Sheep with Partial RXFP2 Knockout Exhibit Normal Horn Phenotype but Unilateral Cryptorchidism1

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.