Sheep with Partial RXFP2 Knockout Exhibit Normal Horn Phenotype but Unilateral Cryptorchidism

doi:10.1016/j.jia.2023.11.045

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GAO Yawei^1*, XI Siyuan^1*, CAI Bei², WU Tingjie¹, WANG Qian³, Peter KALDS¹, HUANG Shuhong¹, WANG Yuhui¹, HAN Saizheng¹, PAN Menghao⁴, YANG Chong³, KOU Qifang⁵, MA Baohua⁴, WANG Xiaolong^1,6#, ZHOU Shiwei^1,4#and CHEN Yulin^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, Yinchuan, 750002, China.

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

⁵Ningxia Tianyuan Tan Sheep Farm, Hongsibu, 751999, China.

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

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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。

Abstract

Comprehending the genetic basis of economically important traits is of significant importance to enhance livestock breeding. In domestic ruminants, hornlessness is becoming a more desirable trait since horns could lead to accidental injuries to animals and producers. Recent studies have identified the relaxin family peptide receptor 2 (RXFP2) as a primary candidate gene associated with the presence and absence of horns in sheep. However, no sufficient molecular biology-based analyses were performed to validate the association and function of RXFP2 in sheep. Noticeably, previous studies in both humans and mice have provided evidence supporting the involvement of RXFP2 in testicular descent. To validate the potential function of the RXFP2 gene in sheep, we used the CRISPR/Cas9 technology to obtain RXFP2-disrupted sheep individuals. Initially, highly efficient sgRNAs, targeting RXFP2, were screening through in vitro cleavage assays and cellular assessments. Then, two RXFP2-disrupted lambs were generated by intracytoplasmic microinjection of CRISPR/Cas9-sg1 ribonucleoprotein, with an efficiency of 81.84 and 37.17%, respectively. No potential off-target events were detected. Western blot analysis showed that RXFP2 expression was significantly reduced in the pedicle skin of edited lambs (P=0.034). Intriguingly, although the partial disruption of RXFP2 did not affect the horn phenotype in sheep, it led to an obvious unilateral cryptorchidism. These results provide evidence for a hitherto ambiguous link between both horn and testicular development. In conclusion, this study represents the first successful generation of cryptorchid sheep models via the disruption of RXFP2 using CRISPR/Cas9. These findings provide new insights into the roles of RXFP2, whose partial disruption is associated with testicular descent rather than horn formation.

Online: 28 November 2023

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

About author: GAO Yawei, E-mail: yawei_gao@nwafu.edu.cn; XI Siyuan, E-mail: huisiyuan@nwafu.edu.cn; #Correspondence WANG Xiaolong, E-mail: xiaolongwang@nwafu.edu.cn; ZHOU Shiwei, E-mail: zhoushiwei@nwafu.edu.cn; CHEN Yulin, E-mail: chenyulin@nwafu.edu.cn *These authors contributed equally to this work.

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GAO Yawei, XI Siyuan, CAI Bei, WU Tingjie, WANG Qian, Peter KALDS, HUANG Shuhong, WANG Yuhui, HAN Saizheng, PAN Menghao, YANG Chong, KOU Qifang, MA Baohua, WANG Xiaolong, ZHOU Shiwei and CHEN Yulin. 2023. Sheep with Partial RXFP2 Knockout Exhibit Normal Horn Phenotype but Unilateral Cryptorchidism. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2023.11.045

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