Extended Data Fig. 5: Generation and validation of loss-of-function alleles of D. sechellia Or genes.

a, Schematic of the strategy for generating mutant alleles of Or genes, through integration of an eye-expressed fluorescent marker (3×P3:DsRed or 3×P3:GFPnls) into the desired locus via CRISPR–Cas9-cleavage induced homologous recombination. Brown triangles, loxP sites for removal of the fluorescent marker via Cre recombination. b, Schematics depicting Or gene organization, the structure of mutant alleles and the location of the sequences that encode antibody epitopes. For DsecOrco¹, the fluorescent marker was integrated into the first coding exon; for DsecOrco², the marker replaces parts of exons 1 and 3 and the whole of exon 2. DsecOr22a^RFP carries the fluorescent marker in the first coding exon close to the start codon. DsecOr35a^RFP lacks most of exons 1 and 2. For DsecOr85b^GFP, the marker was integrated into exon 1; for DsecOr85c/b^RFP, the marker replaces most of the Or85c gene and part of exon 1 of Or85b. c, Immunofluorescence for Orco and Ir25a (as an internal staining control) on whole-mount antennae from wild-type and DsecOrco² flies. Scale bars, 25 μm (c–g, main panels), 5 μm (c–g, insets). d, RNA FISH for Or22a and Or85b on whole-mount antennae from wild-type, DsecOr22a^RFP and DsecOr85b^GFP mutant flies. e, Immunofluorescence for Ir75b and RNA FISH for Or35a on whole-mount antennae from wild-type and DsecOr35a^RFP mutant flies. Arrowheads indicate Or35a-expressing cells. Or35a neurons also pair with Ir75c neurons in ac3II sensilla¹⁵, which is reflected in Or35a-positive cells that are not paired with Ir75b-expressing cells in wild-type antennae. f, Immunofluorescence for Or22a on whole-mount antennae from wild-type and DsecOr22a^RFP mutant flies. Arrowheads indicate sensilla that house Or22a neurons. g, Far left, immunofluorescence for Orco and Ir25a (as an internal staining control) on whole-mount antennae from wild-type (same image as shown in c) (top) and DsecOrco¹ (bottom) flies. Middle and right, electrophysiological responses in the two neurons of the ab3 sensillum (Fig. 2a) to odours present in noni in wild-type D. sechellia and DsecOrco¹ mutants (n = 5–20, female flies). Representative response traces to methyl hexanoate (10⁻⁶ dilution) and 2-heptanone (10⁻⁶ dilution) are shown. Data points represent the solvent-corrected activities per neuron. Responses of wild-type D. sechellia neurons are replotted from Fig. 2a. Even though Orco expression is undetectable by immunofluorescence, weak electrophysiological responses in ab3 sensilla (and other Orco-dependent sensilla (data not shown)) can be detected. These observations suggest that trace levels of functional Orco are produced from this allele, potentially through use of in-frame start codons downstream of the marker insertion site (as shown in h). h, Schematic depicting the location of the Orco start codon, the fluorescent-marker insertion site of the DsecOrco¹ allele and downstream potential alternative in-frame start codons.