(A) Strategy for assessing the base-editing efficiency for the mutation GJB2 R75W. (B) Representative results from direct genome sequencing after base editing via SaABE#1 to #5 or CjABE#6. The sequence at the bottom is the forward sequence. Red arrows indicate correction of the target site, and gray arrows indicate bystander editing. n = 3 technical replicates, representative of 3 independent experiments. (C) Representative images of EGFP⁺ cells expressing CX26 (red) and forming GJPs after base-editing plasmid transfection. Arrowheads indicate extensive GJPs in EGFP-expressing cells after base editing. Scale bar: 10 μm. (D) Representative images of GJPs after base editing by SaABE or CjABE with each sgRNA. HeLa/CX26 R75W cells were transfected with each base-editing plasmid vector. Negative control cells were left untreated. After transfection for 48 hours, EGFP⁺ cells were sorted, cultured for 1 week, and resorted to collect the EGFP^– cells as samples. Samples were seeded into 96-well plates and cultured for 4 days. Samples were then fixed, permeabilized, and incubated with anti-CX26, followed by incubation with Alexa Flour 488–conjugated anti-rabbit IgG and Alexa Flour 555–conjugated cholera toxin subunit B, and then counterstained with DAPI. n = 3 technical replicates, representative of 3 independent experiments. Scale bar: 20 μm. (E–H) Quantitative length (E), area (F), form factor (G), and LAF (H) data presented in D. Box-and-whisker plots show median, interquartile range, and minimum and maximum values; isolated dots beyond the whiskers correspond to outliers defined as a value that is smaller than the lower quartile −1.5 × the interquartile range or larger than the upper quartile +1.5 times the interquartile range. Each value was normalized to the value obtained for HeLa/CX26 R75W cells (untreated). n = at least 70 analyzed per group. Statistical significance was determined with Kruskal-Wallis test with Dunn’s multiple comparisons test. *P < 0.05, **P < 0.01, ****P < 0.0001.