Figure 1. A genome-wide screening for factors that control the balance between NHEJ and HR.

(a) Schematic representation of the SSR. An I-SceI-induced DSB can be repaired by NHEJ, thus reconstructing an active GFP gene, or by homologous recombination using RFP fragments, thus creating a functional RFP gene. (b) Workflow of the screening method. Individual esiRNAs deposited in a 96-well plate format were used to reverse-transfect U2OS cells harbouring a single copy of the SSR system. After 36 h, lentiviral particles bearing I-SceI and the BFP genes were transduced into cells. After 2 days, cells were imaged using a high-throughput microscope for green, red and blue fluorescence. The number of green and red cells was then established for each individual esiRNA. Scale bar, 100 μm (c). The ratio between green and red cells was established for each individual esiRNA and normalized to an internal control (esiRNA targeted against luciferase) that was included in each plate. The individual NHEJ:HR ratio was then ordered and plotted. Cells with a normalized ratio close to one occupy the central dashed rectangle. Genes for which depletion reduced the ratio (that is, HR was increased) are marked in the red ellipse and correspond to proteins that naturally favour NHEJ. In contrast, genes that encode pro-recombination proteins (that is, NHEJ increased when they were downregulated) are included inside the green ellipse. (d) Functional categories enriched among candidates. The list of candidates that alter the NHEJ:HR ratio was analysed using the IPA Software and categorized into functional groups. Those categories in which genes were statistically significantly over-represented in the set of genes that favour NHEJ (left) or HR (right) are plotted. (e) Network of genes involved in cell cycle, DNA repair, replication and recombination and cancer, which appear in the set of candidates isolated in the screening. Genes that favour HR are labelled in green, and those that facilitate NHEJ, in red.