Embryo microinjection and mutation screening

Embryonic collection and CRISPR microinjections were performed following previously established procedures (Aryan et al., 2014; Kistler et al., 2015). The concentration of plasmids used for the U6 promoter screen was 300 ng/µL. Injected G₀ and G₁ progeny were visualized at the larval, pupal, and adult life stages under a dissecting microscope (Olympus SZ51 and Leica M165FC). The heritable mutation rates were calculated as the number of G₁ progeny with the loss-of-function mutation out of the number of all G₁ progeny crossed with the white eye (w–) strain mosquitoes. To integrate each GDe construct at the white locus, a mixture containing 100 ng/µL of synthetic gRNA^w, 100 ng/µL of each U6-GDe plasmid (U6a-GDe, U6b-GDe, U6c-GDe, and U6d-GDe), and 100 ng/µL of Cas9 protein was injected into 500 wt embryos for each plasmid. Synthetic gRNAs (Synthego) and recombinant Streptococcus pyogenes Cas9 protein (PNA Bio Inc, Supplementary file 8a) were obtained commercially and diluted to 1,000 ng/µL in nuclease-free water and stored in aliquots at –80°C. A total of 233, 271, 191, 215 G₀ adults were recovered for U6a-, U6b-, U6c-, and U6d-GDe injections, respectively. Successful integration into the white locus was determined by visually identifying the eye-specific 3xP3-tdTomato fluorescence in G₁ heterozygous mosquito larvae with black eyes (w^U6-GDe/w+) and in G₂ homozygous mosquito larvae with white eyes (w^U6-GDe/w^U6-GDe). In addition, site-specific integration of U6-GDe constructs was confirmed by amplifying and Sanger sequencing both the left and right integration points (Figure 2—figure supplement 2) from a genomic DNA prep of each w^U6-GDe line with the following primers: AE20, AE21, AE22, and AE23 (Supplementary file 8b).