A fast-to-faithful transition shapes the DNA repair landscape during embryogenesis.

Accurate and timely repair of DNA double-strand breaks (DSBs) is essential for genome maintenance in all cells. Embryos are particularly vulnerable to DSBs. During zebrafish development, a single fertilized cell undergoes rapid divisions to form an embryo of 50,000 cells in the first 24 hours, subjecting its genome to intense replication stress and the inevitable formation of genomic DSBs. While we know that failure to repair these breaks can result in embryonic lethality, the kinetics, fidelity, and pathway choice of DSB repair during embryogenesis are not well understood. Here, we used light-activated CRISPR to generate targeted genomic DSBs across zebrafish embryo development. Importantly, DSB induction occurs within seconds after light stimulation, enabling precise measurements of repair kinetics within a single cell cycle. We found that DSBs were repaired within 15 minutes during the early, rapid-division stages. At later stages, the pace of DNA repair declines as the cell cycle slows. By leveraging mathematical modeling and mutants that disrupt DNA repair pathways, we uncovered a developmental transition from error-prone microhomology-mediated end joining to more faithful non-homologous end joining that correlates with the gradual shift in repair kinetics. To our knowledge, this is the first study to resolve DSB repair dynamics with high temporal resolution during embryo development. Our study establishes a framework for systematically interrogating the cellular responses to DNA damage in living model organisms.