CRISPR/Cas9-mediated knock-in of the murine Y chromosomal genes Zfy1 and Zfy2

The Y-linked mouse zinc finger genes Zfy1 and Zfy2 are critical fertility factors in mice but the mechanisms by which they regulate spermatogenesis remain unclear. We recently produced Zfy1/2 double knock-out mice and observed a complete loss in fertility. However, the biochemical mechanism by which Zfy regulates spermatogenesis is unknown, and ZFY expression has not yet been confirmed at the protein level. As both Zfy homologues share ~ 95% sequence similarity, it is difficult to produce an anti-ZFY antibody specific to either homologue.

To overcome this technical challenge, we used CRISPR/Cas9 genome editing to develop tagged Zfy1 knock-in (XY^Zfy1-HA), Zfy2 knock-in (XY^Zfy2-FLAG, XY^Zfy2-3xFLAG, and XY^Zfy2-HA), and Zfy1/2 double knock-in (XY^{Zfy1-HA,Zfy2-MYC}) mice. Successful targeting was confirmed by genotyping and sequencing. The knock-in lines were fertile with normal sperm parameters. Using Western blot on testes, knock-in specific bands were detected matching the predicted ZFY expression patterns. Using immunofluorescence on testis sections from knock-in males, ZFY1 and ZFY2 expression was detected in zygotene spermatocytes, and ZFY2 expression was also detected in spermatids step 7-8 and 9.

These novel knock-in mice can be used in future investigations to determine how ZFY controls spermatogenesis.