To assay the adult sex ratio, 20 transgenic males were crossed to 30 wild-type females and, as a control, 30 transgenic females were crossed to 20 wild-type males. Each transgenic line was assayed for at least four consecutive generations. Eggs were collected three times, with an interval of 2 to 3 days and reared to adulthood. The total number of adult male and female flies from each cross were counted. The survival test was performed, 20 transgenic males were crossed to 30 wild-type female and, as a control, 20 wild-type males were crossed to 30 wild type females. The number of eggs laid and the number of adults hatching were counted. Each transgenic line was assayed in triplicate, i.e. in three cages of 20 males and 30 females.

Three crosses were performed to generate homozygous individuals for the analysis of the effect of copy number of each transgene: (1) 10 Cas9.1e males and 20 Cas9.1a females, (2) 10 Cas9.2c males and 20 Cas9.2c females and (3) 10 Cas9.2a males and 20 Cas9.2a females. Also, transhemizygous individuals were generated with the following three crosses: (1) 10 Cas9.1e males and 20 Cas9.2a females, (2) 10 Cas9.2c males and 20 Cas9.1e females and (3) 10 Cas9.2a males and 20 Cas9.2c females.

From each cross, 10 homozygous males and 10 transhemizygous males were selected based on the intensity of DsRed fluorescence and were then individually crossed to 10 wild-type females. We validated that DsRed fluorescence intensity could be used as an indicator for the number of transgenes using PCR on genomic DNA extracted from 9 putative transhemizygous males. For this, we used primer 3F1 in combination with either primers Rev-Cas9.1e-DNA, Rev-Cas9.2a-DNA or Rev-Cas9.2c-DNA (Additional file 9: Table S4) and amplified regions ranging from the transgene to insertion-specific genomic flanking sequences. Egg collection and the determination of adult sex ratio in the progeny were performed as described above. To analyse the sex ratios observed, we used a generalized linear model in which the sex ratios were modelled as a function of the sex of the transgenic parent and the experimental generation using a binomial error distribution (Additional file 10: Table S5). (R scripts and input data are available at

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