Figure 3

Knockout of ChomOrco by CRISPR/Cas9 induce complete loss of Orco protein in screwworm. (A) Schematic of the Cas9-targeted regions of ChomOrco and sgRNAs tested. Targeted sites are indicated by a scissor, PAM motif in red, and genotyping primers as blue arrows. Band migration patterns expected after T7E1 and dual-targeting assays are indicated below. (B,C) In vivo evaluation of sgR-Orco-E1 and sgR-Orco-E2b, respectively. A small number of surviving larvae (n = 6) were collected and genotyped by T7E1 (leftmost). PCR amplifications spanning targeted sites were pooled, Illumina sequenced, and genome modifications quantified using CRISPResso (rightmost chart). A diversity of allele variations is shown in Supplementary Fig. S3. Cropped images (delineated by black lines) of PCRs and T7E1 experiments are from samples run on the same gels (one for each experiment). Full-length gels are displayed in Supplementary Fig. S5. wt = wildtype sample; NTC = no template control; Ldd = 100 bp DNA Ladder (NEB). (D) Crossing scheme used to develop Orco mutant strains. Briefly, G₀ flies were genotyped by T7E1, and mosaic males (Orco^m) backcrossed to wt females (Orco^wt). Heterozygous individuals (Orco^wt/?) at G₁ were genotyped by sequencing, and siblings harboring a − 16 bp mutation inbred at G₂. Heterozygous (Orco^wt/16) and homozygous (Orco¹⁶) flies at G₃ were identified by in vitro Cas9-assay (Supplementary Fig. S8) and inbred to establish the heterozygous (OR16ko) and homozygous (ChomOrco¹⁶) strains. (E) Sequencing confirmation of the mutant genotypes in comparison with the wt allele. (F) Immunostaining of antennae sections from wt (above) and ChomOrco¹⁶ (KO, below) flies, showing cell nuclei (DAPI) and Orco protein (OR83b-IC3) localization within cell body (white arrows) and dendrites (black arrows) of screwworm’ OSNs. As in control slides (Supplementary Fig. S9), mutant flies display undetectable levels of Orco protein.