Different dCas9-Activator Complexes During Activation of the Neurog3


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

4th International Conference on Applied Engineering and Natural Sciences, Konya, Turkey, 10 - 13 November 2022, pp.78

  • Publication Type: Conference Paper / Summary Text
  • City: Konya
  • Country: Turkey
  • Page Numbers: pp.78
  • Akdeniz University Affiliated: Yes

Abstract

Diabetes mellitus is a metabolic disease characterized by the absence of insulin hormone or inadequate effect, characterized by hyperglycemia. Reprogramming of insulin-producing β cells as an alternative therapy offers new regenerative approaches. It has been shown that the basic transcription factors in β-cell formation and differentiation are NEUROG3, PDX1, and MAFA genes. The CRISPR/Cas system, which has been used in gene editing in recent years, can be used with the dCas9 protein and activator domains to increase/reduce the activation of a gene or to silence the gene. 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 determine the most ideal gRNA promoter regions and the most ideal activator domain (VP64, VPR, and P300) for activation of NEUROG3 gene in the CRISPR/dCas9 activation system. With the CRISPR-ERA (http://crispr-era.stanford.edu/) tool, 8 different sgRNAs targeting different regions of the promoter region of the NEUROG3 gene were designed. The effect of each sgRNA on the activation of the endogenic NEUROG3 gene in the HEK293 cell line was analyzed by Real Time-QPCR. Immunofluorescent staining was performed to determine the protein level differences of NEUROG3. Then, gene transcription and translation were analyzed by simultaneously transfecting the 4 sgRNAs with the highest activation when individually transfected. Among the activators used, the best activation was seen in VPR, p300, and VP64, respectively. As a result of the studies, the most suitable sgRNA sequences and VPR activator domain provided the highest activation for the activation of the NEUROG3 gene were determined. Activation was demonstrated by performing both transcript level and protein level analyses. It was observed that the cell morphology was changed by the activation of the endogenic NEUROG3 gene with CRISPR-dCas9.