Systematic evaluation of 39 testis-enriched genes for male fertility in mice using CRISPR/Cas9

The testis expresses approximately 18,700 genes, more than any other tissue. Among these, 1,262 genes show high and testis-enriched expression, but only a subset is essential for male fertility. Knockout mouse phenotypes provide important information for assessing gene function; however, fertility phenotypes are not always reported, particularly when knockout mice are viable and show no obvious reproductive abnormalities. Reporting such negative fertility phenotypes would help avoid redundant studies and facilitate the prioritization of genes with critical roles in male reproduction. In this study, we generated 38 knockout mouse lines to evaluate the roles of 39 testis-enriched genes in male reproduction using the CRISPR/Cas9 system (1700025B11Rik, 4930444P10Rik, 4930550C14Rik, 4930558K02Rik, 4930590J08Rik, 4933412E24Rik, Acsbg2, Ankmy1, Ankrd61, Apobec4, Arl14epl, Atf7ip2, Ccdc110, Csnka2ip, D11Wsu47e, Ddi1, Efcab8, Fam243, Fam50b, Gm36245, Gtf2a1l, Iqca1l, Litafd, Lrp2bp, Mroh5, Nlrp14, Pebp4, Prss38, Pudp, Slc6a16, Spata1, Spata32, Styxl1, Tcp11, Tcp11x2, Tmem225, Tmem232, Trim52, Trp53tg5). All 38 knockout mouse lines were viable and showed no marked developmental or behavioral abnormalities. We identified 30 fertile KO lines (covering 31 genes) that are dispensable for male fertility under the conditions tested. Reproductive phenotypes for nine of the examined lines had been reported previously; six showed phenotypes consistent with previous reports, whereas three showed discrepant phenotypes. These systematic phenotyping data provide a useful resource for prioritizing candidate genes involved in male fertility and for disseminating both fertility-associated and fertility-unassociated phenotypes to the research community.