Activation of PDX1 gene with different activator domains by CRISPRdCas9


Creative Commons License

Kaba A., Akçakale Kaba F., Akıncı E., Cengiz M. F.

4th International Conference on Applied Engineering and Natural Sciences, Konya, Türkiye, 10 - 13 Kasım 2022, ss.77

  • Yayın Türü: Bildiri / Özet Bildiri
  • Basıldığı Şehir: Konya
  • Basıldığı Ülke: Türkiye
  • Sayfa Sayıları: ss.77
  • Akdeniz Üniversitesi Adresli: Evet

Özet

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.