Fig. 1

DSB repair assays in TLR reporter cells. a Diagram of DSB repair pathway choice and ubiquitination of histone H2A at DNA double-strand breaks (DSB). Upon DSB induction, regulatory proteins bind to ubiquitin at positions K13 and K15 via ubiquitin-binding domains (UBDs). The 53BP1 and BRCA1 regulatory proteins play an important role in DSB repair pathway choice. DSB repair is executed by repair proteins and leads either to non-homologous end ligation (NHEJ) or homology-directed repair (HDR). If the NHEJ pathway is chosen, the DNA ends religate, frequently associated with nucleotide deletions or insertions. The HDR pathway enables precise sequence modifications if a DNA repair template is available. b The ‘traffic light’ reporter (TLR) system indicates the ratio of DSB repair by NHEJ or HDR. Upon induction of DSBs in the target region using CRISPR-Cas9, RFP is expressed when repair by NHEJ results in deletions that shift translation into the RFP reading frame. Venus expression reports for HDR when an intact Venus coding region is cotransfected. c Gating scheme for BFP positive cells in transfected HEK and hiPS reporter cells. Single cells were gated by using a forward scatter (FSC-H vs. FSC-A) plot. Transfected cells were gated based on expression of BFP from transfected plasmids compared to non-transfected control. d TLR assay in HEK or hiPS reporter cells. At least 10,000 cells were analyzed per sample for the Venus or RFP positive population. Double positive cells in the HEK^TLR6 clone are gated using an extra window