Date of Award
Spring 2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Molecular, Cellular, and Developmental Biology
First Advisor
Jacob, Yannick
Abstract
In gene targeting, a double strand DNA break is created in a gene ofinterest, and the break is repaired via homologous recombination using an exogenous DNA donor template (DT) harboring a desired modification flanked by sequences homologous to the target locus. If the cell undergoes homologous repair using the template, the desired modification can be copied onto the gene of interest. This is a highly worthwhile process, given that virtually any modification could potentially be made to an endogenous target. However, in plants, the process of gene targeting is extremely inefficient, often to the point that it is impossible, or at least highly arduous, to identify a plant line with the desired mutation. This inefficiency is largely due to the difficulty of delivering DTs to be readily available at the target locus, in conjunction with the prevalence of an alternate and mutagenic DNA repair pathway, non-homologous end joining. In this work, we have applied T-DNA amplification to deliver multiple copies of a DT into plant cells to stimulate gene targeting. We used a native Arabidopsis retrotransposon (RT), ONSEN, to create a vector capable of amplifying the number of T-DNA copies that are incorporated into the Arabidopsis genome via Agrobacterium-mediated transformation (floral dip). We found that the RT vector with an incorporated DT sequence, as well as the T-DNA sequences surrounding the RT vector within the T-DNA left and right ii borders, were amplified as large concatenations of variable copy number in populations of T1 plants. Interestingly, the number of insertions was still a typical one or two sites, as is commonly observed in Arabidopsis floral transformation. Investigating the mechanism behind the RT-based T-DNA amplification, we observed that the amplification likely occurs in the plant cell at or before the point of insertion into the genome. We also found that the long terminal repeat sequences associated with the RT vector were necessary to create the amplification, and that replacing these repeats with random repeat sequences of the same length and GC content was sufficient to detect a similar level of amplification. Analyzing the junctions between T-DNA copies within the concatenation, we also provide evidence that the hallmarks of DNA polymerase theta-mediated end joining (TMEJ), which plays a major role in the incorporation of T-DNAs into the plant genome, could be detected between the T-DNA copies within the concatenation, pointing to a role for TMEJ in the T-DNA amplification. Finally, given that the RT vector could create multi-copy T-DNA insertions containing a DT, we applied the RT construct to a gene targeting system to assess if the increased DT copies could lead to more efficient gene targeting. We found that lines that had been transformed with the RT system were approximately three times as likely to generate a successful gene targeting edit, indicating the utility of the RT vector. The likelihood of detecting successful gene targeted modifications was also positively correlated with the T-DNA/DT copy number. Thus, in this study we demonstrate that the RT vector can be useful in situations in which multiple copies of a transformed sequence is desirable.
Recommended Citation
Dickinson, Lauren, "Increasing Gene Targeting Efficiency Through T-DNA Amplification in Plants" (2023). Yale Graduate School of Arts and Sciences Dissertations. 1030.
https://elischolar.library.yale.edu/gsas_dissertations/1030
