Plant Genome Engineering With Sequence-Specific Nucleases: Methods For Editing DNA In Whole Plants

Abstract

The development and function of all living organisms, from bacteria to humans, is encoded within a universal blueprint-deoxyribonucleic acid (DNA). The ability to re-write this code of life promises great benefits, ranging from a better understanding of gene function to correcting genetic diseases. Therefore, there is high value for tools and techniques that enable genome editing in living cells. In the last 20 years, multiple classes of enzymes have been developed that can be `rewired' to recognize and break a DNA sequence of interest. These enzymes (sequence-specific nucleases) have proven to be powerful reagents for editing DNA in higher-eukaryotic cells. However, the ability to modify DNA, particularly in plant cells, does not solely depend on the activity of the sequence specific nuclease. Instead, it also depends on the efficiency with which the genome engineering reagents are delivered, the cells they are delivered to, and the effectiveness of selecting (or screening) for cells with the desired modification. Studies within this dissertation seek to develop novel methods for delivering genome engineering reagents to whole plants. First, we focused our attention on geminiviruses--a large family of plant DNA viruses. Prior to these studies, geminiviruses were primarily used as vectors for virus-induced gene silencing or for protein expression; however, their circular DNA genomes, and their ability to replicate extrachromosomally, makes them an attractive vector for delivering genome engineering reagents. Here, we describe proof-of-principle experiments showing that, in Nicotiana tabacum, replicons based on the bean yellow dwarf virus can indeed deliver genome engineering reagents to leaf cells, and that these modified cells could grow into calli and seedlings. Interestingly, we also observed an enhancement in homologous recombination in leaf cells, relative to our non-viral controls. This enhancement appeared to be due to replication of donor molecules and by pleiotropic activity of the virus replication proteins. In addition to DNA viruses, we also explored the use of RNA viruses for the delivery of sequence-specific nucleases in Arabidopsis. And, finally, we expanded the utility of stable integration into plant genomes by applying this approach to additional plants, additional target genes, and additional genome modifications.