Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS), an AIDS-defining cancer in HIV-1-infected individuals or immune-suppressed transplant patients. The prevalence for both KSHV and KS are highest in sub-Saharan Africa where HIV-1 infection is also epidemic. There is no effective treatment for advanced KS; therefore, the survival rate is low. Similar to other herpesviruses, KSHV's ability to establish latent infection in the host presents a major challenge to KS treatment or prevention. Strategies to reduce KSHV episomal persistence in latently infected cells might lead to approaches to prevent KS development. The CRISPR-Cas9 system is a gene editing technique that has been used to specifically manipulate the HIV-1 genome but also Epstein-Barr virus (EBV) which, similar to KSHV, belongs to the Gammaherpesvirus family. Among KSHV gene products, the latency-associated nuclear antigen (LANA) is absolutely required in the maintenance, replication, and segregation of KSHV episomes during mitosis, which makes LANA an ideal target for CRISPR-Cas9 editing. In this study, we designed a replication-incompetent adenovirus type 5 to deliver a LANA-specific Cas9 system (Ad-CC9-LANA) into various KSHV latent target cells. We showed that KSHV latently infected epithelial and endothelial cells transduced with Ad-CC9-LANA underwent significant reductions in the KSHV episome burden, LANA RNA and protein expression over time, but this effect is less profound in BC3 cells due to the low infection efficiency of adenovirus type 5 for B cells. The use of an adenovirus vector might confer potential in vivo applications of LANA-specific Cas9 against KSHV infection and KS.IMPORTANCE The ability for Kaposi's sarcoma-associated herpesvirus (KSHV), the causative agent of Kaposi's sarcoma (KS), to establish and maintain latency has been a major challenge to clearing infection and preventing KS development. This is the first study to demonstrate the feasibility of using a KSHV LANA-targeted CRISPR-Cas9 and adenoviral delivery system to disrupt KSHV latency in infected epithelial and endothelial cell lines. Our system significantly reduced the KSHV episomal burden over time. Given the safety record of adenovirus as vaccine or delivery vectors, this approach to limit KSHV latency may also represent a viable strategy against other tumorigenic viruses and may have potential benefits in developing countries where the viral cancer burden is high.