Abstract
Purpose
Viruses, such as Ebola virus (EBOV), evolve rapidly and threaten the human health. There is a great demand to exploit efficient gene-editing techniques for the identification of virus to probe virulence mechanism for drug development.
Methods
Based on lambda Red recombination in Escherichia coli (E. coli), counter-selection, and in vitro annealing, a high-efficiency genetic method was utilized here for precisely engineering viruses. EBOV trVLPs assay and dual luciferase reporter assay were used to further test the effect of mutations on virus replication.
Results
Considering the significance of matrix protein VP24 in EBOV replication, the types of mutations within vp24, including several single-base substitutions, one double-base substitution, two seamless deletions, and one targeted insertion, were generated on the multi-copy plasmid of E. coli. Further, the length of the homology arms for recombination and in vitro annealing, and the amount of DNA cassettes and linear plasmids were optimized to create a more elaborate and cost-efficient protocol than original approach. The effects of VP24 mutations on the expression of a reporter gene (luciferase) from the EBOV minigenome were determined, and results indicated that mutations of key sites within VP24 have significant impacts on EBOV replication.
Conclusion
This precise mutagenesis method will facilitate effective and simple editing of viral genes in E. coli.
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Acknowledgements
We are grateful to Prof. Hailong Wang from Shandong University, China, for providing E. coli GBred-gyrA462, E. coli GB2005 and plasmid pR6K-neo-ccdB.
Supporting information
Supplementary Figure 1— Restriction analysis of obtained clones during the process of constructing mutants.
Supplementary Figure 2— BamHI＋HindIII restriction analysis of M1* (recombinant plasmids) obtained in LCHR using different amounts of DNA cassette.
Supplementary Figure 3— HindIII restriction analysis of M1* (recombinant plasmids) obtained in LCHR using different lengths of homology arm (ha).
Supplementary Figure 4— HindIII restriction analysis of M1 obtained in vitro annealing using different amounts of linear plasmid.
Supplementary Figure 5— HindIII restriction analysis of M1 obtained in vitro annealing using different lengths of circularized homology arm (ca).
Supplementary Table 1— Primers used in this study.
Supplementary Table 2— Colony numbers per mL of recovery cultures after electroporation and the editing efficiency in different conditions.
Funding
This work was supported by the Anhui Provincial Natural Science Foundation (Grant no. 2208085Y09), the National Natural Science Foundation of China (Grant nos. 32170073 and 31972930), and the Natural Science Research Project of Colleges and Universities in Anhui Province (Grant nos. KJ2021A0077 and 2022AH050063).
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JY: conceptualization, methodology, investigation, and original draft writing. MFZ: methodology and investigation. LBL: formal analysis and data curation. PPW: investigation. HW, LZ, CZX and BCZ: supervision, project administration, and writing-reviewing. All authors have read and approved the final manuscript.
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Yi, J., Zhang, M., Zhu, L. et al. High-efficiency genetic engineering toolkit for virus based on lambda red-mediated recombination. Biotechnol Lett 45, 1327–1337 (2023). https://doi.org/10.1007/s10529-023-03412-9
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DOI: https://doi.org/10.1007/s10529-023-03412-9