a For an MIT nuclease (against the mouse mtDNA tRNA-Ala mutation [MIT 11–12]), a pair of engineered CHO lines were produced to carry either the wild-type (WT) or mutant mtDNA target site in the nuclear DNA. The target site was positioned between direct repeats of a GFP gene such that cleavage of the target site promotes homologous recombination events between repeated regions to yield a functional GFP. In addition, there is a target site for a positive control nuclease (“CHO 23-24”) incorporated next to the MIT target site (left panel). Each of the cell lines was transfected with mRNA encoding MIT 11–12 or CHO 23-24 (control) and cells were assayed by flow cytometry 2, 5, 7, 9, and 12 days post transfection for the percentage of GFP+ cells (the average GFP fluorescence for the different time points is shown in the right panel). As the different time points are not biological replicates, no statistic was applied. b mitoARCUS gene construct for ex vivo expression includes CMV promoter, mitochondrial localization sequence (MLS) of Cox8 or Cox8/Su9, Flag tag for immunological detection, Meganuclease (ARCUS) sequence, and PolyA tail. c Immunofluorescence done on HeLa cells 24 h after transfection with mitoARCUS. MitoTracker stains mitochondria red, Flag stains mitoARCUS green, and merged image shows colocalization (yellow) of mitoARCUS to mitochondria. Images taken at ×40 magnification. This experiment was repeated twice with identical results. (d) Western blot depicting mitoARCUS expression (FLAG) in HEK293T cells 24 h after transfection with either CF or CSF construct. Lanes CF + GFP and CSF + GFP depict protein expression in cells transfected with mitoARCUS constructs in which we added a GFP sequence. Lane Unt represents untransfected cells. Lane GFP represents cells transfected with GFP only. Tubulin (Tub) expression was used as a loading control. This experiment was performed once.