UNG can promote both error-prone and error-free repair of AID-induced lesions. NIH-3T3 cells were co-transduced with mOrange^STOP- and AID-ER–expressing vectors, and Ugi, UNG, or empty vector as control. Transduced cells were cultured in the presence of OHT during 11 d. (A) Percentage of mOrange⁺ cells was monitored by flow cytometry. Representative FACS analyses at day 9 are shown. (B) Time-course analysis of mOrange⁺ cells appearance in co-transduced NIH-3T3 cells, relative to the percentage of mOrange⁺ cells in control cultures after 2 d of OHT treatment. Means from three to seven experiments are represented (P < 0.05 at all days). (C and D) mOrange^STOP transgene was PCR amplified from the total population of co-transduced NIH-3T3 cells after 11 d of OHT treatment and sequenced by NGS. Proportion (C) and absolute mutation frequency (D) of G/C transversions, G/C transitions, and A/T mutations are represented. Frequencies were calculated as the mean of the mutation frequencies in all nucleotides of the amplified mOrange^STOP sequence. Error bars show the mean values of three different experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Complete mutation data are shown in Table S1.