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.

CRISPR（成簇规则间隔短回文重复）/Cas9（CRISPR相关基因9）系统是一种RNA引导的DNA编辑方法，已广泛用于基因编辑，包括人类病毒。卡波西肉瘤相关疱疹病毒（KSHV/HHV8）潜伏感染人体细胞后，可引起多种恶性肿瘤，如卡波西肉瘤（KS）、原发性渗出性淋巴瘤（PEL）和多中心卡斯尔曼病（MCD）。免疫功能低下患者中患病率较高。值得注意的是，KSHV 潜伏感染已被证明会导致抗病毒治疗耐药性增加。MicroRNA (miRNA) 是一组非编码小 RNA 分子，可在转录后和翻译水平调节蛋白质编码基因。KSHV有其miRNA，其中大部分在潜伏感染细胞中表达，在维持KSHV潜伏期中发挥着至关重要的作用。值得注意的是，通过调节宿主细胞中下游靶基因的表达，KSHV miRNA可以与宿主环境相互作用，促进KSHV相关疾病的发展。尽管已有报道CRISPR/Cas9可以编辑KSHV蛋白编码基因，但目前还没有关于CRISPR/Cas9系统是否可以调节KSHV miRNA表达的文献发表。在本研究中，我们使用 CRISPR/Cas9 通过直接编辑潜伏 KSHV 感染的 PEL 细胞中单个 KSHV miRNA 的 DNA 序列或簇状 KSHV miRNA 的启动子来抑制 KSHV miRNA 的表达。我们的结果表明，CRISPR/Cas9 可以消除 KSHV miRNA 的表达，进而导致病毒裂解基因的上调和宿主细胞基因表达的改变。据我们所知，我们的研究是首次报道的 CRISPR/Cas9 系统编辑 KSHV miRNA 的演示，进一步扩展了 CRISPR/Cas9 作为针对 KSHV 潜伏期的新型抗病毒策略的应用。