CRISPR/Cas9-Mediated genome editing in grapevine protoplasts

Yıl 2025, Cilt: 62 Sayı: 1, 117 - 131, 14.03.2025

Hilal Betul Kaya

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

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

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

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.

Kaynakça

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