Highly Efficient CRISPR/Cas9-Mediated Cloning and Functional Characterization of Gastric Cancer-Derived Epstein-Barr Virus Strains

Abstract

The Epstein-Barr virus (EBV) is etiologically linked to approximately 10% of gastric cancers, in which viral genomes are maintained as multicopy episomes. EBV-positive gastric cancer cells are incompetent for progeny virus production, making viral DNA cloning extremely difficult. Here we describe a highly efficient strategy for obtaining bacterial artificial chromosome (BAC) clones of EBV episomes by utilizing a CRISPR/Cas9-mediated strand break of the viral genome and subsequent homology-directed repair. EBV strains maintained in two gastric cancer cell lines (SNU719 and YCCEL1) were cloned, and their complete viral genome sequences were determined. Infectious viruses of gastric cancer cell-derived EBVs were reconstituted, and the viruses established stable latent infections in immortalized keratinocytes. While Ras oncoprotein overexpression caused massive vacuolar degeneration and cell death in control keratinocytes, EBV-infected keratinocytes survived in the presence of Ras expression. These results implicate EBV infection in predisposing epithelial cells to malignant transformation by inducing resistance to oncogene-induced cell death. IMPORTANCERecent progress in DNA-sequencing technology has accelerated EBV whole-genome sequencing, and the repertoire of sequenced EBV genomes is increasing progressively. Accordingly, the presence of EBV variant strains that may be relevant to EBV-associated diseases has begun to attract interest. Clearly, the determination of additional disease-associated viral genome sequences will facilitate the identification of any disease-specific EBV variants. We found that CRISPR/Cas9-mediated cleavage of EBV episomal DNA enabled the cloning of disease-associated viral strains with unprecedented efficiency. As a proof of concept, two gastric cancer cell-derived EBV strains were cloned, and the infection of epithelial cells with reconstituted viruses provided important clues about the mechanism of EBV-mediated epithelial carcinogenesis. This experimental system should contribute to establishing the relationship between viral genome variation and EBV-associated diseases. E pstein-Barr virus (EBV) is one of the most widespread human pathogens. EBV infection is usually asymptomatic, but it sometimes causes severe disorders, such as EBV-related lymphoproliferative disease, B-cell lymphomas, and NK/T-cell lymphomas (1). In addition, causal relationships between EBV infection and epithelial cell-derived cancers, such as nasopharyngeal carcinomas (NPCs) and gastric cancers, have been investigated extensively (2, 3). However, the precise mechanisms underlying EBV-mediated epithelial carcinogenesis remain largely unknown.Recent deep-sequencing studies demonstrated unexpected levels of heterogeneity in EBV genomes derived from various EBV-positive cell lines, including Burkitt's lymphoma-derived cell lines (4), spontaneously established lymphoblastoid cell lines (LCLs), Hodgkin's lymphoma cell lines, NPC-derived cell lines, a gastric cancer-derived cell line (5), and NPC biopsy samples ...

Highly Efficient CRISPR/Cas9-Mediated Homologous Recombination Promotes the Rapid Generation of Bacterial Artificial Chromosomes of Pseudorabies Virus

“…We suppose the reason may be the formation of superhelix of circular donor plasmid, and thus difficult to be incised by CRISPR/Cas9. However, in the recent construction of an Epstein–Barr virus-BAC system, only a circular donor plasmid, rather than a linearized one, produced successful HR though without exact HR efficiency (Kanda et al, 2016). Another study in which the PRV genome was manipulated with CRISPR/Cas9 showed that HR failed when a linearized homologous repair donor plasmid was inserted into the PRV genome, but achieved a recombinant efficiency of almost 50% when a homology-independent DNA repair mechanism was used (Liang et al, 2016).…”

“…CRISPR/Cas9 is emerging as a powerful tool for DNA engineering in diverse organisms, and allows efficient DNA editing (Cong et al, 2013). Gene knock-in of large DNA viruses with CRISPR/Cas9 has been reported, including of adenovirus (Bi et al, 2014), Herpes simplex virus 1 (Bi et al, 2014), PRV (Xu et al, 2015; Liang et al, 2016; Tang et al, 2016), and Epstein–Barr virus (Kanda et al, 2016). However, there is little information about the features of CRISPR/Cas9 that are important in enhancing the efficiency of the HR between PRV and BAC.…”

The implication of CRISPR/Cas9 genome editing technology in combating human oncoviruses

“…EBV genome shows heterogeneity during the progression of infection that generates new pathogenic strains which reduces the effects of antiviral drugs . Kanda et al used CRISPR/Cas9‐mediated cloning of disease‐associated viral strains in gastric cancer cell lines. A plasmid pX330‐sgEBV was constructed to cleave a target site of BVRF1 ORF and triggers the insertion of 12 kb transgenes into the EBV genome via homology‐directed repair.…”

Recent advances in understanding Epstein-Barr virus

“…Recently, it has been shown that CRISPR/Cas9-mediated cleavage for bacterial artificial chromosome (BAC) insertion into EBV episomal DNA in gastric carcinoma (GC) cell lines has facilitated the cloning of these viral genomes with unprecedented efficiency 41 . Subsequent infection of epithelial cells with the BAC clone reconstituted viruses induced resistance to oncogene-induced cell death, providing important clues concerning EBV-mediated epithelial carcinogenesis.…”