Deletion of Y Chromosome Ssty2 Repetitions produced alterations in Mice Sex Ratio

Animal facilities often have surplus unused male mice. To reduce unnecessary mouse sacrifices, we proposed innocuously preselecting sex sperm using CRISPR/Cas9. Specifically, we aimed to significantly decrease the number of copies of the multicopy Ssty2 gene family (repeated over 220 times on the Y chromosome), which plays a pivotal role in regulating X and Y gene expression during sperm differentiation. Transgenic lines were created by microinjecting Cas9 RNA and specific Ssty2 sgRNAs into B6CBAF1 zygotes. Pup genotyping of Ssty2 copies was conducted via qPCR. Epididymal sperm samples were obtained from 2 to 6-month KO and WT males, and after a swim-out, sperm motility was evaluated using the Integrated Semen Analysis System. Motile sperm were categorized by K-means clustering of CASA kinetic parameters. To uncover the impact of Ssty2 depletion on sex-ratio segregation, we selected a line with a substantial decrease in Ssty2 copies, to fewer than 50. Consequently, 70 to 80% of pups were female upon in vivo fertilization, remaining consistent in IVF, but with a normal sex ratio in ICSI. However, we noted a reduction in litter size, declining from an average of 8.5 to 6.4 pups per litter. Testicle size in KO mice exhibited a slight enlargement, with a minor decrease in sperm concentration compared to controls (non-significant). However, over 80% of KO sperm displayed abnormal morphology, showing head, neck, and midpiece defects. Sperm motility comparison revealed a shift, with 60.6% of KO sperm static vs. 35.9% in WT controls, displaying KO sperm as slow and non-progressive motility patterns. In conclusion, by targeting the Ssty2 multicopy gene, we produced CRISPR-mediated KO mice that presented preferential fertilization of sperm carrying the X chromosome via in vivo and IVF.