Dissertation

Strategies for the improvement of genome editing in Arabidopsis thaliana

Increasing the efficiency of gene targeting (GT) as a genome editing tool in plants has been an important goal in plant biotechnology.

Author

Strunks, G.D.

Date

11 September 2019

Links

Thesis in Leiden Repository

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 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.

• crispr/cas9
• dna repair
• double strand break
• gene targeting
• genome editing
• homologous recombination
• non-homologous end joining