Mammalian Cysteine‐RIch Secretory Protein (CRISP) family includes four members present in sperm and reported to regulate Ca2+ channels and fertilization. Based on our previous observations using single knockouts models and suggesting the existence of functional compensation among CRISP proteins, we investigated their relevance for male fertility by generating multiple Crisp gene mutants by CRISPR/Cas9 technology. Whereas targeting of Crisp1 and Crisp3 yielded subfertile males with early embryo developmental defects, the same deletion in zygotes from fertile Crisp2−/−.Crisp4−/− mice led to the generation of both triple and quadruple knockout mice exhibiting a complete or severe disruption of male fertility due to a combination of sperm transport, fertilization, and embryo developmental defects linked to intracellular Ca2+ dysregulation. These observations reveal that CRISP proteins are essential for male fertility and organize in functional modules that contribute distinctly to fertility success, bringing insights into the mechanisms underlying functional redundancy/compensation in protein families and emphasizing the importance of generating multiple and not just single knockout which might be masking the true functional relevance of family genes.

中文翻译：

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功能冗余和补偿：删除多个小鼠 Crisp 基因揭示了它们对男性生育能力的重要作用

哺乳动物富含半胱氨酸的分泌蛋白 (CRISP) 家族包括存在于精子中的四个成员，据报道可调节 Ca2+ 通道和受精。基于我们之前使用单敲除模型的观察结果并表明 CRISP 蛋白之间存在功能补偿，我们通过 CRISPR/Cas9 技术生成多个 Crisp 基因突变体来研究它们与男性生育能力的相关性。虽然靶向 Crisp1 和 Crisp3 产生了具有早期胚胎发育缺陷的不育雄性，但来自可育 Crisp2−/−.Crisp4−/− 小鼠的受精卵中的相同缺失导致三重和四重敲除小鼠的产生表现出完全或严重的破坏由于精子运输、受精和与细胞内 Ca2+ 失调相关的胚胎发育缺陷的综合作用，导致男性生育能力下降。