Genome-wide mapping of Cas9-induced sister chromatid exchange across single and over 200 genomic sites in human cells.

CRISPR/Cas9-induced DNA double-strand breaks (DSBs) are widely used for genome engineering, yet their capacity to provoke sister chromatid exchange (SCE) remains incompletely understood because exchanges between identical sisters leave no sequence change and are invisible to conventional sequencing. Using sci-L3-Strand-seq, a scalable single-cell template-strand sequencing platform, we mapped SCE following targeted Cas9 cleavage. Cas9 targeting of a unique genomic locus strongly enriched SCE at the break site, reaching 41% in the same cell cycle and 17% in the subsequent division, indicating frequent non-local inter-sister repair. Targeting 237 repetitive Alu sites produced mild enrichment of on-target SCE, whereas cells with elevated SCE burden showed significant enrichment at programmed cut sites, suggesting context-dependent recombination at repetitive loci. Reciprocal daughter-cell pair analysis revealed large-scale structural alterations on chromosomes with induced SCEs and at SCE junctions, indicating that Cas9-induced SCE does not universally reflect error-free homologous recombination. Disruption of LIG3, LIG4, XRCC1, and XRCC4 at Cas9 cut sites did not measurably alter SCE frequency, consistent with delayed functional loss following editing. Together, these findings identify Cas9-induced DSBs as potent local triggers of SCE and reveal structural outcomes invisible to conventional sequencing.