Skip to main content
Login | Suomeksi | På svenska | In English

Activation of KCNQ1 expression in HEK293 cells using inducible CRISPR-dCas9-VPR tripartite transcriptional activator domain

Show full item record

Title: Activation of KCNQ1 expression in HEK293 cells using inducible CRISPR-dCas9-VPR tripartite transcriptional activator domain
Author(s): Kuncheva, Ema
Contributor: University of Helsinki, Faculty of Biological and Environmental Sciences
Degree program: Master's Programme in Genetics and Molecular Biosciences
Specialisation: Cell and Developmental Biology
Language: English
Acceptance year: 2022
Mutations in the KCNQ1 gene have been implicated in the onset of hypopituitarism. Regulating KCNQ1 expression would therefore enable future clinical research on the mechanism of the disease. CRISPR offers a flexible toolset for controlling genetic expression via knockout, knock-in, knockdown, and gene activation. Of these approaches, CRISPR activation (CRISPRa) is distinguished by its ability to induce gene overexpression in a cell’s native context, making it a valuable tool in the interrogation of genetic disorder pathogenesis. This thesis therefore tested the efficacy of a CRISPRa subsystem in increasing KCNQ1 expression. The CRISPRa subsystem, VPR, was chosen because of its high activation efficiency and the ease of controlling the activation system of its doxycycline-inducible mode of action. The cell line used for the experiment, HEK293, was similarly chosen because of its ease of culture and transfection. To validate the proper functioning of the activation system, expression rates of the related genes ASCL1 and GHRH were measured as positive controls. The activation system successfully upregulated the expression rates of the two genes. As the dCas9-VPR system is dependent on the Tet-ON operator for inducing activation in a controllable manner, a test for dCas9 leakage was conducted. RT-qPCR analysis showed the upregulation of ASCL1 expression in the uninduced state of the system, confirming the presence of dCas9-VPR leakage. The dCas9-VPR system finally aimed to test the expression rate of KCNQ1. Although one novel guide RNA successfully upregulated KCNQ1 expression, it did so inefficiently and its success was not shared by any of the other tested guide RNAs. Altogether, the dCas9-VPR system was successfully established in HEK293 cells, and the leakage of the inducible system was confirmed, however, KCNQ1 activation by CRISPRa requires further optimization.
Keyword(s): KCNQ1 CRISPR/Cas CRISPRa dCas9-VPR HEK293 Human embryonic kidney 293 cells

Files in this item

Files Size Format View
Kuncheva_Ema_thesis_2022.pdf 868.6Kb PDF

This item appears in the following Collection(s)

Show full item record