Background Kaposi’s sarcoma-associated herpesvirus (KSHV) is a transforming gammaherpesvirus. Like other herpesviruses, KSHV infection is for life long and there is no treatment that can cure patients from the virus. In addition, there is an urgent need to target viral genes to study their role during the infection cycle. The CRISPR-Cas9 technology offers a means to target viral genomes and thus may offer a novel strategy for viral cure as well as for better understanding of the infection process. We evaluated the suitability of this platform for the targeting of KSHV. Methods We have used the recombinat KSHV BAC16 genome, which contains an expression cassette encoding hygromycin-resistance and a GFP marker gene. Three genes were targeted: gfp, which serves as a marker for infection; orf45 encoding a lytic viral protein; and orf73, encoding LANA which is crucial for latent infection. The fraction of cells expressing GFP, viral DNA levels and LANA expression were monitored and viral genomes were sequenced. Results We found that KSHV episomes can be targeted by CRISPR-Cas9. Interestingly, the quantity of KSHV DNA declined, even when target sites were not functionally important for latency. In addition, we show that antibiotic selection, used to maintain infection, interferes with the outcome of targeting. Conclusions Our study provides insights into the use of this fundamental approach for the study and manipulation of KSHV. It provides guidelines for the targeting CRISPR-Cas9 to the viral genome and for outcomes interpretation.

Background: Kaposi’s sarcoma-associated herpesvirus (KSHV) is a transforming gammaherpes. Like other herpesviruses, KSHV infection is for life long and there is no treatment that can cure of patients from the virus. In addition, there is urgent need to target viral genes to study their role during the infection cycle. The CRISPR-Cas9 technology offers a means to target viral genomes and thus may offer a novel strategy for viral cure as well for better understanding of the infection process. We evaluated the suitability of this platform for the targeting of KSHV. Methods: We have used BAC16 genome, which contains an expression cassette encoding hygromycin-resistance and a GFP marker gene. Three genes were targeted: gfp which serves as a marker for infection; orf45 encoding a lytic viral protein; and orf73, encoding LANA which is crucial for latent infection. The fraction of cells expressing GFP as well as viral DNA levels and LANA expression were monitored and viral genomes were sequenced. Results: We found that KSHV episomes can be targeted by CRISPR-Cas9. Interestingly, the quantity of KSHV DNA declined, even when target sites were not functionally important for latency. In addition, we show that antibiotic selection, used to maintain infection, interferes with the outcome of targeting.Conclusions: Our study provides insights to the use of this fundamental approach for the study and manipulation of KSHV. It provides guidelines for the targeting CRISPR-Cas9 to the viral genome and for outcomes interpretation.

Tertiary lymphoid structures (TLSs) are formed in many cancer types and have been correlated with better prognosis and response to immunotherapy. In liver cancer, TLSs have been reported to be pro-tumorigenic as they harbor tumor progenitor cells and nurture their growth. The processes involved in TLS development and acquisition of pro- or anti-tumorigenic phenotype in cancer are largely unknown. RORc expressing immune cells have been previously implicated in TLS formation, however we find that they are not necessary for TLS neogenesis in the liver under chronic inflammation conditions. On the contrary, RORc expressing cells rather negatively regulate TLS formation, since in their absence TLSs form in excess. Importantly, in chronically inflamed livers lacking RORc expressing cells, pro-tumorigenic TLSs become anti-tumorigenic, resulting in reduction of tumor load. Comparing liver pro- and anti-tumorigenic TLSs by transcriptional, proteomic and immunohistochemical analyses, revealed an enrichment of exhausted CD8 cells that retained effector functions and had a progenitor-like proliferative phenotype in anti-tumorigenic TLSs. Similar observations were found when comparing pro- and anti-tumorigenic TLSs in human tissues. Thus, RORc expressing cells negatively regulate CD8 cell abundance, and facilitate the pro-tumorigenic functions of hepatic TLSs.