CRISPR-Cas9 Olfactory Receptor Mutation

After microinjections, all G₀ larvae transiently expressing the ZsGreen marker were selected and raised to adulthood. Non-lethal DNA extractions (Supplementary Fig. S6 and Supplementary Methods) were performed for G₀ adult flies and used as templates for PCR amplifications spanning the targeted site at ChomOrco exon 1. Amplifications were submitted to T7E1 cleavage assays, as previously described^{23 (link)}, and ten mosaic males were individually backcrossed to wt females (1 Cas9 G₀ ♂ × 4 wt ♀). On average, eight G₁ adult males from each crossing were randomly selected and genotyped as before. Nine heterozygous G₁ males (one per obtained mutant line) were selected and individually backcrossed to wt females for a second time, while simultaneously genotyped by Sanger sequencing and CRISP-ID analysis^{42 (link)}. Adult G₂ male and female flies harboring a − 16 bp deletion were identified by T7E1 and let to interbreed freely in cages. Homozygous mutants at G₃ were identified by in vitro Cas9-assay, using the Guide-it Genotype Confirmation Kit (Takara), and inbred to establish the Orco mutant strain at G₄. Further genotyping, when required, was conducted using our custom High Resolution Mobility analysis (HRMob, described in Supplementary Fig. S7).