Asma protoplastlarında CRISPR/Cas9 aracılı genom düzenleme

Hilal Betul Kaya

doi:10.20289/zfdergi.1432614

TR EN

Asma protoplastlarında CRISPR/Cas9 aracılı genom düzenleme

Öz

Amaç: Bu çalışmanın amacı, CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) aracılı genom düzenleme yöntemi kullanılarak asma protoplastlarında hedefe yönelik mutasyonun gerçekleştirilmesidir. Materyal ve Yöntem: Chardonnay asma çeşidinde CRISPR/Cas9 aracılı genom düzenleme için hedef gene uygun gRNA tasarımı yapılarak, hem gRNA’yı hem de Cas9 ve GFP genlerini içeren final CRISPR/Cas9 vektörü elde edilmiştir. Asma yapraklarından protoplast izolasyonu ve transformasyonu gerçekleştirilerek, transformasyon ve mutasyon verimliliği analiz edilmiştir. Araştırma Bulguları: Çalışmada Chardonnay yapraklarından yapılan protoplast izolasyonunda 1 g taze yapraktan 1x107 protoplast elde edilmiştir. VvPDS genini hedef alan vektörün (~10 kb) protoplastlarda transformasyon verimliliği %40-60 iken, sadece GFP genini içeren vektörde (~3 kb), transformasyon verimliliği %80-90 olarak bulunmuştur. Vektör büyüklüğü transformasyon verimliliğini büyük oranda etkilemiş, vektör büyüklüğü arttıkça verimlilik azalmıştır. Transformasyon gerçekleşmesine rağmen, hedeflenen mutasyon doğrulanamamıştır. Sonuç: Çalışmada asma protoplastlarında CRISPR/Cas9 aracılı genom düzenleme için ilk aşama olan gRNA tasarımından son aşama olan protoplast transformasyonuna kadar uygulanan basamaklar başarılı bir şekilde gerçekleştirilerek, sistemin sorunsuz uygulanabilirliği gösterilmiştir. Uygulanan protokoller ve elde edilen sonuçlar asmada hedef mutasyonların gerçekleştirilmesi amacıyla yapılacak olan diğer çalışmalarda kullanılabilecektir.

Anahtar Kelimeler

CRISPR/Cas9, genom düzenleme, PDS geni, protoplast, Vitis vinifera

CRISPR/Cas9-Mediated genome editing in grapevine protoplasts

Öz

Objective: This study aims to perform targeted mutation in grapevine protoplasts using the CRISPR/Cas9-mediated genome editing method. Material and Methods: For CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9)-mediated genome editing in the Chardonnay cultivar, a gRNA design targeting the desired gene was performed, resulting in obtaining a final CRISPR/Cas9 vector containing both the gRNA and Cas9 and GFP genes. Protoplast isolation and transformation were performed using leaves, followed by analysis of transformation and mutation efficiency. Results: In the study, Chardonnay leaf protoplast isolation produced 1x107 protoplasts per 1 g of fresh leaves. The vector targeting the VvPDS gene (~10 kb) achieved a transformation efficiency of 40-60%, while the vector containing only the GFP gene (~3 kb) reached 80-90% efficiency. Vector size notably impacted transformation, with larger vectors reducing efficiency. Despite successful transformation, the presence of the targeted mutation could not be confirmed. Conclusion: The study successfully completed all stages from gRNA design, the initial step of CRISPR/Cas9-mediated genome editing in protoplasts, to the final protoplast transformation stage, showcasing the system's seamless usability. The protocols applied and the results obtained can be utilized in future studies aimed at implementing targeted mutations in grapevines.

Anahtar Kelimeler

CRISPR/Cas9, genom düzenleme, PDS geni, protoplast, Vitis vinifera

Destekleyen Kurum

Bu çalışma Manisa Celal Bayar Üniversitesi Bilimsel Araştırma Projeleri Koordinatörlüğü tarafından finansal olarak desteklenmiştir (BAP, Proje No; 2020-123). Yazar finansal destek için teşekkür eder.

Proje Numarası

2020-123

Teşekkür

Çalışmada kullanılan plazmitleri temin etmemi sağlayan Cornell Üniversitesi’nden Prof. Dr. Adam Bogdanove’a ve Minesota Üniversitesi’nden Dr. Colby Starker’a desteklerinden dolayı teşekkür ederim.

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Ayrıntılar

Birincil Dil

Türkçe

Konular

Biyoteknolojinin Genetik Modifikasyonsuz Kullanımı, Genetiği Değiştirilmiş Bahçe Bitkileri, Tarımsal Biyoteknoloji (Diğer)

Bölüm

Araştırma Makalesi

Yazarlar

Hilal Betul Kaya ^*
0000-0002-2543-7212
Türkiye

Erken Görünüm Tarihi

13 Mart 2025

Yayımlanma Tarihi

14 Mart 2025

Gönderilme Tarihi

8 Şubat 2024

Kabul Tarihi

2 Kasım 2024

Yayımlandığı Sayı

Yıl 2025 Cilt: 62 Sayı: 1

DOI

https://doi.org/10.20289/zfdergi.1432614

IZ

https://izlik.org/JA93BM98DP

APA

Kaya, H. B. (2025). Asma protoplastlarında CRISPR/Cas9 aracılı genom düzenleme. Journal of Agriculture Faculty of Ege University, 62(1), 117-131. https://doi.org/10.20289/zfdergi.1432614

AMA

1.Kaya HB. Asma protoplastlarında CRISPR/Cas9 aracılı genom düzenleme. Journal of Agriculture Faculty of Ege University. 2025;62(1):117-131. doi:10.20289/zfdergi.1432614

Chicago

Kaya, Hilal Betul. 2025. “Asma protoplastlarında CRISPR/Cas9 aracılı genom düzenleme”. Journal of Agriculture Faculty of Ege University 62 (1): 117-31. https://doi.org/10.20289/zfdergi.1432614.

EndNote

Kaya HB (01 Mart 2025) Asma protoplastlarında CRISPR/Cas9 aracılı genom düzenleme. Journal of Agriculture Faculty of Ege University 62 1 117–131.

IEEE

[1]H. B. Kaya, “Asma protoplastlarında CRISPR/Cas9 aracılı genom düzenleme”, Journal of Agriculture Faculty of Ege University, c. 62, sy 1, ss. 117–131, Mar. 2025, doi: 10.20289/zfdergi.1432614.

ISNAD

Kaya, Hilal Betul. “Asma protoplastlarında CRISPR/Cas9 aracılı genom düzenleme”. Journal of Agriculture Faculty of Ege University 62/1 (01 Mart 2025): 117-131. https://doi.org/10.20289/zfdergi.1432614.

JAMA

1.Kaya HB. Asma protoplastlarında CRISPR/Cas9 aracılı genom düzenleme. Journal of Agriculture Faculty of Ege University. 2025;62:117–131.

MLA

Kaya, Hilal Betul. “Asma protoplastlarında CRISPR/Cas9 aracılı genom düzenleme”. Journal of Agriculture Faculty of Ege University, c. 62, sy 1, Mart 2025, ss. 117-31, doi:10.20289/zfdergi.1432614.

Vancouver

1.Hilal Betul Kaya. Asma protoplastlarında CRISPR/Cas9 aracılı genom düzenleme. Journal of Agriculture Faculty of Ege University. 01 Mart 2025;62(1):117-31. doi:10.20289/zfdergi.1432614