Determination of the relationship between diabetes and the CHOP (DDIT3) gene involved in endoplasmic reticulum stress using the CRISPR/Cas9 system

ER stress plays a critical role in the pathogenesis of diabetes. In this thesis, the induction conditions of ER stress in the MIN6 pancreatic β-cell line were determined using MTT and RT-PCR analyses following tunicamycin treatment. Increased ER stress activates the UPR pathway, leading to a strong induction of CHOP protein expression—a transcription factor—and consequently disrupting the cellular stress–apoptosis balance. Based on the hypothesis that CRISPR/Cas9-mediated deletion of the CHOP gene, which encodes the CHOP protein, may alleviate the effects of ER stress, CHOP was successfully knocked out, and changes in the expression of diabetes-related genes were examined under conditions with and without ER stress, as well as before and after stress induction, using RT-PCR analyses. Additionally, the effects of ER stress—which plays a central role in diabetes pathogenesis—on glucose-stimulated insulin secretion were evaluated using ELISA analyses. Through these findings, it was aimed to contribute to the literature regarding the impact of gene-editing approaches targeting CHOP on diabetes pathogenesis. The results demonstrated that tunicamycin-induced ER stress increased the expression of several diabetes-related genes in the MIN6 cell line. Moreover, the findings indicate that CHOP deletion reduces cellular stress burden, stabilizes the expression profiles of stress-associated genes (IGF2BP2, CDKAL1, HNF1A, TCF7L2), and particularly contributes to the preservation of insulin secretion.