利用 Cas9 或 dCas9 靶向常染色体角膜炎变体基因,在不诱导突变的情况下使 XX 人男性化。

IF 2.9 Q2 Biochemistry, Genetics and Molecular Biology
Pasquale Primo, Angela Meccariello, Maria Grazia Inghilterra, Andrea Gravina, Giuseppe Del Corsano, Gennaro Volpe, Germano Sollazzo, Serena Aceto, Mark D Robinson, Marco Salvemini, Giuseppe Saccone
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引用次数: 0

摘要

背景:地中海果蝇(Ceratitis capitata,Medfly)的雌蝇是主要的农业害虫,因为它们在数百种植物的果实中产卵。在地中海果蝇中,雌性性别决定基于 Cctransformer(Cctra)的激活。在 XX 胚胎中,需要母源 Cctra 激活 Cctra 本身,并通过雌性特异性替代剪接启动和维持 Cctra 的正反馈回路,从而导致雌性发育。在 XY 胚胎中,决定雄性的 Maleness-on-the-Y 基因(MoY)阻止了这种激活,Cctra 产生雄性特异性转录本,编码截短的 CcTRA 异构体,从而发生雄性分化:为了在第一个编码外显子中诱导移帧突变,以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 和单导 RNA,sgRNA)。由于这种方法主要导致单拷贝突变,因此预计只有在 G0 注入个体杂交后,携带双拷贝突变的 G1 XX 个体才会男性化。令人惊讶的是,这些注射到只有 XX 胚胎中的 G0 成体不仅包括 XX 雌性,还包括 50%的可育 XX 雄性。这些 G0 XX 雄性表达了雄性特异的 Cctra 转录本,表明它们已完全男性化。有趣的是,在六只 G0 XX 雄性中,有四只显示了 Cctra 野生型序列。这一发现表明,Cas9-sgRNA注射的男性化作用与其诱变活性无关。与这一观察结果相一致的是,在XX胚胎中用死Cas9(无酶活性,dCas9)对Cctra进行胚胎靶向,也有利于Cctra在胚胎和成体中的雄性特异性剪接:我们的数据表明,在 XX 胚胎中,Cctra 基因第一外显子上的 Cas9-sgRNA 瞬时结合抑制了 Cctra 早期胚胎发生过程中雌性特异性自动调节的建立。dCas9 还诱导 Cctra 的剪接从雌性模式转变为雄性模式,这一观察结果也支持了这一假设。总之,本研究结果证实了一个观点,即 Cctra 在胚胎期的短暂失活足以决定雄性性别。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

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

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

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

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

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 G1 XX individuals carrying biallelic mutations, following crosses of G0 injected individuals. Surprisingly, these injections into XX-only embryos led to G0 adults that included not only XX females but also 50% of reverted fertile XX males. The G0 XX males expressed male-specific Cctra transcripts, suggesting full masculinization. Interestingly, out of six G0 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.

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来源期刊
BMC Genetics
BMC Genetics 生物-遗传学
CiteScore
4.30
自引率
0.00%
发文量
77
审稿时长
4-8 weeks
期刊介绍: BMC Genetics is an open access, peer-reviewed journal that considers articles on all aspects of inheritance and variation in individuals and among populations.
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