Large-scale CRISPR/Cas9 deletions within the WFDC gene cluster uncover gene functionality and critical roles in mammalian reproduction

Despite 96 million years of evolution separating humans and rodents, 11 closely related reproductive tract-specific genes in humans-<i>SPINT3</i>, <i>WFDC6</i>, <i>EPPIN</i>, <i>WFDC8</i>, <i>WFDC9</i>, <i>WFDC10A</i>, <i>WFDC11</i>, <i>WFDC10B</i>, <i>WFDC13</i>, <i>SPINT4</i>, and <i>WFDC3</i>-and the 13 reproductive tract-specific orthologous genes in mice, form highly conserved syntenic gene clusters indicative of conserved, combined critical functions. Further, despite significant progress toward a nonhormonal male contraceptive targeting the protein encoded by one of these genes, epididymal peptidase inhibitor (EPPIN), and associations found between mutations in <i>EPPIN</i> and an increased risk of male infertility, neither EPPIN nor any closely related whey acidic protein four-disulfide core (WFDC) gene have been explored functionally. To clarify the involvement of WFDC genes in male fertility, we strategically used CRISPR/Cas9 to generate mice lacking 13, 10, 5, or 4 genes within the cluster and demonstrated that males with deletions of 13, 10, or 4 genes (Wfdc6a, Eppin, Wfdc8, and Wfdc6a) were sterile due to an arrest in spermatogenesis, preventing formation beyond round spermatids. In contrast, the five gene knockout (KO) males (lacking <i>Wfdc16</i>, <i>Wfdc9</i>, <i>Wfdc10</i>, <i>Wfdc11</i>, and <i>Wfdc13</i>), despite normal spermatogenesis and sperm counts, were infertile due to defects in sperm motility and increased sperm death. Similarly to our previously reported <i>Spint3</i> single gene KO, <i>Wfdc3</i> single KO mice were fertile with no obvious reproductive phenotype. Our KO mouse studies to explore the entire WFDC locus of closely related genes have clarified the functional requirements of WFDC locus genes in different aspects of male fertility. Our research has implications for improving clinical diagnoses of male infertility and identifying additional targets for nonhormonal male contraception.