In vivo systematic detection of the outcomes of CRISPR-Cas9-mediated DNA repair in skeletal muscle stem cells.

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRSIPR-associated protein 9 (Cas9) has revolutionized genome editing with broad therapeutic applications, yet its repair patterns in vivo remain poorly understood. Here, we systematically profile CRISPR-Cas9 editing outcomes at 95 loci using our established CRISPR-Cas9/adeno-associated virus (AAV)9-single guide RNA (sgRNA) system in skeletal muscle stem cells (MuSCs). Through comprehensive characterization of the repair outcomes, our findings demonstrate that the general rules governing CRISPR-Cas9-mediated editing in vivo largely align with those observed in vitro. In addition to the anticipated small editing insertions or deletions (indels), such as microhomology-mediated end joining (MMEJ)-mediated deletions and non-homologous end joining (NHEJ)-mediated templated insertions, we uncover a prevalent occurrence of large on-target modifications, including large deletions (LDs) characterized by microhomology (MH) and large insertions (LIs). Notably, the LIs comprise not only exogenous AAV vector integrations but also endogenous genomic DNA fragments (Endo-LIs). Endo-LIs preferentially originate from active genomic regions, with their integration shaped by 3D chromatin architecture. By disrupting key components of the NHEJ and MMEJ repair pathways in vivo, we identify their distinct roles in regulating the large on-target modifications. Together, our work systematically profiles CRISPR-Cas9 repair outcomes in vivo and offers valuable guidance for improving the safety of CRISPR-Cas9-based gene therapies.