1887

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 (HI 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.

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2015-03-01
2021-10-15
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References

  1. Adams A., Lindahl T. 1975; Epstein–Barr virus genomes with properties of circular DNA molecules in carrier cells. Proc Natl Acad Sci U S A 72:1477–1481 [View Article][PubMed]
    [Google Scholar]
  2. Bi Y., Sun L., Gao D., Ding C., Li Z., Li Y., Cun W., Li Q. 2014; High-efficiency targeted editing of large viral genomes by RNA-guided nucleases. PLoS Pathog 10:e1004090 [View Article][PubMed]
    [Google Scholar]
  3. Böttcher R., Hollmann M., Merk K., Nitschko V., Obermaier C., Philippou-Massier J., Wieland I., Gaul U., Förstemann K. 2014; Efficient chromosomal gene modification with CRISPR/cas9 and PCR-based homologous recombination donors in cultured Drosophila cells. Nucleic Acids Res 42:e89 [View Article][PubMed]
    [Google Scholar]
  4. Cai X., Schäfer A., Lu S., Bilello J. P., Desrosiers R. C., Edwards R., Raab-Traub N., Cullen B. R. 2006; Epstein–Barr virus microRNAs are evolutionarily conserved and differentially expressed. PLoS Pathog 2:e23 [View Article][PubMed]
    [Google Scholar]
  5. Chen H., Hutt-Fletcher L., Cao L., Hayward S. D. 2003; A positive autoregulatory loop of LMP1 expression and STAT activation in epithelial cells latently infected with Epstein–Barr virus. J Virol 77:4139–4148 [View Article][PubMed]
    [Google Scholar]
  6. Chen A., Divisconte M., Jiang X., Quink C., Wang F. 2005a; Epstein–Barr virus with the latent infection nuclear antigen 3B completely deleted is still competent for B-cell growth transformation in vitro. J Virol 79:4506–4509 [View Article][PubMed]
    [Google Scholar]
  7. Chen H., Huang J., Wu F. Y., Liao G., Hutt-Fletcher L., Hayward S. D. 2005b; Regulation of expression of the Epstein–Barr virus BamHI-A rightward transcripts. J Virol 79:1724–1733 [View Article][PubMed]
    [Google Scholar]
  8. Cheung S. T., Huang D. P., Hui A. B., Lo K. W., Ko C. W., Tsang Y. S., Wong N., Whitney B. M., Lee J. C. 1999; Nasopharyngeal carcinoma cell line (C666-1) consistently harbouring Epstein–Barr virus. Int J Cancer 83:121–126 [View Article][PubMed]
    [Google Scholar]
  9. Cho S. W., Kim S., Kim Y., Kweon J., Kim H. S., Bae S., Kim J. S. 2014; Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res 24:132–141 [View Article][PubMed]
    [Google Scholar]
  10. Choy E. Y. W., Kok K. H., Tsao S. W., Jin D. Y. 2008a; Utility of Epstein–Barr virus-encoded small RNA promoters for driving the expression of fusion transcripts harboring short hairpin RNAs. Gene Ther 15:191–202 [View Article][PubMed]
    [Google Scholar]
  11. Choy E. Y. W., Siu K. L., Kok K. H., Lung R. W. M., Tsang C. M., To K. F., Kwong D. L., Tsao S. W., Jin D. Y. 2008b; An Epstein–Barr virus-encoded microRNA targets PUMA to promote host cell survival. J Exp Med 205:2551–2560 [View Article][PubMed]
    [Google Scholar]
  12. Edwards R. H., Marquitz A. R., Raab-Traub N. 2008; Epstein–Barr virus BART microRNAs are produced from a large intron prior to splicing. J Virol 82:9094–9106 [View Article][PubMed]
    [Google Scholar]
  13. Feederle R., Bartlett E. J., Delecluse H. J. 2010; Epstein–Barr virus genetics: talking about the BAC generation. Herpesviridae 1:6 [View Article][PubMed]
    [Google Scholar]
  14. Feederle R., Linnstaedt S. D., Bannert H., Lips H., Bencun M., Cullen B. R., Delecluse H. J. 2011; A viral microRNA cluster strongly potentiates the transforming properties of a human herpesvirus. PLoS Pathog 7:e1001294 [View Article][PubMed]
    [Google Scholar]
  15. Geiser V., Cahir-McFarland E., Kieff E. 2011; Latent membrane protein 1 is dispensable for Epstein–Barr virus replication in human embryonic kidney 293 cells. PLoS One 6:e22929 [View Article][PubMed]
    [Google Scholar]
