Department of Biology, University of Naples Federico II, 80126, Naples, Italy.
Department of Molecular Life Sciences and SIB Swiss Institute of Bioinformatics, University of Zurich, Winterthurerstrasse 190, CH-8057, Zurich, Switzerland.
BMC Genet. 2020 Dec 18;21(Suppl 2):150. doi: 10.1186/s12863-020-00941-4.
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.
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 XX individuals carrying biallelic mutations, following crosses of G injected individuals. Surprisingly, these injections into XX-only embryos led to G adults that included not only XX females but also 50% of reverted fertile XX males. The G XX males expressed male-specific Cctra transcripts, suggesting full masculinization. Interestingly, out of six G 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.
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.
地中海实蝇（Medfly）的雌性是主要的农业害虫，因为它们会将卵产入数百种植物物种的果实作物中。在 Medfly 中，雌性性别决定基于 Cctransformer（Cctra）的激活。母体对 Cctra 的贡献对于在 XX 胚胎中激活自身 Cctra 以及通过雌性特异性选择性剪接开始并表观遗传维持 Cctra 阳性反馈回路以启动雌性发育是必需的。在 XY 胚胎中，雄性决定基因 Maleness-on-the-Y 基因（MoY）阻止这种激活，Cctra 产生编码截短的 CcTRA 同种型的雄性特异性转录本，从而发生雄性分化。
为了在第一个编码外显子中诱导移码突变，从而破坏雌性特异性和较短的雄性特异性 CcTRA 开放阅读框（ORF），我们在胚胎中注射了 Cas9 核糖核蛋白（Cas9 和单指导 RNA，sgRNA）。由于这种方法主要导致单等位基因突变，因此预计只有携带双等位基因突变的 G XX 个体在 G 注射个体的杂交后代中才会发生雄性化。令人惊讶的是，这些仅在 XX 胚胎中的注射导致 G 成虫不仅包括 XX 雌性，还包括 50%的可育 XX 雄性。G XX 雄性表达雄性特异性 Cctra 转录本，提示完全雄性化。有趣的是，在六个 G XX 雄性中，有四个显示 Cctra 野生型序列。这一发现表明，Cas9-sgRNA 注射的雄性化与其诱变活性无关。与这一观察结果一致，通过 Cas9 失活（无酶活性，dCas9）在 XX 胚胎中靶向 Cctra 也有利于 Cctra 的雄性特异性剪接，无论是在胚胎中还是在成虫中。
我们的数据表明，在早期胚胎发生过程中，Cctra 雌性特异性自身调节的建立已被 Cas9-sgRNA 在 Cctra 基因第一外显子上的瞬时结合在 XX 胚胎中抑制。这一假设得到了以下观察结果的支持：即 Cctra 剪接从雌性模式向雄性模式的转变也是由 dCas9 诱导的。总之，目前的发现支持这样一种观点，即 Cctra 的瞬时胚胎失活足以决定雄性性别。
