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Article: CRISPR/Cas9-mediated genome editing of Epstein–Barr virus in human cells

TitleCRISPR/Cas9-mediated genome editing of Epstein–Barr virus in human cells
Authors
Issue Date2015
Citation
Journal of General Virology, 2015, v. 96, n. 3, p. 626-636 How to Cite?
Abstract© 2015 The Authors. The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPRassociated 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.
Persistent Identifierhttp://hdl.handle.net/10722/210091
ISSN
2015 Impact Factor: 3.192
2015 SCImago Journal Rankings: 1.741

 

DC FieldValueLanguage
dc.contributor.authorYuen, Kit San-
dc.contributor.authorChan, Chi Ping-
dc.contributor.authorWong, Nok Hei Mickey-
dc.contributor.authorHo, Chau Ha-
dc.contributor.authorHo, Ting Hin-
dc.contributor.authorLei, Ting-
dc.contributor.authorDeng, Wen-
dc.contributor.authorTsao, Sai Wah-
dc.contributor.authorChen, Honglin-
dc.contributor.authorKok, Kin Hang-
dc.contributor.authorJin, Dong Yan-
dc.date.accessioned2015-05-22T06:06:36Z-
dc.date.available2015-05-22T06:06:36Z-
dc.date.issued2015-
dc.identifier.citationJournal of General Virology, 2015, v. 96, n. 3, p. 626-636-
dc.identifier.issn0022-1317-
dc.identifier.urihttp://hdl.handle.net/10722/210091-
dc.description.abstract© 2015 The Authors. The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPRassociated 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.-
dc.languageeng-
dc.relation.ispartofJournal of General Virology-
dc.titleCRISPR/Cas9-mediated genome editing of Epstein–Barr virus in human cells-
dc.typeArticle-
dc.description.natureLink_to_subscribed_fulltext-
dc.identifier.doi10.1099/jgv.0.000012-
dc.identifier.scopuseid_2-s2.0-84923672936-
dc.identifier.hkuros253290-
dc.identifier.volume96-
dc.identifier.issue3-
dc.identifier.spage626-
dc.identifier.epage636-
dc.identifier.eissn1465-2099-

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