Targeted genome editing of Epstein-Barr virus by CRISPR/Cas9 technology in human cells

Abstract

Epstein-Barr virus (EBV)-encoded BART microRNAs (miR-BARTs) are highly expressed in nasopharyngeal carcinoma (NPC), suggesting that they might play a critical role in NPC development. In order to investigate the function of miR-BARTs in nasopharyngeal epithelial (NP) cells, here a novel recombinant EBV construction and NP cell infection system was developed. CRISPR/Cas9 system is an emerging DNA editing technology platform which is powerful in the manipulation of cellular genome. We first demonstrated that not only the human genome but also the genome of latent DNA viruses, which undergoes episomal replication in human cells, are amenable to cleavage and editing by CRISPR/Cas9. By using this novel system, we constructed the miR-BART-deficient EBV by removal of 558 bp in the promoter region of BART transcript which encodes viral microRNAs in latently infected cells. A recombinant virus with the desired deletion was obtained after puromycin selection and the loss of miR-BART expression and activity was verified. Although CRISPR/Cas9-mediated editing of EBV genome was highly efficient, the multicopy episome of EBV in infected cells constituted a major technical challenge to CRISPR/Cas9-medaited editing. In order to overcome this technical barrier, DsRed selectable marker was introduced into EBV genome during the course of CRISPR/Cas9-mediated editing. DsRed incorporated viruses were successfully recovered and introduced into B cells at low moi and further isolated through cell sorting. B cells containing the pure mutant virus served as a good source of recombinant EBV for subsequent introduction into NP cells through co-culture. As one example, the miR-BART-deficient EBV was studied in NP cells. Taken together, our work provides a new and efficient method not only for targeted editing of EBV genome in human cells, but also for functional characterization of recombinant EBVs in NP cells.