Myoblast fusion is essential for muscle development and regeneration. The long-term goal of our research is to identify key molecular regulators of this process, focusing on the role of CHAMP1 gene in CHAMP1 syndrome—a severe developmental disorder characterized by muscle weakness, hypotonia, and motor impairments, often requiring lifelong care. Our CRISPR-based screening, developed with R21 funding, identified CHAMP1 as a novel regulator of human myoblast fusion. We discovered a noncanonical function of CHAMP1 as a MyoD co-factor that promotes fusion by inducing the expression of muscle fusogen Myomaker. Deletion of CHAMP1 in patient cells and mouse models leads to impaired myoblast fusion, reduced Myomaker expression, and phenotypes resembling Myomaker-null conditions. To build on these findings, we aim to thoroughly investigate CHAMP1’s role in muscle development and function in vivo, utilizing tissue- and stage-specific mouse models that target both embryonic muscle precursors and adult myofibers. This involves detailed molecular and phenotypic analyses, assessing muscle morphology and functional performance. We will also leverage an unique transplantation model and employ the newest spatial transcriptomics tools to unbiasedly assess the consequence of patient mutations on human myoblast fusion. By this project, we will also elucidate the biochemical mechanisms by which CHAMP1 regulates Myomaker expression, focusing on its zinc finger motifs and interaction with the MyoD:TCF12:P300 complex. Our approach integrates RNA sequencing, CUT&Tag assays, protein interaction studies, and AlphaFold3-based structural modeling to map protein interfaces at the atomic level. Upon completing this study, we will not only gain critical insights into how CHAMP1 mutations cause fusion myopathy but also establish new animal models and molecular targets for studying and treating muscle symptoms in CHAMP1 syndrome. This project will benefit CHAMP1 patients in three distinct venues: 1)