  16. Hsu P. D., Lander E. S., Zhang F. 2014; Development and applications of CRISPR-Cas9 for genome engineering. Cell 157:1262–1278 [View Article][PubMed]
    [Google Scholar]
  17. Kanda T., Yajima M., Ahsan N., Tanaka M., Takada K. 2004; Production of high-titer Epstein–Barr virus recombinants derived from Akata cells by using a bacterial artificial chromosome system. J Virol 78:7004–7015 [View Article][PubMed]
    [Google Scholar]
  18. Kew C., Lui P. Y., Chan C. P., Liu X., Au S. W. N., Mohr I., Jin D. Y., Kok K. H. 2013; Suppression of PACT-induced type I interferon production by herpes simplex virus 1 Us11 protein. J Virol 87:13141–13149 [View Article][PubMed]
    [Google Scholar]
  19. Kwok H., Wu C. W., Palser A. L., Kellam P., Sham P. C., Kwong D. L. W., Chiang A. K. S. 2014; Genomic diversity of Epstein–Barr virus genomes isolated from primary nasopharyngeal carcinoma biopsy samples. J Virol 88:10662–10672 [View Article][PubMed]
    [Google Scholar]
  20. Lan K., Verma S. C., Murakami M., Bajaj B., Robertson E. S. 2007; Epstein–Barr Virus (EBV): infection, propagation, quantitation, and storage. Curr Protoc Microbiol Chapter 14:2[PubMed]
    [Google Scholar]
  21. Lei T., Yuen K. S., Tsao S. W., Chen H., Kok K. H., Jin D. Y. 2013a; Perturbation of biogenesis and targeting of Epstein–Barr virus-encoded miR-BART3 microRNA by adenosine-to-inosine editing. J Gen Virol 94:2739–2744 [View Article][PubMed]
    [Google Scholar]
  22. Lei T., Yuen K. S., Xu R., Tsao S. W., Chen H., Li M., Kok K. H., Jin D. Y. 2013b; Targeting of DICE1 tumor suppressor by Epstein–Barr virus-encoded miR-BART3* microRNA in nasopharyngeal carcinoma. Int J Cancer 133:79–87 [View Article][PubMed]
    [Google Scholar]
  23. Lieberman P. M. 2013; Keeping it quiet: chromatin control of gammaherpesvirus latency. Nat Rev Microbiol 11:863–875 [View Article][PubMed]
    [Google Scholar]
  24. Lin Z., Wang X., Strong M. J., Concha M., Baddoo M., Xu G., Baribault C., Fewell C., Hulme W.other authors 2013; Whole-genome sequencing of the Akata and Mutu Epstein–Barr virus strains. J Virol 87:1172–1182 [View Article][PubMed]
    [Google Scholar]
  25. Liu P., Speck S. H. 2003; Synergistic autoactivation of the Epstein–Barr virus immediate-early BRLF1 promoter by Rta and Zta. Virology 310:199–206 [View Article][PubMed]
    [Google Scholar]
  26. Liu P., Fang X., Feng Z., Guo Y. M., Peng R. J., Liu T., Huang Z., Feng Y., Sun X.other authors 2011; Direct sequencing and characterization of a clinical isolate of Epstein–Barr virus from nasopharyngeal carcinoma tissue by using next-generation sequencing technology. J Virol 85:11291–11299 [View Article][PubMed]
    [Google Scholar]
  27. Lo A. K. F., Dawson C. W., Jin D. Y., Lo K. W. 2012; The pathological roles of BART miRNAs in nasopharyngeal carcinoma. J Pathol 227:392–403 [View Article][PubMed]
    [Google Scholar]
  28. Lun S. W. M., Cheung S. T., Cheung P. F. Y., To K. F., Woo J. K. S., Choy K. W., Chow C., Cheung C. C. M., Chung G. T. Y.other authors 2012; CD44+ cancer stem-like cells in EBV-associated nasopharyngeal carcinoma. PLoS ONE 7:e52426 [View Article][PubMed]
    [Google Scholar]
  29. Mali P., Esvelt K. M., Church G. M. 2013; Cas9 as a versatile tool for engineering biology. Nat Methods 10:957–963 [View Article][PubMed]
    [Google Scholar]
  30. Marquitz A. R., Mathur A., Chugh P. E., Dittmer D. P., Raab-Traub N. 2014; Expression profile of microRNAs in Epstein–Barr virus-infected AGS gastric carcinoma cells. J Virol 88:1389–1393 [View Article][PubMed]
    [Google Scholar]
  31. Raab-Traub N. 2012; Novel mechanisms of EBV-induced oncogenesis. Curr Opin Virol 2:453–458 [View Article][PubMed]
    [Google Scholar]
  32. Ran F. A., Hsu P. D., Wright J., Agarwala V., Scott D. A., Zhang F. 2013; Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8:2281–2308 [View Article][PubMed]
    [Google Scholar]
