Development of a CRISPR systemfor gene knockouts in Diplomonad parasites
Abstract: This thesis describes the attempt to implement the CRISPR-Cas9 system in the two diplomonad pathogens: Giardia intestinalis and Spironucleus salmonicida. Two approaches of the implementation were done: first the Cas9 was expressed, followed by expression of the guide RNA. Additionally, to extend the understanding of how small nuclear RNAs are processed in Giardia intestinalis, two putative RNA processing enzymes were expressed and localized within the cells. The results show expression of Cas9 in both G. intestinalis and S .salmonicida. However, the Cas9 does not translocate to the nuclei, which is crucial for a functional gene knockout system. The approach where the guide RNA is expressed show a successful expression. In the experiments with the two putative RNA processing enzymes expression is observed in both cases, where one of the enzymes is localized to the nuclei.
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