Fabian Hitz (Universität Bielefeld)
Fabian Hitz's Master thesis was awarded with the Prize for the Best Plant Science Master Thesis, which was carried out at Bielefeld University in the year 2025 with the title:
Establishment of microinjection-based gene editing and enabling natural photonics in the diatom Coscinodiscus
The thesis established a novel transformation method in diatoms using microinjection as an alternative strategy to introduce foreign molecules into cells.
The establishment of microinjection was performed to enable the introduction of DNA, RNA or proteins for gene editing in Coscinodiscus wailesii. Single guide RNAs (sgRNA) were designed to target the adenine phosphoribosyltransferase (APT) gene as biallelic knockout mutations convey resistance to the adenine analog 2-fluoroadenine (2-FA). Firstly, the tolerance of C. wailesii wildtype cells to 2-FA was assessed and it was determined that their susceptibility was sufficient for mutant screening. Secondly, microinjection was successfully established and injected cells had a survival rate of up to 70 %. Thirdly, three different approaches were tested to introduce an APT knockout or convey antibiotic resistance. Plasmid DNA, Cas9 mRNA or protein with the previously mentioned sgRNAs were injected at varying concentrations. Even though this thesis was not able to create biallelic knockouts of the APT gene, it demonstrated that targeting this gene is a feasible method to easily detect knockouts in C. wailesii. Because of its large size, C. wailesii transformation with traditional methods can be difficult and microinjection demonstrated to be a promising alternative. Gene editing in C. wailesii can be used to study the formation of the siliceous diatom cell wall by reverse genetics. The cell wall of C. wailesii is of particular interest as it shows characteristics of slab photonic crystals (i.e. it can be used to manipulate light in a wavelength dependent manner). Studying the underlying mechanisms that convey such optical properties is crucial to understanding how diatom cell walls can be useful for industrial applications, for example optical instruments, in the future.
___
Fabian Hitz conducted this work in the working group Cell, Molecular Biology and Genomics of Atle Magnar Bones at the Norwegian University of Science and Technology. The thesis was supervised by Dr. Tore Brembu and Dr. Romy Schmidt-Schippers.
