Abstract：
Marek\'s disease virus (MDV) is a member of alphaherpesviruses associated with Marek\'s disease, a highly contagious neoplastic disease in chickens. The availability of the complete sequence of the viral genome allowed for the identification of major genes associated with pathogenicity using different techniques, such as bacterial artificial chromosome (BAC) mutagenesis and the recent powerful clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based editing system. Thus far, most studies on MDV genome editing using the CRISPR/Cas9 system have focused on gene deletion. However, analysis of the expression and interactions of the viral proteins during virus replication in infected cells and tumor cells is also important for studying its role in MDV pathogenesis. The unavailability of antibodies against most of the MDV proteins has hindered the progress in such studies. This prompted us to develop pipelines to tag MDV genes as an alternative method for this purpose. Here we describe the application of CRISPR/Cas9 gene-editing approaches to tag the phosphoprotein 38 (pp38) gene of the MDV vaccine strain CVI988 with both V5 and green fluorescent protein (GFP). This rapid and efficient viral-gene-tagging technique can overcome the shortage of specific antibodies and speed up the MDV gene function studies significantly, leading to a better understanding of the molecular mechanisms of MDV pathogenesis.
摘要：
马立克氏病病毒（MDV）是与马立克氏病相关的α疱疹病毒的成员，鸡的高度传染性肿瘤疾病。病毒基因组的完整序列的可用性允许使用不同的技术鉴定与致病性相关的主要基因。例如细菌人工染色体（BAC）诱变和最近强大的成簇规则间隔短回文重复序列（CRISPR）/CRISPR相关蛋白9（Cas9）的编辑系统。到目前为止,使用CRISPR/Cas9系统进行MDV基因组编辑的大多数研究都集中在基因缺失上。然而,分析病毒在感染细胞和肿瘤细胞中复制过程中病毒蛋白的表达和相互作用对于研究其在MDV发病机理中的作用也很重要。针对大多数MDV蛋白的抗体的不可用性阻碍了此类研究的进展。这促使我们开发管道来标记MDV基因作为用于此目的的替代方法。在这里，我们描述了CRISPR/Cas9基因编辑方法的应用，用V5和绿色荧光蛋白（GFP）标记MDV疫苗株CVI988的磷蛋白38（pp38）基因。这种快速高效的病毒基因标记技术可以克服特异性抗体的不足，显著加快MDV基因功能研究，从而更好地了解MDV发病的分子机制。
