PROJECT SUMMARY/ABSTRACT Kaposi’s sarcoma (KS) is an HIV/AIDS-associated malignancy, that remains prevalent, despite antiretroviral therapy, in areas where there is high incidence of KS-associated herpesvirus (KSHV) infection. KSHV seroprevalence is geographically uneven but is high in sub-Saharan Africa (SSA) and in parts of the United States, such as Southern Louisiana, where HIV-1 infection rates are high. In this region, both HIV and KS disproportionately affect people of color who also experience other health disparities. Because HIV- and KSHV- infected individuals remain at risk to develop KS and other KSHV-associated malignances despite HIV viral load suppression, novel research strategies are needed for people living with HIV (PLWH). Chronic oncogenic viral infections possess mechanisms that engender immune avoidance and viral persistence that leads to neoplastic growth. The KSHV latency locus encodes a small number of viral genes that are involved in viral persistence. The latency-associated nuclear antigen (LANA) plays a key role in the maintenance of latency and help the virus to evade host immune surveillance by inhibiting MHC-1 antigen presentation, while other latency genes, such as kaposin (K12) was reported to have oncogenic properties, as well as microRNAs that could be essential for tumorigenesis. Although the individual function of KSHV latency-associated genes have been studied, there remain unresolved questions regarding their specific contribution to latency, immune evasion and neoplasia. It is also critical to know, which host genes and pathways interact with the viral genes to elicit the latency program, and to answer how this virus-host interplay contributes to malignant transformation. We recently demonstrated that our unique CRISPR/Cas9 vector, specifically designed to target a single KSHV LANA nucleotide sequence, reduced ~70-80% of KSHV episomal copies, and attenuated the growth of KSHV-positive cells. Our major objective here is to utilize this new technology to disrupt/eliminate multiple genes of the KSHV latency locus, and to investigate their individual and combined effects on KSHV latency. We will seek to identify cellular genes and pathways affected by, and possibly contributing to the development and maintenance of the malignant phenotype. We hypothesize that the components of the KSHV latency locus individually dysregulate expression of specific sets of cellular genes to promote KSHV persistence and synergistically to promote growth dysregulation and tumorigenesis. We will test this concept by editing multiple viral gene sequences within the latency locus with CRISPR/Cas9 using KS and PEL cell culture models. Through bioinformatics analyses of transcriptome profiles of the CRISPR/Cas9 edited KSHV infected cells, we will identify unique host genes/cellular pathway and their roles/interaction with viral latency genes in viral latency, growth dysregulation and tumorigenesis.