Measuring DNA Double-Strand Break Repair Using the DR-white Assay in Drosophila melanogaster

Traditional studies in DNA double-strand break (DSB) repair are frequently limited to biochemical investigations or unicellular systems (e.g., cells, yeast). To facilitate studies of DSB repair in a multicellular organism, we use the DR-white assay in Drosophila melanogaster. The DR-white assay is a simple yet versatile DSB repair reporter assay that carries a single I-SceI recognition sequence, which can be cleaved by expression of the I-SceI meganuclease. Expression of I-SceI can be induced constitutively, via heat shock, or under other promoters, enabling investigations of tissue- or developmentally specific repair. A primary advantage of the assay is that it can distinguish repair outcomes involving homologous recombination (HR), nonhomologous end joining (NHEJ), and single-strand annealing (SSA). This article provides a detailed protocol for the established DR-white assay, including a step-by-step workflow for inducing DSBs in the premeiotic germline and analyzing repair events phenotypically by scoring progeny. It also describes molecular analysis of repair events by polymerase chain reaction (PCR), sequencing, and the Tracking of Indels by DEcomposition (TIDE) R script. This protocol provides a useful tool for investigating the genetic and molecular mechanisms of DNA repair in an organismal model.