Fig. 4: The effects of deleting HOX13 binding sites.

a On top are the HOX13 motifs in the II1 element, identified in Fig. 3b, with their positions indicated below (light blue boxes with orientations). The dark blue boxes below the DNA sequence indicate the positions and orientations of the CRISPR guides. Combinations of guides were used to generate small deletions (g2 + g3 or g2 + g6). The yellow crosses indicate the approximate cutting position of the Cas9. b E12.5 F0 LacZ stained embryos containing these deletions. The left panel is a II1 T-DOM control limb (no CRISPR cutting), with weak staining in the distal digit mesenchyme (dozens were stained). The two central panels are representative embryos (sample size n indicated in upper left corners) with the indicated deletions (see Supplementary Fig. 4-1 for images of all embryos including these two #5083 and #5146). Embryos carrying either the g2 + g3 or g2 + g6 deletions lost all staining in distal limb cells. In the same litters, we observed some embryos with strong distal limb staining (as shown in the right panel, see also Supplementary Fig. 4-1b, c). They contained a deletion that extends from the native II1 element in C-DOM to the II1 transgenic element in T-DOM, due to the guide RNA sequence presence at both sites (scheme in Supplementary Fig. 4-1c). Deletions in all embryos were sequenced (Supplementary Data 2). Embryos with ambiguous sequencing results or mosaicism were not used in this analysis. c DNA sequence, mapping to the putative HOX13 binding sites in the II1 enhancer elements (see a). II1 transgenic embryos were generated using these variants as the transgenic material. The top track is the wild-type sequence used in control embryos (II1 TgN ctrl). The Del 2 × 13 sequence lacks the two centromeric motifs (see a), while the Del 3 × 13 sequence lacks all three motifs for putative HOX13 binding. d On top are LacZ stained E12.5 transgenic embryos, the table below reports the number of embryos positive for the transgene by PCR (PCR + ), the second column is the number of embryos that stained in the distal forelimb (DFL), followed by the embryos with no staining in the DFL (No DFL Stain). The p values are determined by two-sided Fisher’s exact tests. Representative embryos are shown for each transgene. See also Supplementary Fig. 4-2. Scale bars are 0.5 mm.