Detection of Epstein-Barr virus encoded RNA in fixed cells and tissues using CRISPR/Cas-mediated RCasFISH

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. EBER in situ hybridization 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 mediated in situ RNA 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.