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Figure 1. Experimental design for identifying conditions that increase gene disruption efficiency. (a) Semi-quantitative image analysis of the pigment phenotype (upper panels) and representative embryos with specific percentages of pigmented regions (lower panels). (b) Flow chart of the step-by-step analyses of the five experimental parameters expected to influence gene disruption efficiency.
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Figure 2.Effects of varying the injection timing and the amount of injected Cas9 mRNA per embryo. A box-and-whisker plot showing pigmentation ratios of the uninjected embryos (Uninj.) and the embryos generated under the experimental conditions shown in the Table (I, II, III, IV, and V) as dots. In each sample group, vertical lines indicate the minimum, first quartile, median (Med.), third quartile, and maximum values excluding outliers beyond the whisker end, respectively, from left to right. The upper limit of the whisker is 1.5 times the box length. Only significant differences in the rates of normally developed embryos are shown in this and other tables. ***p < .001; *p < .05; and “n.s.” (not significant).
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Figure 3. Effects of varying the culture temperature of the injected embryos, the volume of the injected reagents, and the number of sites injected. (a) A box-and-whisker plot showing the pigmentation ratio of uninjected embryos cultured at 16°C and the embryos generated under the experimental conditions shown in the Table (IV, VI, and VII). (b) A box-and-whisker plot showing the pigmentation ratios of the uninjected embryos and the embryos generated under the experimental conditions shown in the Table (VI, VIII, and IX). **p < .01.
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Figure 4. Reassessment of culture conditions for injected embryos. (a) A box-and-whisker plot showing the pigmentation ratios of the uninjected embryos cultured at 22°C and the embryos generated under the experimental conditions shown in the Table (IX, X, and XI). (b) The left and right box-and-whisker plots show the disruption ratios of tyr.L and tyr.S in embryos generated under the conditions IX, X, and XI, as dots, respectively. Since the plots were generated only with the disruption ratios whose certainty (R-squared) were greater than the recommended value (0.8) in the ICE analysis, the plotted dot numbers for tyr.L and tyr.S are different. The raw output data of the ICE analysis corresponding to black-lined dots are shown in Figure S4.
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Fig. S1. Pigmentation ratios of uninjected embryos cultured at different temperatures.
A bar graph showing the pigmentation ratios of the uninjected embryos cultured at a constant temperature of 16, 22, or 25°C after fertilization, or initially at 12°C for 6 hours and subsequently at 22°C. The data are shown as in Figure 2. There were no significant differences in the pigmentation ratios among the sample groups.
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Fig. S2. Reproducibility of the relationship in pigmentation ratios, culture conditions, and the rates of normally developed embryos.
(A) In the bar graph, each
dot indicates the mean
pigmentation ratio for
each group of embryos
generated under Condition
IX, X, or XI shown in
Figure 4A. The sample
groups were generated by
three rounds of
experiments, including the
one shown in Figure 4A,
and an additional two
independent experiments
using embryos obtained
from different female and
male pairs (Exp. 1, Exp. 2,
and Exp. 3 shown in the
table in (B), respectively).
Each bar length represents
the mean of the three
means obtained from these
three rounds of
experiments. The error
bars indicate SEM. Statistical analysis was performed using the Tukey-Kramer test (Tukey HSD) in R. (B) A table showing the number of embryos with normal external morphology and the total number of analyzed embryos for each sample group shown in the bar graph in A, along with the percentage of the embryos with normal external morphology. The bar graph represents the means of the percentages of normally developed embryos obtained from the three rounds of experiments for the sample groups shown in the table. The error bars indicate SEM. No significant differences were detected among the sample groups (shown as n.s.) by the Tukey- Kramer test.
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Fig. S3. Effects of varying the amount of injected Cas9 protein per embryo.
A box-and-whisker plot showing the pigmentation ratios of the uninjected embryos (Uninj.) and the embryos generated under the experimental conditions shown in the table (XII, XIII, and XIV) as dots. The data are shown as in Figures 2–4. There were no significant differences in the rates of normally developed embryos among the four sample groups.
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Fig. S4. Output data of the ICE analyses performed to analyze tyr.L and tyr.S disruption ratios in representative embryos generated under Conditions IX, X, and XI.
The genomic sequences around the target regions of tyr.L-sgRNA and tyr.S-sgRNA are shown on the top, from left to right. The target sequences are underlined, and the PAM sequences are boxed in red. The tables in the left and right rows are the outputs of the ICE analyses that were performed for tyr.L and tyr.S loci, respectively, using representative embryos generated under the Conditions IX, X, or XI. A sum of the percentages of the indel patterns shown in each table indicates a total disruption ratio of the target gene in the analyzed representative embryo. The total disruption ratios of those embryos are indicated by black-lined dots in Figure 4B.
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