Figure 1.

Construction and validation of the repair reporter system. (A) Schematic representation of reporter system and plausible outcomes. Outcome signal 1 produces mCherry fluorescence from plasmids that are not edited or are repaired by homology-directed repair (HDR), splicing an unedited fragment from another copy of the plasmid in the cell. Note that for output signal 2, both mCherry and GFP fluorescence would occur because during transient transfection only a fraction of transfected copies of sequences will likely be spliced to eliminate mCherry and express GFP. For output signal 3, if the construct remains unrepaired no fluorescence will be produced for GFP or mCherry. (B) Validation of reporter system by fluorescent microscopy identifies transfected mCherry positive cells and reveals repair-induced GFP protein expression when both TALENs are co-expressed. Representative images from triplicate experiments are shown. (C) Western blot confirmation of clonal selection of XRCC4 knockout from HeLa cells in clone 2G3** revealed no XRCC4 expression. ** indicates clone used for experiments. (D) The XRCC4 gene sequence corresponding to the target region in the 1st exon (nucleotide position 179–212th) of XRCC4 for wild-type cells and XRCC4 knockout 2G3 clone. Deletions are indicated by “-”. Clone 2G3 contains a frameshift indel on all alleles of XRCC4. Abbreviations: TBS = TALEN binding site; CMV = cytomegalovirus promoter. Scale bar = 100 μm.