Indel pattern-guided repair mapping reveals genome-wide DNA repair networks in CRISPR/Cas9 editing.

CRISPR/Cas9-induced DNA double-strand breaks (DSBs) trigger diverse repair outcomes, yet the dynamic regulatory networks governing these outcomes remain incompletely understood. Here, we develop indel pattern-guided repair mapping, an integrative framework that deciphers DSB repair mechanisms by integrating repair outcome spectra, kinetic dynamics, and functional gene regulation. Our analysis categorizes Cas9-mediated repair outcomes into seven distinct patterns based on their frequency and sequence characteristics, revealing differential repair kinetics among these subtypes. Functional clustering identifies three regulatory pillars: (i) microhomology-mediated end joining (MMEJ)-driven MH deletions form a cohesive module defined by a shared regulatory network of protein-coding genes and miRNAs, rather than by the core repair enzymes themselves; (ii) non-homologous end joining coordinates 1 bp insertions and non-MH deletions, with RFC4/5 stabilizing repair templates to suppress large deletions; (iii) Atypical repair outcomes show distinct genetic signatures: large insertions are associated with polymerase-related regulators, whereas mutations are associated with a signature enriched for chromatin-associated regulators. Strikingly, S100A8 emerges as a potent MMEJ suppressor via direct interaction with PARP1, revealing unappreciated cross-talk between inflammatory signaling and DSB repair pathway choice. By linking repair outcome patterns to molecular determinants, our work provides a transformative platform to interrogate DNA repair mechanisms for precise genome editing optimization and therapeutic genome stabilization.