Efficient Mutagenesis of Marek’s Disease Virus-Encoded microRNAs Using a CRISPR/Cas9-Based Gene Editing System
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cells and Virus
2.2. Construction of sgRNAs Plasmids
2.3. Generation of Viral miRNA Mutated RB-1B Viruses
2.4. Characterization of miR-KO RB-1B Viruses
2.5. Immunofluorescence Assay (IFA)
2.6. qRT-PCR Analysis for MDV-1 miRNA Expressions
2.7. The Growth Kinetics of the miR-KO RB-1B Viruses
2.8. Growth Kinetics of RB-1B∆M11 Virus in CEFs Overexpressing miR-M11
3. Results
3.1. The Efficacy of sgRNAs for Editing the Viral miRNA Using CRISPR/Cas9 System
3.2. Cloning and Purification of the Viral miRNA-Deletion Mutant RB-1B Viruses
3.3. Effects of miR-KO on the Expression of MDV Protein-Coding and Non-Coding Genes
3.4. Growth Kinetics of the miR-KO Mutant Viruses
3.5. Compensatory miR-M11 Overexpression Suppresses the Improved Growth of RB-1B∆M11 Virus
4. Discussion
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Luo, J.; Teng, M.; Zai, X.; Tang, N.; Zhang, Y.; Mandviwala, A.; Reddy, V.R.A.P.; Baigent, S.; Yao, Y.; Nair, V. Efficient Mutagenesis of Marek’s Disease Virus-Encoded microRNAs Using a CRISPR/Cas9-Based Gene Editing System. Viruses 2020, 12, 466. https://doi.org/10.3390/v12040466
Luo J, Teng M, Zai X, Tang N, Zhang Y, Mandviwala A, Reddy VRAP, Baigent S, Yao Y, Nair V. Efficient Mutagenesis of Marek’s Disease Virus-Encoded microRNAs Using a CRISPR/Cas9-Based Gene Editing System. Viruses. 2020; 12(4):466. https://doi.org/10.3390/v12040466
Chicago/Turabian StyleLuo, Jun, Man Teng, Xusheng Zai, Na Tang, Yaoyao Zhang, Ahmedali Mandviwala, Vishwanatha R. A. P. Reddy, Susan Baigent, Yongxiu Yao, and Venugopal Nair. 2020. "Efficient Mutagenesis of Marek’s Disease Virus-Encoded microRNAs Using a CRISPR/Cas9-Based Gene Editing System" Viruses 12, no. 4: 466. https://doi.org/10.3390/v12040466
APA StyleLuo, J., Teng, M., Zai, X., Tang, N., Zhang, Y., Mandviwala, A., Reddy, V. R. A. P., Baigent, S., Yao, Y., & Nair, V. (2020). Efficient Mutagenesis of Marek’s Disease Virus-Encoded microRNAs Using a CRISPR/Cas9-Based Gene Editing System. Viruses, 12(4), 466. https://doi.org/10.3390/v12040466