Fig. 3. Tyrosinase and VAChT loci and sgRNA validation results.

A) Selected Tyrosinase sgRNA target sites are indicated, along with Amplicon-EZ (Genewiz) indel plots and automated mutagenesis rate estimations. Mutagenesis rates are approximated for plots in which naturally occurring indels confounded automatic rate estimation as described before. B) Left: diagram of simple pigmentation assay (see text for details). Right: results of pigmentation assay of Tyr CRISPR larvae using sgRNAs 1.185 and 5.16 together (40 μg each) in combination with 50 μg Sox1/2/3>Cas9::Geminin^Nterminus. (Pennati et al., 2024; Stolfi et al., 2014). CRISPR larvae (n = 45) were compared to negative control larvae (n = 29) electroporated with 80 μg of the “control” sgRNA plasmid instead (Stolfi et al., 2014). Plasmid DNA amounts indicated per 700 μl of total electroporation volume. Statistical significance determined using Fisher’s exact test. C) Selected VAChT sgRNA target sites indicated, along with indel plots. Dashed red line indicates alternative splicing of first exon with remaining exons encoding choline acetyltransferase (ChAT) out of view, as part of a conserved cholinergic locus. Red arrows indicate CRISPR-generated indel peaks. Grey asterisks indicate naturally-occurring indels.