Mutagenesis and Genome Engineering of Epstein–Barr Virus in Cultured Human Cells by CRISPR/Cas9

  • Kit-San Yuen
  • Chi-Ping Chan
  • Kin-Hang Kok
  • Dong-Yan Jin
Part of the Methods in Molecular Biology book series (MIMB, volume 1498)


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 words

RNA-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


Double-strand break


Epstein–Barr virus


Green fluorescent protein


Guide RNA


Multiplicity of infection


Protospacer adjacent motif


BamHI-A region rightward transcript promoter


Cytomegalovirus 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).


  1. 1.
    Delecluse HJ, Hilsendegen T, Pich D, Zeidler R, Hammerschmidt W (1998) Propagation and recovery of intact, infectious Epstein-Barr virus from prokaryotic to human cells. Proc Natl Acad Sci U S A 95:8245–8250CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Zhou FC, Zhang YJ, Deng JH, Wang XP, Pan HY, Hettler E, Gao SJ (2002) Efficient infection by a recombinant Kaposi’s sarcoma-associated herpesvirus cloned in a bacterial artificial chromosome: application for genetic analysis. J Virol 76:6185–6196CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Umene K (1999) Mechanism and application of genetic recombination in herpesviruses. Rev Med Virol 9:171–182CrossRefPubMedGoogle Scholar
  4. 4.
    Cotter MA, Robertson ES (1999) Molecular genetic analysis of herpesviruses and their potential use as vectors for gene therapy applications. Curr Opin Mol Ther 1:633–644PubMedGoogle Scholar
  5. 5.
    Messerle M, Crnkovic I, Hammerschmidt W, Ziegler H, Koszinowski UH (1997) Cloning and mutagenesis of a herpesvirus genome as an infectious bacterial artificial chromosome. Proc Natl Acad Sci U S A 94:14759–14763CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    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 78:7004–7015CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Feederle R, Bartlett EJ, Delecluse HJ (2010) Epstein-Barr virus genetics: talking about the BAC generation. Herpesviridae 1:6–11CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Zhou F, Gao SJ (2011) Recent advances in cloning herpesviral genomes as infectious bacterial artificial chromosomes. Cell Cycle 10:434–440CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Cho SW, Kim S, Kim JM, Kim JS (2013) Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 31:230–232CrossRefPubMedGoogle Scholar
  10. 10.
    Cong L, Zhang F (2015) Genome engineering using CRISPR-Cas9 system. Methods Mol Biol 1239:197–217CrossRefPubMedGoogle Scholar
  11. 11.
    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 72:1477–1481CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Yuen KS, Chan CP, Wong NH, Ho CH, Ho TH, Lei T, Deng W, Tsao SW, Chen H, Kok KH et al (2015) CRISPR/Cas9-mediated genome editing of Epstein-Barr virus in human cells. J Gen Virol 96:626–636CrossRefPubMedGoogle Scholar
  13. 13.
    Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8:2281–2308CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Kit-San Yuen
    • 1
  • Chi-Ping Chan
    • 1
  • Kin-Hang Kok
    • 2
  • Dong-Yan Jin
    • 1
  1. 1.School of Biomedical SciencesThe University of Hong KongPokfulamHong Kong
  2. 2.Department of MicrobiologyThe University of Hong KongPokfulamHong Kong

Personalised recommendations