CRISPR/Cas9-Mediated Biallelic Knockout of IRX3 Reduces the Production and Survival of Somatic Cell-Cloned Bama Minipigs

SIMPLE SUMMARY: Bama minipigs are a local pig breed that is unique to China and possess several negative features, including high fat content, low feed utilization rate, and slow growth rate. The iroquois homeobox 3 (IRX3) gene has been implicated in human obesity and controls body mass and composition in mouse. In this study, we successfully generated IRX3 biallelic knockout Bama minipigs using CRISPR/Cas9-mediated gene editing combined with somatic cell cloning. The results show that the use of IRX3(-/-) cells as donor cells for the production of somatic cell-cloned pigs induces a significant decrease in the average live litter size and a significant increase in the average number of stillbirths. Moreover, the birth weight of surviving IRX3(-/-) somatic cell-cloned pigs is significantly lower, and viability is poor such that all piglets die shortly after birth. Therefore, the preliminary results of this study suggest that IRX3 may have important biological functions in pigs, and IRX3 should not be used as a gene editing target to reduce fat content in Bama minipigs. ABSTRACT: Bama minipigs are a local pig breed that is unique to China and has a high development and utilization value. However, its high fat content, low feed utilization rate, and slow growth rate have limited its popularity and utilization. Compared with the long breeding cycle and high cost of traditional genetic breeding of pigs, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) endonuclease 9 system (CRISPR/Cas9)-mediated gene editing can cost-effectively implement targeted mutations in animal genomes, thereby providing a powerful tool for rapid improvement of the economic traits of Bama minipigs. The iroquois homeobox 3 (IRX3) gene has been implicated in human obesity. Mouse experiments have shown that knocking out IRX3 significantly enhances basal metabolism, reduces fat content, and controls body mass and composition. This study aimed to knock out IRX3 using the CRISPR/Cas9 gene editing method to breed Bama minipigs with significantly reduced fat content. First, the CRISPR/Cas9 gene editing method was used to efficiently obtain IRX3(-/-) cells. Then, the gene-edited cells were used as donor cells to produce surviving IRX3(-/-) Bama minipigs using somatic cell cloning. The results show that the use of IRX3(-/-) cells as donor cells for the production of somatic cell-cloned pigs results in a significant decrease in the average live litter size and a significant increase in the average number of stillbirths. Moreover, the birth weight of surviving IRX3(-/-) somatic cell-cloned pigs is significantly lower, and viability is poor such that all piglets die shortly after birth. Therefore, the preliminary results of this study suggest that IRX3 may have important biological functions in pigs, and IRX3 should not be used as a gene editing target to reduce fat content in Bama minipigs. Moreover, this study shows that knocking out IRX3 does not favor the survival of pigs, and whether targeted regulation of IRX3 in the treatment of human obesity will also induce severe adverse consequences requires further investigation.