Fig. 4

Molecular partial masculinization of XX embryos by dCas9 targeting Cctra. (A) RT-PCR analysis of Cctra sex-specific transcripts in 15 h old XX embryos, following injections of various samples (Table 1, set n. 6–8) at 1 h after egg laying (Panel 1). The XX embryos were injected respectively with Cas9 protein+sgtraEx1 (first lane), with sgtraEx1 + Cas9-encoding plasmid (pIE1-Cas9; second lane) and with dCas9-encoding plasmid (pAct-dCas9) (third lane). Male-specific Cctra transcripts were detected (500 bp cDNA fragment) in all three samples of injected XX embryos, in addition to the female-specific transcripts (160 bp cDNA fragment). RT-PCR analysis of the Y-linked MoY gene indirectly confirmed the absence of Y chromosome in all 3 samples and in XX females, and its presence in a mixed XX/XY embryos sample and in adult XY flies (Panel 2). CcSOD positive control is shown in panel 3. (B) Negative controls. RT-PCR of Cctra sex-specific transcripts in 15 h old XX embryos, following injections at 1 h after egg laying (Table 1, set n. 9–13). The XX embryos were injected respectively with Cas9 protein, Cas9-encoding plasmid, dCas9-encoding plasmid, with sgtraEx1 and with buffer alone. Only female-specific Cctra transcripts were detected (160 bp) in all samples of injected XX embryos (Panel 1). In Panel 2 and 3, the control of the karyotype and positive controls, conducted as in A, are shown