(a) Sequence comparison of the dsx intron 4-exon 5 boundary and the dsx female-specific exon 5 within the 16 anopheline species16. The sequence of the intron 4-exon 5 boundary is completely conserved within the six species that form the Anopheles gambiae species complex (noted in bold). The gRNA used to target the gene is underlined and the PAM is highlighted in blue. Changes in the DNA sequence are shaded grey and codon silent and missense substitutions are noted in blue and red respectively. (b) SNP frequencies obtained from 765 Anopheles gambiae mosquitoes captured across Africa17. Across the dsx female-specific Exon 5 there are only 2 SNP variants (noted in yellow) with frequencies of 2.9% (the SNP in the gRNA-complementary sequence) and 0.07%.