Histone acetylation and deacetylation are key epigenetic modifications or heritable alterations of gene expression without changes in DNA sequence. Deregulation of histone deacetylase (HDAC) activity, showing either tumor-promoting or suppressive function, has been demonstrated in many cancers. The HDAC inhibitors currently in clinical trials have disappointing results in solid tumors, likely due to their broad specificity, toxicity and net effect of tumor-promoting and suppressive functions of HDACs. Therefore, there remains a need to identify new drug targets that are specific to the activity of HDACs with anti-tumor effects. Embryonal rhabdomyosarcoma (ERMS) is one of the most common and lethal pediatric cancers of soft tissue and is pathologically characterized by myogenic differentiation arrest. Most children with metastatic or recurrent ERMS will die from their cancer despite intensive chemotherapy, radiation, and surgery, illustrating the pressing need for innovative therapy. Our data characterizing the effects of pan HDAC inhibitors in vitro and in vivo demonstrate anti-tumor phenotypes such as increased tumor cell differentiation and reduced self-renewal potential. The biological function specific to each HDAC in ERMS remains unknown. Using the CRISPR (clustered regularly interspersed short palindrome repeats)/Cas9 genome engineering technology, we have targeted class I and class II HDAC genes in an ERMS cell line and demonstrated either tumor promoting or suppressive roles of selected HDACs in ERMS. The goals of the proposed study are to determine the role of HDAC genes in human ERMS tumorigenesis in vitro and in vivo by characterizing loss-of-function phenotypes, generated by the CRISPR (clustered regularly interspersed short palindrome repeats)/Cas9 genome engineering technology and to determine the mechanisms by which selected HDACs exert their tumor suppressive or promoting function. Key genes and pathways epigenetically regulated by selected HDACs will be identified through an integrated approach of Chromatin Immunoprecipitation Sequencing (ChIP-seq) and expression profiling studies. Potential interacting co-factor(s) of selected HDACs will also be identified by co- immunoprecipitation and characterized by loss-of-function studies. The innovation of the study is demonstrated by systemic targeting of the HDAC genes by the CRISPR-Cas9 technology, as delineation of functions specific to each HDAC gene by this genome editing technology has not been accomplished in any cancer. In addition, Our proposed approach integrating ChIP, expression profiling and candidate interacting gene studies to delineate functions specific to selected HDACs in modulating tumor cell phenotypes of ERMS has not been done previously. Results from this study will have high clinical and translational impact because insights gained from this study on functions of HDACs will provide mechanistic insights into disease pathogenesis for ERMS and other cancers linked to deregulated HDAC activity. In addition, potential new drug targets specific to the activity of selected HDACs identified from the study will improve treatment of ERMS patients with advanced or relapsed disease.