Department of Microbiology and Immunology, Stony Brook University, Stony Brook, New York, United States of America.
HIV and AIDS Malignancy Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
PLoS One. 2021 Jun 4;16(6):e0252313. doi: 10.1371/journal.pone.0252313. eCollection 2021.
Epstein-Barr virus (EBV) and Kaposi sarcoma herpesvirus (KSHV) are cancer-causing viruses that establish lifelong infections in humans. Gene editing using the Cas9-guideRNA (gRNA) CRISPR system has been applied to decrease the latent load of EBV in human Burkitt lymphoma cells. Validating the efficacy of Cas9-gRNA system in eradicating infection in vivo without off-target effects to the host genome will require animal model systems. To this end, we evaluated a series of gRNAs against individual genes and functional genomic elements of murine gammaherpesvirus 68 (MHV68) that are both conserved with KSHV and important for the establishment of latency or reactivation from latency in the host. gRNA sequences against ORF50, ORF72 and ORF73 led to insertion, deletion and substitution mutations in these target regions of the genome in cell culture. Murine NIH3T3 fibroblast cells that stably express Cas9 and gRNAs to ORF50 were most resistant to replication upon de novo infection. Latent murine A20 B cell lines that stably express Cas9 and gRNAs against MHV68 were reduced in their reactivation by approximately 50%, regardless of the viral gene target. Lastly, co-transfection of HEK293T cells with the vector expressing the Cas9-MHV68 gRNA components along with the viral genome provided a rapid read-out of gene editing and biological impact. Combinatorial, multiplex MHV68 gRNA transfections in HEK293T cells led to near complete ablation of infectious particle production. Our findings indicate that Cas9-gRNA editing of the murine gammaherpesvirus genome has a deleterious impact on productive replication in three independent infection systems.
EB 病毒 (EBV) 和卡波西肉瘤疱疹病毒 (KSHV) 是导致癌症的病毒，它们在人类中引发终身感染。使用 Cas9-guideRNA (gRNA) CRISPR 系统进行基因编辑已被应用于降低人类伯基特淋巴瘤细胞中 EBV 的潜伏负荷。为了验证 Cas9-gRNA 系统在体内消除感染而不产生对宿主基因组的脱靶效应的疗效，需要动物模型系统。为此，我们评估了一系列针对小鼠γ疱疹病毒 68 (MHV68) 的单个基因和功能基因组元件的 gRNA，这些基因和元件与 KSHV 保守，并且对于在宿主中建立潜伏或从潜伏中重新激活至关重要。针对 ORF50、ORF72 和 ORF73 的 gRNA 序列导致这些基因组靶区域在细胞培养中发生插入、缺失和取代突变。稳定表达 Cas9 和 gRNA 针对 ORF50 的小鼠 NIH3T3 成纤维细胞在重新感染时最能抵抗复制。稳定表达针对 MHV68 的 Cas9 和 gRNA 的潜伏小鼠 A20 B 细胞系的再激活减少了约 50%，无论病毒基因靶标如何。最后，将表达 Cas9-MHV68 gRNA 组件的载体与病毒基因组共转染 HEK293T 细胞可快速提供基因编辑和生物学影响的读数。在 HEK293T 细胞中进行组合、多重 MHV68 gRNA 转染可导致传染性颗粒产生几乎完全消除。我们的研究结果表明，Cas9-gRNA 编辑小鼠γ疱疹病毒基因组对三种独立感染系统中的有效复制具有有害影响。
