Extended Data Fig. 3: Distribution of 23 ultraconserved enhancers among the three groups of enhancer mutagenesis outcomes.

a, Images of embryos and enhancer activity results for all 23 enhancers. Bar plots under the embryo images show the strength and reproducibility of LacZ staining (serving as a proxy for enhancer activity) among individual transgenic embryos from each enhancer allele. Enhancer activity was scored by five independent reviewers blind to the embryo genotype after the images were randomized (see Methods, Extended Data Fig. 2 and Supplementary Information for details). Embryo images for CRISPR-assisted transgenic experiments for the reference alleles of enhancers hs200 and hs215 were published previously²⁴. All other transgenic assays were newly performed for this study. All of the mutation alleles tested harbored sequence changes at 2, 5, or 20% of ultraconserved bases, as indicated above, with the exception of three of the alleles grouped with the 20% variants (marked with an asterisk [*] for hs122, hs200, and hs267). For the hs122 allele, 25% of bp were actually mutated. For hs200 (16% mutation) and hs267 (12% mutation), fewer than 20% of base pairs are well-conserved among ~100 vertebrates, so lower levels of mutagenesis were used. CN, cranial nerve; DRG, dorsal root ganglia; EY, eye; FB, forebrain; HB, hindbrain; HT, heart; LB, limb; MB, midbrain; NT, neural tube. b, Length of ultraconserved sequences for all 23 enhancers arranged by enhancer mutagenesis outcomes with mean and median shown per group as solid and dashed lines, respectively. No statistically significant differences detected between groups (two-tailed t-tests). bp, base pairs.