University of Twente Student Theses


Early Detection of Cervical Cancer via Cas13a Mediated microRNA Detection

Rops, Joyce (2022) Early Detection of Cervical Cancer via Cas13a Mediated microRNA Detection.

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Abstract:Deregulated microRNAs (miRNAs) have recently been implicated in the development of cervical cancer, and therefore could be potential biomarkers. Validation by qPCR is complicated by the presence of miRNA length variant called isomiRs. Cas13a has shown to offer high-fidelity and rapid quantification of miRNA. Due to its high specificity, efficiency and ease of programming, CRISPR-Cas13a is an alternative candidate for isomiR discrimination. This thesis explores the possibility to create an isomiR discrimination tool using CRISPR-Cas13a in combination with strand displacement.\\ A Cas13a nucleic acid detection assay was optimized, reaching a LOD of 40 fM. It has been shown the association of the crRNA with Cas13a is the limiting factor in Cas13a activation. Next to that, it has been discovered that two isomiRs with one nucleotide length difference at the 3' end activate Cas13a with different efficiencies. Placeholders have been designed to use in a strand displacement reaction. However, these did not function as hoped. More research should be put into fundamental properties of this Cas13a homologue (LwaCas13a) to effectively design such placeholder RNA molecules. An attempt was made to optimize a Cas13a-based digital droplet assay to lower the LOD of the platform to levels found in urine, however future research should finish this. Once working, placeholder strand displacement in digital droplets using Cas13a holds great potential to become a extremely sensitive isomiR discrimination tool.
Item Type:Essay (Master)
Faculty:EEMCS: Electrical Engineering, Mathematics and Computer Science
Subject:35 chemistry, 42 biology, 44 medicine
Programme:Biomedical Engineering MSc (66226)
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