Skeletal muscle satellite cells (SCs) are stem cells responsible for muscle development and regeneration. Although CRISPR/Cas9 has been widely used, its application in endogenous SCs remains elusive. Here, we generate mice expressing Cas9 in SCs and achieve robust editing in juvenile SCs at the postnatal stage through AAV9-mediated short guide RNA (sgRNA) delivery. Additionally, we reveal that quiescent SCs are resistant to CRISPR/Cas9-mediated editing. As a proof of concept, we demonstrate efficient editing of master transcription factor (TF) Myod1 locus using the CRISPR/Cas9/AAV9-sgRNA system in juvenile SCs. Application on two key TFs, MYC and BCL6, unveils distinct functions in SC activation and muscle regeneration. Particularly, we reveal that MYC orchestrates SC activation through regulating 3D genome architecture. Its depletion results in strengthening of the topologically associating domain boundaries thus may affect gene expression. Altogether, our study establishes a platform for editing endogenous SCs that can be harnessed to elucidate the functionality of key regulators governing SC activities.

骨骼肌卫星细胞（SC）是负责肌肉发育和再生的干细胞。尽管 CRISPR/Cas9 已被广泛应用，但其在内源性 SC 中的应用仍然难以捉摸。在这里，我们生成了在 SC 中表达 Cas9 的小鼠，并通过 AAV9 介导的短向导 RNA (sgRNA) 传递在出生后阶段在幼年 SC 中实现了强大的编辑。此外，我们发现静止的 SC 对 CRISPR/Cas9 介导的编辑具有抵抗力。作为概念证明，我们展示了使用 CRISPR/Cas9/AAV9-sgRNA 系统在幼年 SC 中有效编辑主转录因子 (TF) Myod1位点。 MYC 和 BCL6 这两个关键 TF 的应用揭示了 SC 激活和肌肉再生中的不同功能。特别是，我们揭示了 MYC 通过调节 3D 基因组结构来协调 SC 激活。它的耗尽导致拓扑关联域边界的加强，因此可能影响基因表达。总而言之，我们的研究建立了一个编辑内源性 SC 的平台，可用于阐明管理 SC 活动的关键监管机构的功能。