Fig. 1.

CRISPR-Cas9–directed mutagenesis of M. sexta Orco. (A) The M. sexta Orco protein has 7 transmembrane domains. The first transmembrane domain was mutated generating a predicted frameshift (yellow) and translational stop signals (red) (B, Top). The M. sexta 10-exon Orco gene (18.85 kb) was accessed through the annotated official gene set (OGS) (gene ID Msex2.12779), with scaffold and base pair coordinate location on the positive strand (12,214 to 31,059 bp) within scaffold JH668978.1. A proximal region to the start of exon 2 was targeted for CRISPR single-guide RNA (sgRNA) design. Two sgRNAs (target1 and target2) on opposite DNA strands were multiplexed and injected. Target 1 was designed with a mismatch nucleotide, indicated in red to raise in vitro transcription efficiency. PAM sites indicated in purple, Cas9 cleavage sites 3 nucleotides from the PAM site indicated by arrows. (B, Bottom) Three germ-line insertion deletion (indel) mutations were recovered and sequenced (mutations 1, 2, and 3). All indels were generated by sgRNA target 1, insertion indicated in red, deletions indicated as dash lines. (C, Top) Mutation 1 was used for downstream functional analysis. Sanger sequencing chromatogram of wild-type (WT) individual and mutation 1 allele shows insertion event, indicated by box. (C, Bottom) Indel generated a frameshift mutation introducing 2 stop codons downstream, giving rise to a truncated 72-residue protein, indicated through the predicted amino acid sequence.