High-frequency mutagenesis by other ZFNs in the smc6b background. (A) TT4-ZFN-mediated mutagenesis in Col and smc6 mutant backgrounds. (B) MPK8-ZFN-mediated mutagenesis in Col and smc6 mutant backgrounds. In both cases, DNA was prepared from 1-wk-old pooled T1 seedlings in Col or smc6 backgrounds. The seedlings were grown on 20 μM estradiol MS medium. Target sites were PCR-amplified and digested with NspI (for TT4-ZFN) or MslI (for MPK8-ZFN). Four biological replicates of each sample were used to calculate mutation frequencies (see Supplemental Fig. S2). (Error bars) Standard errors. (*) Statistically significant differences (P < 0.005, t-test).