Figure EV1. Schematic summary of Y‐line generation.

1. Mouse genomic fragments containing homology arms to the Uty gene located on mouse chromosome Y were amplified from a BAC clone and sequentially assembled into a targeting vector along with negative and positive selection markers (DTA and Neo, respectively). There are only few mice reported with a transgene on the Y chromosome, one of which is on the Uty gene 28, and thus, we chose to target the KI cassette into this gene. The KI cassette, encoding the gRNAs targeting genes Atp5b, Cdc20, and Casp8, each expressed from a U6 promoter, was inserted into the targeting vector, which was designed to integrate in reverse orientation of the 2^nd exon of the Uty gene.
2. The KI cassette integrated into the Uty gene and the Neo cassette was self‐deleted due to the loxP sites. Details on the linearization of the targeting vector, its transfection into C57BL/6N ES cells, as well as PCR and Southern blot analyses and ES implantation procedures, are provided in the Appendix.