  33. Riley K. J., Rabinowitz G. S., Yario T. A., Luna J. M., Darnell R. B., Steitz J. A. 2012; EBV and human microRNAs co-target oncogenic and apoptotic viral and human genes during latency. EMBO J 31:2207–2221 [View Article][PubMed]
    [Google Scholar]
  34. Sarisky R. T., Hayward G. S. 1996; Evidence that the UL84 gene product of human cytomegalovirus is essential for promoting oriLyt-dependent DNA replication and formation of replication compartments in cotransfection assays. J Virol 70:7398–7413[PubMed]
    [Google Scholar]
  35. Seto E., Moosmann A., Grömminger S., Walz N., Grundhoff A., Hammerschmidt W. 2010; Micro RNAs of Epstein–Barr virus promote cell cycle progression and prevent apoptosis of primary human B cells. PLoS Pathog 6:e1001063 [View Article][PubMed]
    [Google Scholar]
  36. Shen B., Zhang W., Zhang J., Zhou J., Wang J., Chen L., Wang L., Hodgkins A., Iyer V.other authors 2014; Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat Methods 11:399–402 [View Article][PubMed]
    [Google Scholar]
  37. Shimizu N., Yoshiyama H., Takada K. 1996; Clonal propagation of Epstein–Barr virus (EBV) recombinants in EBV-negative Akata cells. J Virol 70:7260–7263[PubMed]
    [Google Scholar]
  38. Siu K. L., Yeung M. L., Kok K. H., Yuen K. S., Kew C., Lui P. Y., Chan C. P., Tse H., Woo P. C. Y.other authors 2014; Middle east respiratory syndrome coronavirus 4a protein is a double-stranded RNA-binding protein that suppresses PACT-induced activation of RIG-I and MDA5 in the innate antiviral response. J Virol 88:4866–4876 [View Article][PubMed]
    [Google Scholar]
  39. Tang H. M. V., Gao W. W., Chan C. P., Siu Y. T., Wong C. M., Kok K. H., Ching Y. P., Takemori H., Jin D. Y. 2013; LKB1 tumor suppressor and salt-inducible kinases negatively regulate human T-cell leukemia virus type 1 transcription. Retrovirology 10:40 [View Article][PubMed]
    [Google Scholar]
  40. Terns R. M., Terns M. P. 2014; CRISPR-based technologies: prokaryotic defense weapons repurposed. Trends Genet 30:111–118 [View Article][PubMed]
    [Google Scholar]
  41. Tsai M. H., Raykova A., Klinke O., Bernhardt K., Gärtner K., Leung C. S., Geletneky K., Sertel S., Münz C.other authors 2013; Spontaneous lytic replication and epitheliotropism define an Epstein–Barr virus strain found in carcinomas. Cell Rep 5:458–470 [View Article][PubMed]
    [Google Scholar]
  42. Tsang C. M., Zhang G., Seto E., Takada K., Deng W., Yip Y. L., Man C., Hau P. M., Chen H.other authors 2010; Epstein–Barr virus infection in immortalized nasopharyngeal epithelial cells: regulation of infection and phenotypic characterization. Int J Cancer 127:1570–1583 [View Article][PubMed]
    [Google Scholar]
  43. Tsang C. M., Yip Y. L., Lo K. W., Deng W., To K. F., Hau P. M., Lau V. M., Takada K., Lui V. W.other authors 2012; Cyclin D1 overexpression supports stable EBV infection in nasopharyngeal epithelial cells. Proc Natl Acad Sci U S A 109:E3473–E3482 [View Article][PubMed]
    [Google Scholar]
  44. Tso K. K., Yip K. Y., Mak C. K., Chung G. T., Lee S. D., Cheung S. T., To K. F., Lo K. W. 2013; Complete genomic sequence of Epstein–Barr virus in nasopharyngeal carcinoma cell line C666-1. Infect Agent Cancer 8:29 [View Article][PubMed]
    [Google Scholar]
  45. Umbach J. L., Cullen B. R. 2009; The role of RNAi and microRNAs in animal virus replication and antiviral immunity. Genes Dev 23:1151–1164 [View Article][PubMed]
    [Google Scholar]
  46. Westphal E. M., Sierakowska H., Livanos E., Kole R., Vos J. M. 1998; A system for shuttling 200-kb BAC/PAC clones into human cells: stable extrachromosomal persistence and long-term ectopic gene activation. Hum Gene Ther 9:1863–1873 [View Article][PubMed]
    [Google Scholar]
  47. Yip Y. L., Pang P. S., Deng W., Tsang C. M., Zeng M., Hau P. M., Man C., Jin Y., Yuen A. P., Tsao S. W. 2013; Efficient immortalization of primary nasopharyngeal epithelial cells for EBV infection study. PLoS One 8:e78395 [View Article][PubMed]
    [Google Scholar]
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