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Fig. 2 The effect of low-temperature incubation on on-target and off-target mutation rates. After the introduction of RGEN, embryos were cultured at 28 °C or 16 °C until Stage 25. Amplicon sequencing was performed for each region, and the mutation rate was calculated. A DJ-1 and Off-D (n = 10) B p4hb and Off-P (n = 10) C avt and Off-A (n = 8). While no significant difference in on-target mutation rates was observed between temperatures, off-target mutation rates were significantly reduced at 16 °C compared to 28 °C. N.S.: no significant difference, **: p < 0.01, ***: p < 0.001 Columns and error bars represent mean ± SE