title: CRISPR/Cas9-mediated genome editing of Epstein-Barr virus in human cells.
abstract: The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated 9) system is a highly efficient and powerful tool for RNA-guided editing of the cellular genome. Whether CRISPR/Cas9 can also cleave the genome of DNA viruses such as Epstein-Barr virus (EBV), which undergo episomal replication in human cells, remains to be established. Here, we reported on CRISPR/Cas9-mediated editing of the EBV genome in human cells. Two guide RNAs (gRNAs) were used to direct a targeted deletion of 558 bp in the promoter region of BART (BamHI A rightward transcript) which encodes viral microRNAs (miRNAs). Targeted editing was achieved in several human epithelial cell lines latently infected with EBV, including nasopharyngeal carcinoma C666-1 cells. CRISPR/Cas9-mediated editing of the EBV genome was efficient. A recombinant virus with the desired deletion was obtained after puromycin selection of cells expressing Cas9 and gRNAs. No off-target cleavage was found by deep sequencing. The loss of BART miRNA expression and activity was verified, supporting the BART promoter as the major promoter of BART RNA. Although CRISPR/Cas9-mediated editing of the multicopy episome of EBV in infected HEK293 cells was mostly incomplete, viruses could be recovered and introduced into other cells at low m.o.i. Recombinant viruses with an edited genome could be further isolated through single-cell sorting. Finally, a DsRed selectable marker was successfully introduced into the EBV genome during the course of CRISPR/Cas9-mediated editing. Taken together, our work provided not only the first genetic evidence that the BART promoter drives the expression of the BART transcript, but also a new and efficient method for targeted editing of EBV genome in human cells.
authors.affiliation: Department of Biochemistry, University of Hong Kong, Pokfulam, Hong Kong.
authors.forename: Kit-San
authors.initials: KS
authors.lastname: Yuen
authors.affiliation: Department of Biochemistry, University of Hong Kong, Pokfulam, Hong Kong.
authors.forename: Chi-Ping
authors.initials: CP
authors.lastname: Chan
authors.affiliation: Department of Biochemistry, University of Hong Kong, Pokfulam, Hong Kong.
authors.forename: Nok-Hei Mickey
authors.initials: NM
authors.lastname: Wong
authors.affiliation: Department of Biochemistry, University of Hong Kong, Pokfulam, Hong Kong.
authors.forename: Chau-Ha
authors.initials: CH
authors.lastname: Ho
authors.affiliation: Department of Biochemistry, University of Hong Kong, Pokfulam, Hong Kong.
authors.forename: Ting-Hin
authors.initials: TH
authors.lastname: Ho
authors.affiliation: Department of Pathology, School of Medicine, Xi'an Jiaotong University, Xi'an, PR China.
authors.forename: Ting
authors.initials: T
authors.lastname: Lei
authors.affiliation: Department of Anatomy, University of Hong Kong, Pokfulam, Hong Kong.
authors.forename: Wen
authors.initials: W
authors.lastname: Deng
authors.affiliation: Department of Anatomy, University of Hong Kong, Pokfulam, Hong Kong.
authors.forename: Sai Wah
authors.initials: SW
authors.lastname: Tsao
authors.affiliation: Department of Microbiology, University of Hong Kong, Pokfulam, Hong Kong.
authors.forename: Honglin
authors.initials: H
authors.lastname: Chen
authors.affiliation: Department of Microbiology, University of Hong Kong, Pokfulam, Hong Kong.|Department of Biochemistry, University of Hong Kong, Pokfulam, Hong Kong.
authors.forename: Kin-Hang
authors.initials: KH
authors.lastname: Kok
authors.affiliation: Department of Biochemistry, University of Hong Kong, Pokfulam, Hong Kong.
authors.forename: Dong-Yan
authors.initials: DY
authors.lastname: Jin
mesh_terms: D064130:CRISPR-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D005819:Genetic Markers; D057809:HEK293 Cells / Q000145:classification; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D017393:RNA Editing / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D016865:Reassortant Viruses; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D054334:Viral Regulatory and Accessory Proteins
chemical_list: D064130:CRISPR-Associated Proteins; D005819:Genetic Markers; D012367:RNA, Viral; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins