Fig 2. CRISPR/Cas9-mediated clearance of EBV genomes from latently infected Burkitt’s lymphoma cells.

a) Anti-EBV gRNAs induce a potent loss of EBV genomes from latently infected cells. Burkitt’s lymphoma Akata-Bx1 cells latently infected with eGFP-EBV (endogenously driving eGFP expression) were transduced with anti-EBV gRNAs targeting EBNA1, OriP, or control genes and selected with puromycin for 2 days. Subsequently, the cells were monitored for the presence of EBV-eGFP by flow cytometry 21 days post transduction. The percentage of eGFP negative cells as measure for EBV-eGFP loss is indicated. b) Combinatorial anti-EBV gRNA treatment of Akata-Bx1 cells causes increased loss of EBV genomes from latently infected cells. Similar experimental set-up as in a), but with a larger set of anti-EBV gRNAs and combinations thereof introduced through sequential application of two separate CRISPR vectors. The percentage of EBV-eGFP negative cells is presented. c) Samples from b) were subjected to qPCR to quantify the relative EBV genome content in the indicated gRNA-expressing Akata-Bx1 cells. Since the amplified region in the qPCR lies outside the genomic region that is targeted by the gRNAs, the qPCR can also detect mutated, yet repaired EBV variants. For all bar diagrams, measurements for (at least) triplicate experiments + STD are presented.