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

Background Females of the Mediterranean fruit flyCeratitis 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 ofCctransformer(Cctra). A maternal contribution ofCctrais required to activateCctraitself in the XX embryos and to start and epigenetically maintain aCctrapositive feedback loop, by female-specific alternative splicing, leading to female development. In XY embryos, the male determiningMaleness-on-the-Ygene (MoY) blocks this activation andCctraproduces 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 G1XX individuals carrying biallelic mutations, following crosses of G0injected individuals. Surprisingly, these injections into XX-only embryos led to G0adults that included not only XX females but also 50% of reverted fertile XX males. The G0XX males expressed male-specificCctratranscripts, suggesting full masculinization. Interestingly, out of six G0XX males, four displayed theCctrawild type sequence. This finding suggests that masculinization by Cas9-sgRNA injections was independent from its mutagenic activity. In line with this observation, embryonic targeting ofCctrain XX embryos by a dead Cas9 (enzymatically inactive, dCas9) also favoured a male-specific splicing ofCctra, in both embryos and adults. Conclusions Our data suggest that the establishment ofCctrafemale-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 theCctragene. This hypothesis is supported by the observation that the shift ofCctrasplicing from female to male mode is induced also by dCas9. Collectively, the present findings corroborate the idea that a transient embryonic inactivation ofCctrais sufficient for male sex determination.