MicroRNAs (miRNAs) are small non-coding RNAs with profound regulatory roles in many areas of biology, including cancer. MicroRNA 155 (miR-155), one of the extensively studied multifunctional miRNAs, is important in several human malignancies such as diffuse large B cell lymphoma and chronic lymphocytic leukemia. Moreover, miR-155 orthologs KSHV-miR-K12-11 and MDV-miR-M4, encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) and Marek's disease virus (MDV) respectively, are also involved in oncogenesis. In MDV-induced T-cell lymphomas and lymphoblastoid cell lines derived from them, MDV-miR-M4 is highly expressed. Using excellent disease models of infection in natural avian hosts, we showed previously that MDV-miR-M4 is critical for the induction of T-cell lymphomas as mutant viruses with precise deletions were significantly compromised in their oncogenicity. However, these studies did not elucidate whether continued expression of MDV-miR-M4 is essential for maintaining the transformed phenotype of tumor cells. Here using an in situ CRISPR/Cas9 editing approach, we deleted MDV-miR-M4 from the MDV-induced lymphoma-derived lymphoblastoid cell line MDCC-HP8. Precise deletion of MDV-miR-M4 was confirmed by PCR, sequencing, quantitative RT-PCR and functional analysis. Continued proliferation of the MDV-miR-M4-deleted cell lines demonstrated that MDV-miR-M4 expression is non-essential for maintaining the transformed phenotype, despite its initial critical role in the induction of lymphomas. Ability to examine the direct role of oncogenic miRNAs in situ in tumour cell lines is valuable in delineating distinct determinants and pathways associated with the induction or maintenance of transformation in cancer cells and will also contribute significantly to gain further insights into the biology of oncogenic herpesviruses.ImportanceMarek's disease virus (MDV) is an alphaherpesvirus associated with Marek's disease, a highly contagious neoplastic disease of chickens. MD serves as an excellent model for studying virus-induced T-cell lymphomas in the natural chicken hosts. Among the limited set of genes associated with MD oncogenicity, MDV-miR-M4, a highly expressed viral ortholog of the oncogenic miR-155, has received extensive attention due to its direct role in the induction of lymphomas. Using a targeted CRISPR-Cas9-based gene editing approach in MDV-transformed lymphoblastoid cell lines, we show that MDV-miR-M4, despite its critical role in the induction of tumours, is not essential for maintaining the transformed phenotype and continuous proliferation. As far as we know, this is the first study where precise editing of an oncogenic miRNA has been carried out in situ in MD lymphoma-derived cell lines to demonstrate that it is not essential in maintaining the transformed phenotype.