Improved Protoplast Production Protocol for Fungal Transformations Mediated by CRISPR/Cas9 in Botrytis cinerea Non-Sporulating Isolates
Abstract
:1. Introduction
2. Results
2.1. Optimization of the Protocol for Protoplasts Isolation from Non-Sporulating B. cinerea Isolates
2.2. Optimized Protocol for Protoplast Regeneration
2.3. Protoplast Suitability for Transformation by CRISPR/Cas9
3. Discussion
4. Materials and Methods
4.1. Microorganisms and Growth Conditions
4.2. Time Optimization for Protoplast Obtention
4.3. Type of Culture Optimization for Protoplast Isolation
- -
- Five mycelium plugs over 100 mL of HA medium in 500 mL flasks and 180 rpm orbital shaking (classical method for protoplasts isolation from the isolate B05.10 from freshly germinated spores).
- -
- Five mycelium plugs over 20 mL of HA medium in Petri dishes and 40 rpm orbital shaking.
- -
- Five mycelium plugs on 20 mL of HA medium supplemented with 2% agar and covered with a layer of cellophane.
4.4. Enzyme Optimization for Protoplast Isolation
4.5. Protoplast Preparation
4.6. Protoplast Regeneration
4.7. Evaluation of Protoplast Usefulness by CRISPR/Cas9 Transformation
4.8. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Isolate | Condition | False Positive | No. of Colonies with hph Insertion | No. of Heterokaryon Colonies | No. of Homokaryon Colonies | Total Number of Colonies Screened | Transformation Efficiency (Percentage of Mutants) | Transformation Efficiency (Percentage of Homokaryon) |
---|---|---|---|---|---|---|---|---|
B05.10 | No DNA | 12 | 0 | - | - | 12 | 0% | - |
pNDH-OGG | 13 | 2 | - | - | 15 | 13% | - | |
No DNA + Cas9 | 10 | 0 | - | - | 10 | 0% | - | |
pNDH-OGG + Cas9 | 21 | 4 | - | - | 25 | 16% | - | |
No DNA + Cas9 + BcniaD sgRNA | 10 | 0 | - | - | 10 | 0% | - | |
pNDH-OGG + Cas9 + BcniaD sgRNA | 2 | 49 | 40 | 9 | 51 | 96% | 17% | |
B371 | No DNA | 12 | 0 | - | - | 12 | 0% | 0% |
pNDH-OGG | 15 | 1 | - | - | 16 | 6.3% | - | |
No DNA + Cas9 | 10 | 0 | - | - | 10 | 0% | - | |
pNDH-OGG + Cas9 | 20 | 3 | - | - | 23 | 13.3% | - | |
No DNA + Cas9 + BcniaD sgRNA | 10 | 0 | - | - | 10 | 0% | ||
pNDH-OGG + Cas9 + BcniaD sgRNA | 1 | 51 | 44 | 7 | 52 | 98% | 13.5% | |
B459 | No DNA | 13 | 0 | - | - | 13 | 0% | 0% |
pNDH-OGG | 14 | 2 | - | - | 16 | 12.5% | - | |
No DNA + Cas9 | 10 | 0 | - | - | 10 | 0% | - | |
pNDH-OGG + Cas9 | 18 | 4 | - | - | 22 | 18.1% | - | |
No DNA + Cas9 + BcniaD sgRNA | 9 | 0 | - | - | 9 | 0% | - | |
pNDH-OGG + Cas9 + BcniaD sgRNA | 2 | 52 | 46 | 8 | 54 | 96.3% | 14.8% |
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Coca-Ruiz, V.; Cabrera-Gómez, N.; Collado, I.G.; Aleu, J. Improved Protoplast Production Protocol for Fungal Transformations Mediated by CRISPR/Cas9 in Botrytis cinerea Non-Sporulating Isolates. Plants 2024, 13, 1754. https://doi.org/10.3390/plants13131754
Coca-Ruiz V, Cabrera-Gómez N, Collado IG, Aleu J. Improved Protoplast Production Protocol for Fungal Transformations Mediated by CRISPR/Cas9 in Botrytis cinerea Non-Sporulating Isolates. Plants. 2024; 13(13):1754. https://doi.org/10.3390/plants13131754
Chicago/Turabian StyleCoca-Ruiz, Víctor, Nuria Cabrera-Gómez, Isidro G. Collado, and Josefina Aleu. 2024. "Improved Protoplast Production Protocol for Fungal Transformations Mediated by CRISPR/Cas9 in Botrytis cinerea Non-Sporulating Isolates" Plants 13, no. 13: 1754. https://doi.org/10.3390/plants13131754
APA StyleCoca-Ruiz, V., Cabrera-Gómez, N., Collado, I. G., & Aleu, J. (2024). Improved Protoplast Production Protocol for Fungal Transformations Mediated by CRISPR/Cas9 in Botrytis cinerea Non-Sporulating Isolates. Plants, 13(13), 1754. https://doi.org/10.3390/plants13131754