Fig. 1: Engineering a Y chromosome-linked Cas9 in D. melanogaster.

a The Cas9 transgene design for Y chromosome insertion via CRISPR/Cas9−mediated cleavage and homology-directed repair (HDR). Homology arms (HA) flank two insulator sequences, GypSy and CTCF, and a vasa-controlled Cas9−T2A-eGFP. An eye-specific marker (Tdtomato) allows for the identification of transgenic flies carrying the transgene. For microinjection, a source of Cas9 and gRNA were provided to cleave the Y chromosome. The SGyA template was provided and inserted into the Y chromosome through HDR. b Karyotype of transgenic SGyA males and non-transgenic females. c The number of embryos injected for the SGyA, SGyB, and SGyC transgenes, survival to the larval stage of injected embryos, and rate (no. of independent transgenic individuals found/no. of embryos injected) is shown. No transformants were obtained for SGyB and SGyC. d White light and fluorescent images of the SGyA line compared to WT. There is an overall faint yet distinguishable expression of the fluorescent marker in SGyA individuals. Please refer to Fig. S1 to see the range of fluorescence in the SGyA eye marker. e PCR confirmation of transgenic male flies harboring the SGyA transgene. Primers corresponding to the left and right genomic insertion regions were used to amplify both sides to ensure the transgene was present. Amplification was performed at least twice by two independent scientists. The expected band size for the left side primer pair is 1.690 kb. The expected band size for the right side primer pair is 1.893 kb.