Humanized 3D-tissue-engineered-skeletal-muscles (3D-TESMs) offer advanced disease modelling, but their application often require isogenic controls and involves laborious and time consuming methodology. Here, by combining 3D-TESM and shRNA approaches, we developed a “one-line-fits-all” disease modelling strategy to rapidly induce distinct genetic deficiencies in a single hiPSC-derived cell line. As proof-of-principle, we recapitulated disease-associated pathology of Duchenne Muscular Dystrophy and LGMD2A caused by loss of function of DMD and CAPN3, respectively. shRNA-mediated knockdown of DMD or CAPN3 induced a loss of contractile function, disruption of tissue architecture, and disease-specific proteomes within a seven days period. Pathology in DMD 3D-TESMs was partially rescued by a candidate gene therapy treatment using microdystrophin, with similar efficacy compared to mouse models. These results show that isogenic shRNA-based humanized 3D-TESM models provide a fast and efficient tool to model muscular dystrophies and are useful for the preclinical evaluation of novel therapies.