FIG. 3.

Demonstrating the editing activity of FusXTBE system in vitro for mtDNA. (A) Potential target sites favorable for base editing in the protospacer region of the targeted human MT-ND4 locus. (B) Editing efficiency of the mitochondrial FusXTBE and DdCBE systems in HEK293T cells for the C₅ and C₈ positions, assessed by Sanger sequencing. Data are represented from independent experiments. Red and green colored data points represent editing efficiency by FusXTBE and DdCBE systems, respectively. Black colored data points represent the wild-type heteroplasmy in the target locus in the nontransfected cells. Error bars are represented as standard error of the mean. No significant difference was observed in the editing efficiencies between the FusXTBE and DdCBE systems (p > 0.05 as determined by Student's t-test). (C) Representative chromatogram of the control (nontransfected) and cells transfected with FusXTBE and DdCBE plasmids. Asterisk (*) denotes the site of edit (C₅ position) with corresponding editing percentage (C-to-T or G-to-A). Chromatograms and editing table plot were obtained using EditR. (D) Editing frequency of MT-ND4 alleles estimated by deep sequencing of the target amplicon. Edited cytosine (antisense strand) residue (or guanine on the sense strand) is highlighted in red and circled. Frequency reflects the mean percentage ± standard deviation from independent biological replicates (N = 6 for FusXTBE and N = 3 for DdCBE). A combination of left arm- pKTol2C-FusXTBE-C and right arm- pKTol2C-FusXTBE-N was used to obtain the edits. Color images are available online.