Browsing by Subject "Viruses"

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    Development of RNA Viral Vectors for Plant Genome Engineering
    (2022-02) Ellison, Evan
    Genetic variation is a key principle in the improvement of agricultural crops. For thousands of years, crop productivity, resilience and adaptability has been slowly improved by selection of favorable alleles. An increasing understanding of molecular genetics underlying key traits has contributed to the continuing progress in crop development. Dissecting and exploiting plant molecular genetics is greatly enhanced by the ability to precisely create genetic variation at pre-selected sites. Creating novel genetic variation through gene editing is reliant on technology that creates the desired modification at the target site and delivery of the reagents to plant cells. RNA guided endonucleases, such as CRISPR/Cas9, have enabled an unprecedented ability for site-specific genetic modification. Delivery of reagents, however, is still largely reliant on tissue culture regeneration to fix targeted genetic modifications in the genome. Tissue culture regeneration is a technically difficult process that can easily take several months or years to complete. The work described here outlines approaches to deliver genome editing reagents using RNA viruses. Chapter 1 discusses background information related to plant viruses and viral vectors. Chapter 2 describes a collaborative effort to develop a novel gene editing reagent delivery vector, Foxtail Mosaic Virus (FoMV), and its application in gene editing of monocot and dicot plant species. Genetic modifications obtained in chapter 2 only occurred in somatic cells, and still require laborious tissue-culture to fix in the germline. Chapter 3 describes my approaches to improve Tobacco Rattle Virus (TRV) for highly efficient heritable genome editing in the model species Nicotiana benthamiana. This work was improved further in chapter 4 by using viral vectors and an improved method for heritable genome editing in Solanum lycopersicum (tomato). Together, these improvements to RNA viral vectors provide an efficient and rapid means for delivery of genome engineering reagents. The time to generate targeted modifications that are fixed in the genome, for both model and crop species, is reduced from years to only a few months which enables genome editing at scale. The ease at which targeted genetic modifications can now be generated will enable important progress in crop improvement.