CRISPR/Cas9 ablating viral microRNA promotes lytic reactivation of Kaposi’s sarcoma-associated herpesvirus

Highlights

• CRISPR/Cas9 can robustly and precisely regulate KSHV miRNAs in KSHV-infected PEL cells.

• CRISPR/Cas9 downregulating KSHV miRNAs can alter the expression of their downstream target genes and KSHV lytic genes in host PEL cells.

• CRISPR/Cas9 is a novel antiviral strategy targeting KSHV latency.

Abstract

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system is an RNA-guided, DNA editing method that has been widely used for gene editing, including human viruses. Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV8), following latent infection in human cells, can cause a variety of malignancies, such as Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD), with a high prevalence in immunocompromised patients. Of significant concern, the latent infection with KSHV has been shown to lead to increased resistance to antiviral therapies. MicroRNAs (miRNAs) are a set of non-coding, small RNA molecules that regulate protein-coding genes at the post-transcriptional and translational levels. KSHV has its miRNAs, most of which are expressed in latently infected cells and play a crucial role in maintaining KSHV latency. Notably, by regulating the expression of the downstream target genes in host cells, KSHV miRNAs can interact with the host environment to promote the development of KSHV-related diseases. Although CRISPR/Cas9 has been reported to edit KSHV protein-coding genes, there is no published literature on whether the CRISPR/Cas9 system can regulate the expression of KSHV miRNAs. In this study, we used CRISPR/Cas9 to inhibit the expression of KSHV miRNAs by directly editing the DNA sequences of individual KSHV miRNAs, or the promoter of clustered KHSV miRNAs, in latent KSHV-infected PEL cells. Our results show that CRISPR/Cas9 can ablate KSHV miRNAs expression, which in turn leads to the upregulation of viral lytic genes and alteration of host cellular gene expression. To the best of our knowledge, our study is the first reported demonstration of the CRISPR/Cas9 system editing KSHV miRNAs, further expanding the application of CRISPR/Cas9 as a novel antiviral strategy targeting KSHV latency.

Introduction

Kaposi’s sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8 (HHV8), has been documented as a causative agent for Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD), which are the malignancies prevalent in immunocompromised populations [1]. Like other herpesviruses, KSHV has two phases in its life cycle: the latent and lytic phases. When the latent infection is established in host cells, KSHV expresses only a few essential latent genes to maintain the viral extrachromosomal episomes without producing infectious virions, which allow KSHV to escape from the host’s immune surveillance and remain inactive throughout its life status [2]. Notably, many antiviral drugs only effectively target lytic KSHV, but cannot specifically eliminate latent KSHV [3].

Recent studies have revealed that microRNAs (miRNAs) are a group of non-coding small RNA molecules that play a significant role in the viral life cycle [4]. KSHV expresses 25 mature miRNAs, encoded by 12 pre-miRNA genes, which are pivotal in developing KSHV-related pathogenesis and tumorigenesis [[4], [5], [6]]. Most of KSHV miRNAs are expressed during the latent phase; however, they can be encapsulated into the virions when KSHV is reactivated and switches to the lytic phase [4]. Previous studies already extensively reported the critical role of viral miRNAs in maintaining KSHV latency; however, the regulatory function of these miRNAs in KSHV reactivation and switching to the lytic phase has not been well-studied.

The clustered regularly interspaced short palindromic repeats/CRISPR-associated gene 9 (CRISPR/Cas9) system is an RNA-guided DNA editing method that has been widely used in gene editing [7]. Over the past five years, the application of CRISPR/Cas9 as a robust and convenient gene-editing tool has rapidly expanded to almost all biomedical fields, including the study of human viruses [[8], [9], [10], [11]]. A recent study reported that CRISPR/Cas9 could disrupt KSHV latency by targeting latency-associated nuclear antigen (LANA) and ORF57 in KSHV-infected cells [12,13]. However, to the best of our knowledge, there are no reports of studying KSHV miRNAs with CRISPR/Cas9.

Our previous study has demonstrated the capacity and efficacy of CRISPR/Cas9 to edit human miRNAs in both in vitro and in vivo models [14]. In this study, we utilized CRISPR/Cas9 to target KSHV miRNAs in BCBL-1 and BCP-1, two primary effusion lymphoma (PEL) cell lines with latent KSHV infection. After editing the promoter of the KSHV-miRNA cluster with CRISPR/Cas9, we found ten individual KSHV miRNAs in the cluster were ultimately downregulated. We further targeted two individual KSHV miRNAs, miR-K12-1 and miR-K12-9, for CRISPR/Cas9 editing. Our data show that not only their expression was downregulated, but also their downstream target genes were altered accordingly, demonstrating that CRISPR/Cas9 is a robust and precise tool for modulating KSHV miRNAs. Of significance, the use of CRISPR/Cas9 to target viral miRNAs led to the lytic reactivation of KSHV, resulting from the upregulation of lytic genes, along with metabolic alterations and increased oxygen consumption in host PEL cells. Thus, our results provide novel insights into the development of precision medicine with CRISPR/Cas9 editing viral miRNAs to eliminate KSHV latent infection.