title: Mutagenesis and Genome Engineering of Epstein-Barr Virus in Cultured Human Cells by CRISPR/Cas9.
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.
authors.affiliation: School of Biomedical Sciences, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong.
authors.forename: Kit-San
authors.initials: KS
authors.lastname: Yuen
authors.affiliation: School of Biomedical Sciences, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong.
authors.forename: Chi-Ping
authors.initials: CP
authors.lastname: Chan
authors.affiliation: Department of Microbiology, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong.
authors.forename: Kin-Hang
authors.initials: KH
authors.lastname: Kok
authors.affiliation: School of Biomedical Sciences, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong. dyjin@hku.hk.
authors.forename: Dong-Yan
authors.initials: DY
authors.lastname: Jin
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics*; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D004267:DNA Viruses / Q000235:genetics; D020031:Epstein-Barr Virus Infections / Q000821:virology*; D005818:Genetic Engineering / Q000379:methods; D015894:Genome, Human / Q000235:genetics*; D057809:HEK293 Cells; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D016296:Mutagenesis / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D017393:RNA Editing / Q000235:genetics*
keywords: Cure of Epstein–Barr virus infection; Episomal viral DNA genome; Epstein–Barr virus; Genetic analysis of Epstein–Barr virus; RNA-guided genome editing