Proximity determines donor candidacy during DNA double-stranded break homology directed repair

DNA double-stranded breaks (DSBs) are especially toxic events that can be reversed by homology-directed repair (HDR), wherein information is copied from an intact template molecule. RAD51 mediates initial DSB/template pairing during homology search. A major challenge in understanding homology search in cells is the lack of tools to monitor this process. We developed RAD51 proximity identification sequencing (RaPID-seq), a sensitive method that marks all candidate templates searched by RAD51. We find that HDR is hierarchical, such that DSB proximity determines template candidacy and subsequent recombination is unlocked by DSB/template homology. Sequences that lie outside the proximal window are not efficiently searched, even if identical in sequence. Our data reveal the invisible process of homology search and shed new light on fundamental mechanisms underlying genome editing.