Mutagenesis and Genome Engineering of Epstein–Barr Virus in Cultured Human Cells by CRISPR/Cas9
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.
Key wordsRNA-guided genome editing Episomal viral DNA genome Epstein–Barr virus Genetic analysis of Epstein–Barr virus Cure of Epstein–Barr virus infection
Bacterial artificial chromosome
CRISPR associated protein 9 nuclease
Clustered regularly interspaced short palindromic repeats
Green fluorescent protein
Multiplicity of infection
Protospacer adjacent motif
BamHI-A region rightward transcript promoter
Polymerase chain reaction
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).