Guide it genotype confirmation kit

crRNA was designed to target exon 2 of the Mus musculus Ager gene (target sequence, 5ʹ-GATTGGAGAGCCACTTGTGC-3ʹ). To prepare Cas9 gRNAs, equimolar amounts of Alt-R™ crRNA and Alt-R tracrRNA (Integrated DNA Technologies, US) were mixed in IDT Duplex Buffer (30 mM HEPES, pH 7.5, 100 mM potassium acetate; Integrated DNA Technologies), heated to 95 °C, and then slowly cooled to room temperature. To generate the RNP complex, a mixture of gRNAs, diluted Cas9 enzyme, and Opti-MEM^® (Thermo Fisher Scientific) was incubated for 5 min at room temperature. Lipofection was then performed in 96-well plates. Opti-MEM^® containing Lipofectamine^® RNAiMAX (Thermo Fisher Scientific) was combined with an equal volume of Opti-MEM containing RNP and incubated for 20 min at room temperature. After lipoplex formation, 3 × 10⁵ mProx cells resuspended in 1 mL of DMEM + 10% FBS were added to the transfection complex. Transfection plates were incubated at 37 °C under 5% CO₂/95% air. Mutation was confirmed using a Guide-it™ Genotype Confirmation Kit (TAKARA, Japan) and Sanger sequences. Fourteen bases of the target gene were deleted and the terminal codon was inserted (5ʹ-GCCACTTGTGCTAAGCTGTAA-3ʹ).

After microinjections, all G₀ larvae transiently expressing the ZsGreen marker were selected and raised to adulthood. Non-lethal DNA extractions (Supplementary Fig. S6 and Supplementary Methods) were performed for G₀ adult flies and used as templates for PCR amplifications spanning the targeted site at ChomOrco exon 1. Amplifications were submitted to T7E1 cleavage assays, as previously described^{23 (link)}, and ten mosaic males were individually backcrossed to wt females (1 Cas9 G₀ ♂ × 4 wt ♀). On average, eight G₁ adult males from each crossing were randomly selected and genotyped as before. Nine heterozygous G₁ males (one per obtained mutant line) were selected and individually backcrossed to wt females for a second time, while simultaneously genotyped by Sanger sequencing and CRISP-ID analysis^{42 (link)}. Adult G₂ male and female flies harboring a − 16 bp deletion were identified by T7E1 and let to interbreed freely in cages. Homozygous mutants at G₃ were identified by in vitro Cas9-assay, using the Guide-it Genotype Confirmation Kit (Takara), and inbred to establish the Orco mutant strain at G₄. Further genotyping, when required, was conducted using our custom High Resolution Mobility analysis (HRMob, described in Supplementary Fig. S7).

The blastocysts which were presumed to be mutated were further analysed for type of mutation using Guide-it genotype confirmation kit (632611, Takara Bio, San Jose, CA, USA). The PCR product of the edited blastocysts spanning the targeted sites (50 ng/μL) was incubated with guide-it recombinant Cas9 nuclease (500 ng/μL) at 37 °C for 5 min. In addition, the manufacturer’s instructions were followed when mixing the PCR product with Cas9 nuclease to create a reaction solution with BSA and 15× Cas9 reaction buffer. This solution was then incubated at 80 °C for 5 min and 37 °C for 1 h. The gel documentation system was used to further investigate the data.