Increasing the efficiency of gene targeting (GT) as a genome editing tool in plants has been an important goal in plant biotechnology. Improvements have been made using sequence-specific nucleases... Show moreIncreasing the efficiency of gene targeting (GT) as a genome editing tool in plants has been an important goal in plant biotechnology. Improvements have been made using sequence-specific nucleases such as CRISPR/Cas9 to induce DNA double strand breaks at target loci and activate repair via homologous recombination (HR). GT can then be achieved by HR-mediated integration of an artificial repair template, sharing homology with the target locus. Further improvements have been made with the in planta GT method, in which the repair template is pre-inserted in the genome and can be excised by nucleases. Although these improvements led to substantial increases in GT efficiency, GT is still not efficient enough to be feasible for crop biotechnology. This thesis describes strategies to further improve GT efficiency in the model plant Arabidopsis thaliana. One of these strategies was to perform in planta GT in meiocytes, cells that already have a higher rate of HR. Another strategy was to find new Arabidopsis mutants with increased GT frequencies and to identify genes involved in this phenotype. In the end, this may lead to a better understanding of the mechanisms underlying GT and these may be used to realize higher GT frequencies in plants. Show less
Double-strand breaks (DSBs) are one of the most lethal forms of DNA damage. To prevent this, cells have evolved complex and highly conserved systems to detect these lesions, signal their presence,... Show moreDouble-strand breaks (DSBs) are one of the most lethal forms of DNA damage. To prevent this, cells have evolved complex and highly conserved systems to detect these lesions, signal their presence, trigger various downstream events and finally bring about repair. Two main pathways are used for DNA DSB repair: Homologous Recombination (HR) and Non-Homologous End-Joining (NHEJ). Both of them function together to maintain genome integrity. At least two NHEJ pathways have been identified: the classic NHEJ pathway (c-NHEJ) and the backup-NHEJ pathway (b-NHEJ) also called alternative-NHEJ (a-NHEJ) or microhomology-mediated end-joining (MMEJ). Agrobacterium tumefaciens is widely used as a vector to produce genetically modified plants. Agrobacterium-mediated genetic transformation involves the transfer of T-DNA from its tumor-inducing plasmid to the host cell nucleus, where it integrates into the plant genome. However, the molecular mechanism of T-DNA integration is still unclear. T-DNAs can integrate at artificially induced DSBs, which suggests that DSB repair mechanisms are probably involved in T-DNA integration in plants. Arabidopsis NHEJ mutants have subsequently been studied for T-DNA integration. However, the results obtained by different research groups were variable and revealed either no or limited negative effects. Show less
Agrobacterium tumefaciens is a phytopathogen which is known to be the causal agent of crown gall disease in dicotyledonous plants. Virulent Agrobacterium strains are always carrier of a tumor... Show moreAgrobacterium tumefaciens is a phytopathogen which is known to be the causal agent of crown gall disease in dicotyledonous plants. Virulent Agrobacterium strains are always carrier of a tumor-inducing (Ti) plasmid. As a result of the expression of Vir proteins, a section of this plasmid termed the T-DNA is transferred via a type four secretion system (T4SS) as a T-strand to the recipient cell where it can integrate into the genomic DNA of the recipient cell. In this thesis the prerequisites of T-DNA circle formation using yeast as a model were assayed. It was found that the homologous repair (HR) protein Rad52 is of importance to the formation of T-DNA circles. In addition to this, the effects of double strand break (DSB) induction on the efficiency of T-DNA integration via the HR pathway in yeast and the role the nucleosome occupancy of the target locus has on DSB induction were assayed. It was found that a decrease in the nucleosome occupancy of a target locus had no measurable impact on the efficiency of T-DNA integration at this locus. However, the combined translocation of nuclease proteins aimed at the target locus and T-strands during AMT greatly facilitated HR-mediated integration of T-DNA. Show less
