Comparison of dCas9-activator complexes for the activation of PDX1 and NGN3 pancreatic genes using the CRISPR system

Fatma Akçakale Kaba; Ersin Akıncı; Mehmet Fatih Cengiz; Adem Kaba

Makale detayları

Comparison of dCas9-activator complexes for the activation of PDX1 and NGN3 pancreatic genes using the CRISPR system

Yazarlar:

Fatma Akçakale Kaba | Ersin Akıncı | Mehmet Fatih Cengiz | Adem Kaba

Yayıncı:

Trakya University

Ana Başlıklar:

Cell Development | Proliferation and Death | Synthetic Biology | Biochemistry and Cell Biology (Other) | Epigenetics

Dergi:

Trakya University Journal of Natural Sciences Vol.26 Issue 1, pp.49-59

Belge Tipi:

Makale

Yayın Tarihi:

4/2025

Belge Dili:

İngilizce

Document ID (Org No.):

2001334436

pISSN:

2528-9691

Anahtar kelimeler:

Özet:

 Diabetes mellitus is a prevalent metabolic disorder characterized by persistently high blood glucose levels due to insufficient insulin production or response. Although significant progress has been made in symptom management, a definitive cure remains unavailable. This study presents a novel approach to generate insulin-producing β cells from non-β cell sources using the CRISPRdCas9 gene activation system. We focused on enhancing β-cell differentiation by activating PDX1 and NGN3, two key transcription factors in pancreatic development. To optimize this process, we compared three activator domains VP64, VPR, and p300 and found VPR to be the most effective. Specifically, the VPR activator led to a 19-fold increase in PDX1 expression and a 256-fold increase in NGN3 expression when combined with four gRNAs. This superiority is likely due to its stronger transcriptional activation capability, which enhances gene expression more efficiently than VP64 and p300. Gene and protein expression were confirmed through RT-qPCR and immunostaining, respectively. Our findings demonstrate that CRISPRdCas9-mediated gene activation can effectively induce β-cell differentiation, offering a promising approach for type 1 diabetes therapy, where β-cell loss is a major challenge. Future studies should explore the long-term functionality and stability of these β-like cells in preclinical models to further assess their therapeutic potential. By optimizing transcription factor activation, our study provides new insights into β-cell regeneration, advancing the field of gene-based diabetes treatments.

 Diabetes mellitus is a prevalent metabolic disorder characterized by persistently high blood glucose levels due to insufficient insulin production or response. Although significant progress has been made in symptom management, a definitive cure remains unavailable. This study presents a novel approach to generate insulin-producing β cells from non-β cell sources using the CRISPRdCas9 gene activation system. We focused on enhancing β-cell differentiation by activating PDX1 and NGN3, two key transcription factors in pancreatic development. To optimize this process, we compared three activator domains VP64, VPR, and p300 and found VPR to be the most effective. Specifically, the VPR activator led to a 19-fold increase in PDX1 expression and a 256-fold increase in NGN3 expression when combined with four gRNAs. This superiority is likely due to its stronger transcriptional activation capability, which enhances gene expression more efficiently than VP64 and p300. Gene and protein expression were confirmed through RT-qPCR and immunostaining, respectively. Our findings demonstrate that CRISPRdCas9-mediated gene activation can effectively induce β-cell differentiation, offering a promising approach for type 1 diabetes therapy, where β-cell loss is a major challenge. Future studies should explore the long-term functionality and stability of these β-like cells in preclinical models to further assess their therapeutic potential. By optimizing transcription factor activation, our study provides new insights into β-cell regeneration, advancing the field of gene-based diabetes treatments.

 Diabetes mellitus is a prevalent metabolic disorder characterized by persistently high blood glucose levels due to insufficient insulin production or response. Although significant progress has been made in symptom management, a definitive cure remains unavailable. This study presents a novel approach to generate insulin-producing β cells from non-β cell sources using the CRISPRdCas9 gene activation system. We focused on enhancing β-cell differentiation by activating PDX1 and NGN3, two key transcription factors in pancreatic development. To optimize this process, we compared three activator domains VP64, VPR, and p300 and found VPR to be the most effective. Specifically, the VPR activator led to a 19-fold increase in PDX1 expression and a 256-fold increase in NGN3 expression when combined with four gRNAs. This superiority is likely due to its stronger transcriptional activation capability, which enhances gene expression more efficiently than VP64 and p300. Gene and protein expression were confirmed through RT-qPCR and immunostaining, respectively. Our findings demonstrate that CRISPRdCas9-mediated gene activation can effectively induce β-cell differentiation, offering a promising approach for type 1 diabetes therapy, where β-cell loss is a major challenge. Future studies should explore the long-term functionality and stability of these β-like cells in preclinical models to further assess their therapeutic potential. By optimizing transcription factor activation, our study provides new insights into β-cell regeneration, advancing the field of gene-based diabetes treatments.

