Abstract
The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 nuclease (Cas9) system is a powerful genome-editing tool for both chromosomal and extrachromosomal DNA. DNA viruses such as Epstein–Barr virus (EBV), which undergoes episomal replication in human cells, can be effectively edited by CRISPR/Cas9. We have demonstrated targeted editing of the EBV genome by CRISPR/Cas9 in several lines of EBV-infected cells. CRISPR/Cas9-based mutagenesis and genome engineering of EBV provides a new method for genetic analysis, which has some advantages over bacterial artificial chromosome-based recombineering. This approach might also prove useful in the cure of EBV infection. In this chapter, we use the knockout of the BART promoter as an example to detail the experimental procedures for construction of recombinant EBV in human cells.
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Abbreviations
- BAC:
-
Bacterial artificial chromosome
- Cas9:
-
CRISPR associated protein 9 nuclease
- CRISPR:
-
Clustered regularly interspaced short palindromic repeats
- DSB:
-
Double-strand break
- EBV:
-
Epstein–Barr virus
- GFP:
-
Green fluorescent protein
- gRNA:
-
Guide RNA
- MOI:
-
Multiplicity of infection
- PAM:
-
Protospacer adjacent motif
- pBART:
-
BamHI-A region rightward transcript promoter
- pCMV:
-
Cytomegalovirus promoter
- PCR:
-
Polymerase chain reaction
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Acknowledgments
This work was supported by Hong Kong Health and Medical Research Fund (11100602 and 12110962), S.K. Yee Medical Research Fund (2011), and Hong Kong Research Grants Council (AoE/M-06/08, HKU1/CRF/11G, C7011-15R, and T11-707/15-R).
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Yuen, KS., Chan, CP., Kok, KH., Jin, DY. (2017). Mutagenesis and Genome Engineering of Epstein–Barr Virus in Cultured Human Cells by CRISPR/Cas9. In: Reeves, A. (eds) In Vitro Mutagenesis. Methods in Molecular Biology, vol 1498. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6472-7_2
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DOI: https://doi.org/10.1007/978-1-4939-6472-7_2
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