ZFN-mediated mutagenesis at the ADH1 locus in different DNA repair mutants. (A) ADH1-ZFN-mediated mutagenesis in Col (WT), ku70, lig4, and smc6 mutant backgrounds. DNA from 1-wk-old pooled seedlings grown on 20 μM estradiol MS medium was used for PCR amplification of the ZFN target site. The PCR products were digested with NlaIII. This experiment was repeated six independent times with similar results. (B) High-frequency mutagenesis in the smc6b mutant. Mutation frequencies were measured by dividing the signal intensity of the uncut band by the total signal from uncut and cut bands. Four biological replicates of ADH1-ZFN-4 and ADH1-ZFN-4 smc6b were used to calculate mutation frequencies.