Coral Orel Haddad1, Inna Kalt1, Yehuda Shovman2, Lei Xia2, Yehuda Schlesinger2, Ronit Sarid3, Oren Parnas4. 1. The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, 5290002, Ramat-Gan, Israel. 2. The Concern Foundation at the Lautenberg Center for Immunology and Cancer Research, IMRIC, Hebrew University Faculty of Medicine, 91120, Jerusalem, Israel. 3. The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, 5290002, Ramat-Gan, Israel. saridr@mail.biu.ac.il. 4. The Concern Foundation at the Lautenberg Center for Immunology and Cancer Research, IMRIC, Hebrew University Faculty of Medicine, 91120, Jerusalem, Israel. oren.parnas@gmail.com.
Abstract
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 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.