E toxate water

HT-29 cells were seeded onto 96-well plates at a density of 10⁴ cells/well and cultured until confluency of each well reached 80%. Nucleotides (12 mM of ATP and UDP) were pretreated at 37°C for 1 h with or without wheat phytase (286 mU/mL) in distilled water (E-Toxate Water; Sigma-Aldrich, USA). Then 10 μL of aliquot was taken from the reaction mixture and applied to cells in each well. After 24 h of incubation, 10 μL of EZ-CYTOX was used to treat cells in each well. Cell viabilities were then measured at OD 450 nm using the Synergy 2 microplate reader (BioTek, Winooski, VT, USA) according to the manufacturer’s instructions.

For injection of mosquito embryos, each gRNA was diluted to 100 ng/μl with E-Toxate water (Sigma) and filtered through a 0.2-μm, 1.5-ml filter (Millipore). The mosquito embryo microinjection was done according to our previously established protocol (39 (link)). For each gRNA or gRNA mix, 200 to 400 exu-Cas9 eggs were injected.
A leg PCR was used to screen the surviving G0 adults for mutations using a pair of primers outside ∼100 bp of each gRNA (Table S2). In brief, one hind leg of the adult mosquito was pulled and transferred to a PCR tube with 20 μl Phire reaction buffer (Thermo Fisher Scientific), then heated at 95°C for 5 min, and 1 μl was used as a template for PCR amplification. PCR mixtures were heated to 95°C for 2 min; followed by 40 cycles at 95°C for 10 s, 58°C for 20 s, and 68°C for 30 s. PCR products were checked on a 2% agarose gel, and an extra band was considered to indicate a mutation induced by gRNA. The mutated PCR products were purified using a DNA Clean & Concentrator kit (Zymo Research), sequenced, and compared to the WT gene to confirm the mutation. The mutated G0 mosquito was then outcrossed with exu-Cas9 mosquitoes. After outcrossing of the Obp mutants with exu-Cas9 mosquitoes for several generations, heterozygous mutants were incrossed, and the mutated homozygotes were identified by leg PCR and confirmed by DNA sequencing.