Recruitment and Rejoining of Remote Double-Strand DNA Breaks for Enhanced and Precise Chromosome Editing

Chromosomal rearrangements, such as translocations, deletions, and inversions, underlie numerous genetic diseases and cancers, yet precise engineering of these rearrangements remains challenging. Here, we present a CRISPR-based homologous recombination-mediated rearrangement (HRMR) strategy that leverages homologous donor templates to align and repair broken chromosome ends. HRMR significantly enhances the efficiency of chromosome translocation, with increases of 2~20 fold at most genomic loci and up to 80-fold at specific loci, compared to non-homologous end joining (NHEJ). Additionally, it achieves >95% homologous recombination in all recombination events. Validated across multiple loci and cell lines, HRMR enables efficient and accurate chromosomal rearrangements. Live-cell imaging reveals that homologous donors mediate chromosome end proximity, enhancing rearrangement efficiency. Thus, HRMR provides a powerful tool for disease modeling, chromosomal biology, and therapeutic applications.