Plant Genome Engineering using DNA Replicons

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.