Detection of Epstein-Barr virus encoded RNA in fixed cells and tissues using CRISPR/Cas-mediated RCasFISH.
Detection of Epstein-Barr virus encoded RNA in fixed cells and tissues using CRISPR/Cas-mediated RCasFISH.
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DOI：
10.1016/j.ab.2021.114211
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发表时间：
2021-04
影响因子：
2.9
通讯作者：
Kun Chen;Meng Wang;Rui Zhang;Jinming Li
中科院分区：
文献类型：
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作者：
Kun Chen;Meng Wang;Rui Zhang;Jinming Li
EB病毒(EBV)感染细胞的鉴定对于EBV相关疾病的诊断和临床治疗至关重要。EBV编码的RNA(EBER)位于细胞核内，由于其在组织标本中的高水平表达和固有的稳定性，是一种可靠的标记。EBERin原位杂交长期以来一直是检测肿瘤相关潜伏EBV感染的金标准，在确定EBV相关恶性肿瘤的原发部位和辐射范围方面具有重要价值。然而，该方法的信号扩散和费时费力在一定程度上限制了可靠的检测，特别是当蛋白质和RNA需要同时标记的时候。在这里，我们优化和验证了我们的CRISPR-dCas9介导的原位RNA成像工具-RCasFISH，该工具使我们能够快速检测EBER，并在固定细胞和组织切片中与IHC方法兼容。我们的方法可以为潜在的EBV感染的分子诊断提供一个有吸引力的替代方法。
Identification of Epstein-Barr virus (EBV)-infected cells is critical for the diagnosis and clinical management of EBV-associated diseases. EBV-encoded RNA (EBER) located in the nucleus is a reliable marker due to its high levels of expression and inherent stability in tissue specimens. EBERin situhybridization has long been the gold standard for detecting tumor-associated latent EBV infection and is valuable in determining the primary site and radiation fields of EBV-related malignancies. However, reliable detection is somewhat restricted by diffused signal and time-consuming procedure of this method, especially when proteins and RNA needed to be labeled simultaneously. Here, we optimized and validated our CRISPR-dCas9 mediatedin situRNA imaging tool—RCasFISH that enabled us to detect EBER rapidly and was compatible with IHC methods in fixed cells and tissue sections. Our approach could provide an attractive alternative for the molecular diagnosis of latent EBV infection.