Comparison of different dCas9-activator complexes during activation of the pancreatic genes by CRISPR method

Diabetes mellitus is a metabolic disease characterized by the absence of insulin hormone or inadequate effect, characterized by hyperglycemia. There are two types of insulin-dependent type 1 and insulin-independent type 2 diabetes. In both diabetes, insulin deficiency is seen as the pancreatic beta cells that produce insulin are damaged. As an alternative to today's treatment methods, it is planned to produce β cells from different cell types and to permanently treat diabetes disease as a result of the transplantation of these cells to the person. Reprogramming non- β cells to insulin-producing cells offer potentially new regenerative approaches for the treatment of diabetes. Ngn3 and MafA genes, along with the Pdx1 gene, have been shown to be essential transcription factors in beta cell formation and differentiation. With the CRISPR/Cas system, which has been used in gene regulation in recent years, it has become much easier to increase/decrease the activation of a gene or to silence the gene. To increase activation of a gene, dCas9 protein and activator domain structures are used. VP64, VPR and p300 are the most frequently used activator domains. However, the activation efficiencies of these activators differ for each gene. The distance of the promoter region from which the activators are directed to the transcription start point is another parameter that affects the activation efficiency and again differs for each gene. The aim of this thesis is to determine the most ideal gRNA promoter regions and the most ideal activator domain (VP64, VPR and P300) for activation of Pdx1 and Ngn3 genes in the CRISPR/dCas9 activation system. Then the most ideal gRNA and activator domain combination is to program the β cell through the activation of endogenic genes. As a result of the studies, the promoter regions to be targeted for the activation of the Pdx1 and Ngn3 gene were determined. In addition, it was determined that the activator domain that increases activation most for both genes is VPR. It has been observed that β cell-like cells that can produce insulin are formed through the activation of endogenic genes with CRISPR-dCas9.