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Background  Females of the Mediterranean fruit fly  Ceratitis capitata  (Medfly) are major agricultural pests, as they lay eggs into the fruit crops of hundreds of plant species. In Medfly, female sex determination is based on the activation of  Cctransformer  ( Cctra ). A maternal contribution of  Cctra  is required to activate  Cctra  itself in the XX embryos and to start and epigenetically maintain a  Cctra  positive feedback loop, by female-specific alternative splicing, leading to female development. In XY embryos, the male determining  Maleness-on-the-Y  gene ( MoY ) blocks this activation and  Cctra  produces male-specific transcripts encoding truncated CcTRA isoforms and male differentiation occurs.    Results  With the aim of inducing frameshift mutations in the first coding exon to disrupt both female-specific and shorter male-specific CcTRA open reading frames (ORF), we injected Cas9 ribonucleoproteins (Cas9 and single guide RNA, sgRNA) in embryos. As this approach leads to mostly monoallelic mutations, masculinization was expected only in G 1  XX individuals carrying biallelic mutations, following crosses of G 0  injected individuals. Surprisingly, these injections into XX-only embryos led to G 0  adults that included not only XX females but also 50% of reverted fertile XX males. The G 0  XX males expressed male-specific  Cctra  transcripts, suggesting full masculinization. Interestingly, out of six G 0  XX males, four displayed the  Cctra  wild type sequence. This finding suggests that masculinization by Cas9-sgRNA injections was independent from its mutagenic activity. In line with this observation, embryonic targeting of  Cctra  in XX embryos by a dead Cas9 (enzymatically inactive, dCas9) also favoured a male-specific splicing of  Cctra , in both embryos and adults.    Conclusions  Our data suggest that the establishment of  Cctra  female-specific autoregulation during the early embryogenesis has been repressed in XX embryos by the transient binding of the Cas9-sgRNA on the first exon of the  Cctra  gene. This hypothesis is supported by the observation that the shift of  Cctra  splicing from female to male mode is induced also by dCas9. Collectively, the present findings corroborate the idea that a transient embryonic inactivation of  Cctra  is sufficient for male sex determination.    Supplementary Information  The online version contains supplementary material available at 10.1186/s12863-020-00941-4.

Targeting the autosomal Ceratitis capitata transformer gene using Cas9 or dCas9 to masculinize XX individuals without inducing mutations