Sheep with partial RXFP2 knockout exhibit normal horn phenotype but unilateral cryptorchidism

doi:10.1016/j.jia.2023.11.045

Journal of Integrative Agriculture

2025, Vol. 24

Issue (9): 3698-3702 DOI: 10.1016/j.jia.2023.11.045

Letter

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Sheep with partial RXFP2 knockout exhibit normal horn phenotype but unilateral cryptorchidism

Yawei Gao^1*, Siyuan Xi^1*, Bei Cai², Tingjie Wu¹, Qian Wang³, Peter Kalds¹, Shuhong Huang¹, Yuhui Wang¹, Saizheng Han¹, Menghao Pan⁴, Chong Yang³, Qifang Kou⁵, Baohua Ma⁴, Xiaolong Wang^{1, 6#}, Shiwei Zhou^{1, 4#}, Yulin Chen^{1, 6#}

¹Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China

²Key Laboratory of Ruminant Molecular and Cellular Breeding of Ningxia Hui Autonomous Region, College of Animal Science and Technology, Ningxia University, Yinchuan 750021, China

³Animal Husbandry Workstation of Ningxia Hui Autonomous Region, Jinfeng 750002, China

⁴College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China

⁵Ningxia Tianyuan Tan Sheep Farm, Wuzhong 751900, China

⁶Key Laboratory of Livestock Biology, Northwest A&F University, Yangling 712100, China

Highlights
● CRISPR/Cas9 RNP complex-based strategy demonstrates robustness and accuracy in generating gene-edited sheep.
● Sheep horn development remains unaffected by partial RXFP2 knockout.
● Partial RXFP2 knockout results in unilateral cryptorchidism in sheep.

Abstract
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摘要

深入了解重要经济性状的遗传基础对家畜育种至关重要。由于角对动物、养殖设备及饲养者的伤害性，无角动物新品种的培育已成为家畜育种的一个重要方向。许多组学研究表明，松弛素家族肽受体2（RXFP2）基因是与绵羊角有关的主要候选基因。然而，目前缺乏足够的分子生物学分析来验证RXFP2基因在绵羊中的功能，尤其是其对角的影响。值得注意的是，前人研究已经表明，在人类与小鼠中，RXFP2基因的主要功能与睾丸下降有关，即其功能缺失会导致隐睾症（睾丸不能正常下降到阴囊中）。因此，为了验证RXFP2基因在绵羊中的潜在功能，本研究使用CRISPR/Cas9基因编辑技术创制了RXFP2基因敲除绵羊。

首先，针对绵羊RXFP2基因第一二外显子，设计了4条sgRNA（sg1, sg2, sg3及sg4），并通过体外切割试验筛选了3条具有切割活性的sgRNA（sg1, sg2及sg3）。然后，在细胞水平测试了sgRNA的靶向编辑效率，并确定使用编辑效率最高的sg1（51%）创制基因编辑羔羊。最后，通过向一细胞期受精卵显微注射CRISPR/Cas9-sg1核糖核蛋白（RNP），成功创制了两只RXFP2基因敲除羔羊（雄性），血液编辑效率分别为81.84%、37.17%，皮肤组织编辑效率分别为98.0%、32.0%，且两只基因编辑羔羊体内均未检测到任何脱靶现象。Western blot分析发现，两只基因敲除羔羊的角基部皮肤中RXFP2蛋白表达量显著降低（P=0.034），但角长度（2~11月龄）与野生型个体无差异。有趣的是，这两只编辑羔羊均具有明显的单侧隐睾现象（非遗传因素），与其他物种中RXFP2基因缺失表型一致。这些结果说明，本研究中的RXFP2基因不完全敲除与绵羊角的生长发育无关，但会导致雄性单侧隐睾。当然，绵羊RXFP2基因对角的影响还有待通过更大规模的纯合敲除个体进行验证，以提供更加详细的证据。

综上所述，本研究通过CRISPR/Cas9技术成功创制了RXFP2基因敲除绵羊模型，这是首次成功制备单侧隐睾绵羊模型的尝试，为RXFP2基因在绵羊中的的功能提供了新的见解。

Received: 25 July 2023 Online: 28 November 2023 Accepted: 30 October 2023

Fund: This work was supported by the National Key Research and Development Program of China (2022YFD1300200), the National Natural Science Foundation of China (32161143010, 32202646, and 32272848), the China Agriculture Research System (CARS-39), the Key Special Project of Ningxia Science and Technology Department, China (2021BEF02024), and the local grants, China (NXTS2021-001, 2022GD-TSLD-46, NK2022010207, and NXTS2022-001).

About author: Yawei Gao, E-mail: yawei_gao@nwafu.edu.cn; Siyuan Xi, E-mail: huisiyuan@nwafu.edu.cn; #Correspondence Xiaolong Wang, E-mail: xiaolongwang@nwafu.edu.cn; Shiwei Zhou, E-mail: zhoushiwei@nwafu.edu.cn; Yulin Chen, E-mail: chenyulin@nwafu.edu.cn * These authors contributed equally to this study. *These authors contributed equally to this work.

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Yawei Gao, Siyuan Xi, Bei Cai, Tingjie Wu, Qian Wang, Peter Kalds, Shuhong Huang, Yuhui Wang, Saizheng Han, Menghao Pan, Chong Yang, Qifang Kou, Baohua Ma, Xiaolong Wang, Shiwei Zhou, Yulin Chen. 2025. Sheep with partial RXFP2 knockout exhibit normal horn phenotype but unilateral cryptorchidism. Journal of Integrative Agriculture, 24(9): 3698-3702.

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