Fig. 3: A CRISPR/Cas9 screen identifies factors involved in RT-DSBR.

a Schematic representation of a flow-based CRISPR/Cas9 screen performed using the BFP-to-GFP reporter in HEK293T cells. Cells were transduced with Cas9 and sgRNAs from a DNA damage library. After 10 days of sgRNA selection, the BFP-to-GFP assay was carried out using the DNA^GFP or DNA/RNA^6R donor respectively. b The CRISPR/Cas9 screen data were analyzed using the MAGeCK algorithm by comparing the GFP⁺ sorted cells with the GFP⁻ BFP⁻ cells. A heatmap highlights selected genes with high-ranking scores, indicating factors that promote or suppress single-strand template repair. Lower ranks denote stronger hits. c BFP-to-GFP assay results using DNA^GFP and DNA/RNA^6R donors after knockdown of two top hits that promote (HELQ) or suppress (TP53BP1) RT-DSBR (n = 3–7 biological replicates). sgRNA targeting the AAVS1 locus was used as a control. Statistical significance was assessed using unpaired two-tailed t-tests. Error bars represent the standard error of the mean (± SEM). d Heatmap of the 5 top hits that promote or suppress RT-DSBR. e Comparison of the rank position of major DNA polymerases identified in DNA^GFP vs. DNA/RNA^6R CRISPR/Cas9 screens. Schematic in Fig. 3a was Created in BioRender. (2025) https://BioRender.com/9tmc1xb. Source data are provided as a Source Data file. See also Supplementary Fig. 3.