DSB repair reporter assays

The activity of DSB pathways was measured using GFP reporter assays for homologous recombination (DR-GFP), single-strand annealing (SSA-GFP), alternative end-joining (EJ2-GFP), total NHEJ (EJ5-GFP), and distal NHEJ events without indels (EJ7-GFP). The reporters DR-GFP, SSA-GFP, EJ2-GFPand EJ5-GFP were co-transfected with a plasmid encoding the I-SceI endonuclease to introduce a DSB at I-SceI sites in the reporter constructs. The EJ7-GFP reporter was co-transfected with a CRISPR-Cas9 plasmid to induce blunt DSBs at the recognition site of guide RNAs (sgRNA 7a and 7b) in the construct. All reporters were co-transfected with control or NIHCOLE-targetting gapmers. After 48 hours post-transfection GFP was measured in 1×10⁵ cells by FACS. A GFP expression vector was co-transfected in parallel to measure transfection efficiency. DR-GFP (Addgene: 26475), SSA-GFP (Addgene: 41594) and I-SceI (Addgene: 26477) plasmids were a gift from Maria Jasin. EJ2-GFP (Addgene: 44025), EJ5-GFP (Addgene: 44026) and EJ7-GFP reporter, 7a and 7b sgRNA vectors (Addgene: 113617, 113620 and 113624, respectively) were a gift from Jeremy Stark. S. pyogenes CRISPR-Cas9 plasmid (Addgene: 52961) was a gift from Feng Zhang.