Akademik Veri Yönetim Sistemi
4th International Conference on Applied Engineering and Natural Sciences, Konya, Türkiye, 10 - 13 Kasım 2022, ss.77
Diabetes is a lifelong disease that develops when the secretory gland called the pancreas does
not produce enough insulin hormone in your body or the insulin hormone it produces cannot be used
effectively. Reprogramming insulin-producing beta cells may be a new treatment approach. It has been
reported that NEUROG3, PDX1 and MAFA genes are the most important transcription factors in beta cell
formation. CRISPR-Cas9 genome editing technology has recently been used in genome modifications.
Activation of the target gene is provided by targeting specific genes with dCas9-activator domains. VP64,
VPR, and p300 are the most commonly used activator domains. However, the activation efficiency of these
activators may differ for each gene.
The aim of this study is to perform gene activation by scanning the upstream region of the PDX1 gene with
10 different gRNAs and three different activation domains (VP64, VPR and P300). The effect of each
sgRNA designed with the CRISPR-ERA (http://crispr-era.stanford.edu/) program on the activation of the
endogenic PDX1 gene in the HEK293 cell line was analyzed by Real Time-QPCR. Immunofluorescent
staining was performed to determine the protein level differences of PDX1. The 4 sgRNAs with the highest
activation were selected and transfection was performed simultaneously. The best activation was seen in
VPR, p300 and VP64, respectively.
As a result of the studies, the most suitable sgRNA sequences for the activation of the PDX1 gene were
determined. The VPR activator domain provided the highest activation of the PDX1 gene.