Figure 3. CRISPR/Cas9-induced mutations created null alleles of the D. novamexicana and D. americana ebony genes.

(A) A schematic of the ebony gene is shown with grey boxes indicating exons; coding sequence is indicated in the darker shade of grey. Locations of the five guide RNAs targeting the second exon of ebony are shown with solid lines below scissor symbols. Mutations observed in the two ebony mutants (e^Δ10 and e^Δ7) isolated in D. novamexicana (“N”) and the one ebony mutant (e^Δ46) isolated in D. americana (“A”) are shown. All three alleles included deletions that caused frameshifts. (B) Western blotting showed that the D. americana e^Δ46 and D. novamexicana e^Δ10 mutants (lanes 2 and 4, respectively) lacked a ~100 kDa protein (arrows) recognized by an antibody raised against D. melanogaster Ebony protein (Wittkopp et al. 2002) that is present in wild-type (wt) D. americana and D. novamexicana (lanes 1 and 3, respectively). Relative abundance of total protein loaded into each lane can be seen by the relative intensities of the shorter proteins also detected by the Ebony antibody (Wittkopp et al. 2002) as well as the relative intensities of ~55kDa bands detected by an antibody recognizing alpha Tubulin (Abcam ab52866). The solid black line shows where the membrane was cut prior to incubation with primary antibodies during the western blotting procedure; the top half was incubated with anti-Ebony antibodies whereas the bottom half was incubated with anti-Tubulin antibodies. The two halves were realigned by hand for imaging, using the shape of the cut and the ladder staining as a guide. An un-annotated image of this blot is shown in Supplementary Figure 4.