This research investigates the genetic aspects of skeletal myogenesis in Mus musculus, intending to broaden our comprehension of the matter. Skeletal muscle, a major tissue type in mammals, relies heavily on muscle stem cells for efficient regeneration and homeostasis. Commonly referred to as satellite cells, these stem cells are located between the basal lamina and myofibers' plasma membrane in adult muscle tissue, reminiscent of a satellite's positioning. They typically remain quiescent in healthy tissues but can quickly activate in response to injury, proceeding through cell cycles, proliferating, and differentiating. A subset returns to a quiescent state, reserving themselves for future tissue regeneration.Maintenance of the stem cell pool under aging or disease conditions necessitates strict regulation of these cells' stemness. In-depth study of in vivo molecular mechanisms of muscle development and regeneration involves specific gene deletion from muscle stem cells. A powerful tool for this is the Cre/loxP system, facilitating precise, controlled gene deletion. Despite its robustness, creating each conditional knockout model requires substantial effort and faces technical challenges. We propose an alternative approach using a unique CRISPR donor design combined with the i-GONAD technique. Demonstrating its potential and simplicity, we created floxed alleles for five genes (Fosl1, Plagl1, Ak040954, Clcf1, and Gm44386) in a single attempt with reduced costs and minimal equipment. Alongside the conditional alleles, we also obtained constitutive knockout alleles. Fosl1 is of particular interest, an AP-1 family member, with enriched mRNA in fresh- isolated SCs. Other AP-1 family members like Fos, ATF3, and Fosl2 have been revealed to play vital roles in muscle regeneration. Fosl1's function in muscle development and regeneration is, however, yet to be understood. Thus, we chose to study Fosl1 and created a specific Fosl1 gene deletion mouse model, Fosl1-cKO. After tamoxifen treatment and regeneration assay, we found that Fosl1 loss in muscle satellite cells significantly disrupted muscle regeneration. Furthermore, we found its induction rapidly when satellite cells are activated by injury or dissociations. Interestingly, loss or over-expression of Fosl1 doesn't influence human myoblast proliferation, differentiation, or fusion. However, RNA-seq analysis showed significant downregulations of muscle stem cell markers and myogenic differentiation markers in the Fosl1-cKO group. This study thus reveals Fosl1's critical role in muscle stem cell activation and tissue regeneration.