As the world population continues to increase to unsustainable levels, the importance of birth control and the development of new contraceptives are emerging. To date, male contraceptive options have been lagging behind those available to women, and those few options available are not satisfactory to everyone. To solve this problem, we have been searching for new candidate target proteins for non-hormonal contraceptives. Testis-specific proteins are appealing targets for male contraceptives because they are more likely to be involved in male reproduction and their targeting by small molecules is predicted to have no on-target harmful effects on other organs. Using in silico analysis, we identified Erich2, Glt6d1, Prss58, Slfnl1, Sppl2c, Stpg3, Tex33, and Tex36 as testis-abundant genes in both mouse and human. The genes, 4930402F06Rik and 4930568D16Rik, are testis-abundant paralogs of Glt6d1 that we also discovered in mice but not in human, and were also included in our studies to eliminate the potential compensation. We generated knockout (KO) mouse lines of all listed genes using the CRISPR/Cas9 system. Analysis of all of the individual KO mouse lines as well as Glt6d1/4930402F06Rik/4930568D16Rik TKO mouse lines revealed that they are male fertile with no observable defects in reproductive organs, suggesting that these 10 genes are not required for male fertility nor play redundant roles in the case of the 3 Glt6D1 paralogs. Further studies are needed to uncover protein function(s), but in vivo functional screening using the CRISPR/Cas9 system is a fast and accurate way to find genes essential for male fertility, which may apply to studies of genes expressed elsewhere. In this study, although we could not find any potential protein targets for non-hormonal male contraceptives, our findings help to streamline efforts to find and focus on only the essential genes.

中文翻译：

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CRISPR/Cas9 介导的基因组编辑小鼠揭示了 10 个富含睾丸的基因对于雄性繁殖力是必不可少的。


随着世界人口继续增加到不可持续的水平，节育和开发新型避孕药具的重要性日益凸显。迄今为止，男性避孕选择一直落后于女性，而且这少数几种选择并不能让所有人都满意。为了解决这个问题，我们一直在寻找非激素避孕药的新候选靶蛋白。睾丸特异性蛋白质是男性避孕药颇具吸引力的靶标，因为它们更有可能参与男性生殖，并且小分子靶向它们预计不会对其他器官产生有害影响。通过计算机分析，我们确定了Erich2 、 Glt6d1 、 Prss58 、 Slfnl1 、 Sppl2c 、 Stpg3 、 Tex33和Tex36为小鼠和人类的睾丸丰富基因。基因4930402F06Rik和4930568D16Rik是Glt6d1睾丸丰富的旁系同源基因，我们也在小鼠中发现了这些基因，但在人类中没有发现，并且也将其纳入我们的研究中以消除潜在的补偿。我们使用 CRISPR/Cas9 系统生成了所有列出的基因的敲除 (KO) 小鼠品系。对所有个体 KO 小鼠品系以及Glt6d1/4930402F06Rik / 4930568D16Rik TKO 小鼠品系的分析表明，它们具有雄性可育性，生殖器官没有可观察到的缺陷，这表明这 10 个基因不是雄性生育力所必需的，也不是在雄性生育力中发挥冗余作用。 3 个Glt6D1旁系同源物的情况。 需要进一步的研究来揭示蛋白质功能，但使用 CRISPR/Cas9 系统进行体内功能筛选是一种快速而准确的方法，可以找到男性生育力所必需的基因，这可能适用于其他地方表达的基因的研究。在这项研究中，虽然我们无法找到非激素男性避孕药的任何潜在蛋白质靶点，但我们的发现有助于简化寻找和关注必需基因的工作。