 Türkçe Diabetes mellitus, yetersiz insülin üretimi veya yanıtı nedeniyle kalıcı olarak yüksek kan glukoz seviyeleriyle karakterize edilen yaygın bir metabolik bozukluktur. Belirtilerin yönetimi konusunda önemli ilerlemeler kaydedilmiş olsa da kesin bir tedavi halen mevcut değildir. Bu çalışma, CRISPRdCas9 gen aktivasyon sistemini kullanarak insülin üreten β-benzeri hücrelerin β hücresi olmayan kaynaklardan elde edilmesine yönelik yeni bir stratejiyi araştırmaktadır. Pankreas gelişiminde kilit rol oynayan iki transkripsiyon faktörü olan PDX1 ve NGN3ün aktivasyonu yoluyla β hücre farklılaşmasını artırmaya odaklandık. Bu süreci optimize etmek için üç farklı aktivatör bölgesini VP64, VPR ve p300 karşılaştırdık ve VPRnin en etkili aktivatör olduğunu belirledik. Özellikle VPR aktivatörü, dört gRNA ile birlikte kullanıldığında PDX1 ekspresyonunda 19 kat, NGN3 ekspresyonunda ise 256 kat artış sağladı. Bu üstünlüğün, VPRnin VP64 ve p300e kıyasla daha güçlü transkripsiyon aktivasyonu sağlamasından kaynaklandığını düşünüyoruz. Gen ve protein ekspresyonu sırasıyla RT-qPCR ve immün boyama teknikleriyle doğrulandı. Bulgularımız, CRISPRdCas9 aracılı gen aktivasyonunun β hücre farklılaşmasını etkili bir şekilde indükleyebileceğini ve β hücre kaybının büyük bir sorun olduğu tip 1 diyabet tedavisi için umut vadeden bir yaklaşım sunduğunu göstermektedir. Gelecekteki çalışmalar, bu β-benzeri hücrelerin uzun vadeli fonksiyonelliğini ve stabilitesini preklinik modellerde inceleyerek terapötik potansiyellerini daha kapsamlı bir şekilde değerlendirmelidir. Transkripsiyon faktörü aktivasyonunu optimize eden bu çalışma, β hücre rejenerasyonu hakkında yeni içgörüler sunarak gen temelli diyabet tedavileri alanına önemli bir katkı sağlamaktadır.

Makale detayları

Comparison of dCas9-activator complexes for the activation of PDX1 and NGN3 pancreatic genes using the CRISPR system

Yazarlar:

Fatma Akçakale Kaba | Ersin Akıncı | Mehmet Fatih Cengiz | Adem Kaba

Yayıncı:

Trakya University

Ana Başlıklar:

Cell Development | Proliferation and Death | Synthetic Biology | Biochemistry and Cell Biology (Other) | Epigenetics

Dergi:

Trakya University Journal of Natural Sciences Vol.26 Issue 1, pp.49-59

Belge Tipi:

Makale

Yayın Tarihi:

4/2025

Belge Dili:

İngilizce

Document ID (Org No.):

2001334436

pISSN:

2528-9691

CRISPR/dCas9Activation

Insulin-Producing β-like Cells

PDX1 and NGN3 Gene Regulation

β-cell Differentiation and Regeneration

Diabetes Gene Therapy

Sayı

Makale detayları

Comparison of dCas9-activator complexes for the activation of PDX1 and NGN3 pancreatic genes using the CRISPR system

Yazarlar: Fatma Akçakale Kaba | Ersin Akıncı | Mehmet Fatih Cengiz | Adem Kaba

Yayıncı: Trakya University

Ana Başlıklar: Cell Development | Proliferation and Death | Synthetic Biology | Biochemistry and Cell Biology (Other) | Epigenetics

Dergi: Trakya University Journal of Natural Sciences Vol.26 Issue 1, pp.49-59

Belge Tipi: Makale

Yayın Tarihi: 4/2025

Belge Dili: İngilizce

Document ID (Org No.): 2001334436

pISSN: 2528-9691

CRISPR/dCas9Activation

Insulin-Producing β-like Cells

PDX1 and NGN3 Gene Regulation

β-cell Differentiation and Regeneration

Diabetes Gene Therapy

Sayı

Yazarlar:

Fatma Akçakale Kaba | Ersin Akıncı | Mehmet Fatih Cengiz | Adem Kaba

Yayıncı:

Trakya University

Ana Başlıklar:

Cell Development | Proliferation and Death | Synthetic Biology | Biochemistry and Cell Biology (Other) | Epigenetics

Dergi:

Trakya University Journal of Natural Sciences Vol.26 Issue 1, pp.49-59

Belge Tipi:

Makale

Yayın Tarihi:

4/2025

Belge Dili:

İngilizce

Document ID (Org No.):

2001334436

pISSN:

2528-9691