and reagents for highly efficient plant genome engineering that will be deposited in public repositories to provide unrestricted access. With regard to outreach and training, the project will conduct annual workshops to teach scientists how to use DNA replicons and CRISPR/Cas9 reagents to efficiently create targeted genome modifications. Undergraduate and graduate students will also be trained in plant molecular biology. Graduate students will have the opportunity to study in laboratories around the world, and thereby become part of the global effort to implement genome engineering for the advancement of plant biology. Recent advances in genome engineering make it possible to precisely alter an organism's genetic blueprint. Applications of the technology are expansive, ranging from correcting genetic defects in humans (gene therapy) to understanding gene function in model organisms. In plants, genome engineering promises to not only advance basic plant research but also enable the biosynthetic capacity of plants to be harnessed to produce the many agricultural products required by an expanding world population. In this project, methods will be developed to efficiently engineer plant genomes. One focus is to overcome a primary barrier for implementing this technology, namely the challenge in delivering genome engineering reagents to plant cells. Here, geminiviruses - plant DNA viruses - will be developed as vectors for reagent delivery. Because they have DNA genomes, geminiviruses are ideally suited to deliver the molecules needed to create targeted sequence alterations, including the sequence-specific nucleases that stimulate targeted genome modifications and the DNA templates that incorporate sequence alterations into the genome. Geminivirus-based replicons will first be optimized using tobacco as a model to undertake a variety of genomic modifications, including targeted mutagenesis, gene replacement and gene insertion. The replicons will then be used to carry out a similar spectrum of modifications in the closely related species, tomato and potato, with the intent of developing new crop varieties in these species with valuable traits. In addition, a newly described class of sequence-specific nucleases - the CRISPR/Cas9 system - will be assessed for its utility for plant genome engineering. CRISPR/Cas9 reagents will be optimized for expression and activity in plants, and protocols will be developed to modify single and multiple plant genes simultaneously.