eTLR is based on C-terminally truncated (Trunc.) mScarlet-I followed by a stop codon and a concatenation via P2A sites of three different frames coding for the green fluorescent protein eUnaG T52S . Out-of-frame ATGs were removed to prevent unintended translation initiation. Disruption of the stop codon via, for example, NHEJ/MMEJ/SSA results in readthrough and the activation of one of the eUnaG frames (eUnaG (+1/2/3)) (green fluorescence). Instead, successful repair via PE or HDR results in full-length mScarlet-I (red fluorescence). Cells with a biallelic integration of eTLR were analyzed via FACS, where red fluorescence reports successful HDR or PE, while green fluorescence indicates a mutagenic end-joining event. A signal on both the red and green channels (shown in orange) indicates that both events have occurred, each on a separate allele.
