CRISPR mediated microinjections

Embryonic collection and CRISPR microinjections were performed following the procedure described by Li et al.⁵². Briefly, Rockefeller mosquitoes were blood-fed 5 days before egg collection. An ovicup filled with ddH₂O and lined with filter paper was placed into a cage and female mosquitoes were allowed to lay eggs in the ovicup in the dark. After 15–30 min, the ovicup was taken out and unmelanized eggs were transferred onto a glass slide. The eggs were quickly aligned on a wet piece of filter paper. Aluminosilicate needles were pulled on a Sutter P-1000 needle puller and beveled using a Sutter BV-10 beveler. An Eppendorf Femotojet was used for power injections under a compound microscope at 100× magnification. About 50 eggs were injected each time immediately after fresh eggs were collected. The concentration of components used in the study was as follows; Cas9 protein at 300 ng μl⁻¹, each sgRNA at 40 ng μl⁻¹. After injection, eggs were placed in a cup filled with water and allowed to hatch and developed into adults. To identify mutants, we performed genotyping at each generation of mosquitoes after injection by cutting one hind leg off for genomic DNA isolation. Genomic DNA was extracted using the DNeasy blood & tissue kit (QIAGEN) following the manufacturer’s protocol. Target loci were amplified by PCR using the primers AaOR31F (5’-ATTGGCATGCGCTACTTTTATT-3’) and AaOR31R (5’-ATAACATCCTTTAGCCAGTGCC-3’) (also in Supplementary Table ²). PCR products were gel purified and sent directly for Sanger sequencing using the forward primer used in PCR reactions.