Engineering a genetic system for female-biased, genetically unmodified progeny in mice without affecting litter size

The ability to influence the sex ratio of mammalian offspring has applications in agriculture and animal welfare. Here we describe a genetic system in mice that produces predominantly female progeny, reaching approximately ninety percent, without affecting litter size or introducing genetic modifications into the offspring. The system consists of a doxycycline-regulated cassette inserted into the Y chromosome, encoding dCas9 and RNA guides intended to impair the function of Y bearing sperm. While the cassette was originally intended to repress a spermatid maturation gene, our analyses did not detect meaningful changes in gene expression or sperm function that would account for the phenotype. Nevertheless, male mice carrying the cassette consistently produced female biased litters, and the effect was reversed by doxycycline, confirming that it depends on cassette expression. Similar female bias was observed following in vitro fertilization using sperm from transgenic males. These findings demonstrate a reproducible, genetically confined, and conditionally regulated system for sex ratio bias in mice. The strategy may prove adaptable across species, even without a complete mechanistic understanding. Graphical Abstract A single line genetic system producing female biased, genetically unmodified progeny in mice by conditionally impairing Y bearing sperm. One sentence summary Y chromosome manipulation yields mostly female mice, preserving litter size without genetic alteration in females. Significance statement This study describes a genetic approach for producing predominantly female offspring in mice without altering litter size or transmitting genetic modifications to the progeny. A cassette inserted into the Y chromosome of males induces a strong female bias in offspring through a mechanism that remains unclear. The female offspring do not inherit the transgene and are genetically unmodified. This system provides a simple and conditional platform for sex ratio control in mammals, with potential applications in animal breeding and welfare.