Developing genetic manipulation platforms for Naegleria gruberi

Naegleria gruberi, is a free-living microbial eukaryote, which belongs to the group of Excavates. The organism is widely distributed especially in aquatic environments and it is famous for its ability to transform from an amoeba to flagellate and cyst forms depending on its surroundings. In silico examination of the published Naegleria gruberi genome opened up the possibility of functional exploration of the organism by molecular cell biology. Despite this, several attempts to genetically transfect or genetically manipulate the organism have been unsuccessful so far due to the unique morphological and cellular adaptations of the organisms, but also due to its resistance to certain basic antibiotics. Using a series of protocols and combination of cell biological tools, we attempted to genetic manipulate Naegleria using both CRISPR/Cas9 and traditional genetic transformation protocols. Preliminarily data from these investigations will be discussed. This work is going to provide traits found in the last eukaryotic common ancestor and provide a model for investigating the cell biology of other free-living eukaryotes.