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

Abstract

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 o...

Application of the modified cytosine base-editing in the cultured cells of bama minipig

“…Design and construction of the CBE plasmid were performed according to our previous studies (Wei et al, 2020;Zhu et al, 2020a;b). The sgRNAs used in this study were designed as described below, and were produced by BGI company.…”

“…Procedures used for the isolation, cultivation, and transfection of kidney broblasts from newborn Bama minipigs were based upon our previous studies (Wei et al, 2020;Zhu et al, 2020a;b). After deep anesthetization, the newborn piglet was euthanized and both kidneys were removed and minced in Dulbecco's phosphate-buffered saline (DPBS; Gibco, Grand Island, NY, USA).…”

“…Potential OTSs were predicted by the CRISPR Design Tool (http://crispor.tefor.net) according to our previous studies (Wei et al, 2020;Zhu et al, 2020a;b). Six sites with potential genomic off-target effects for MSTN locus were selected for detection of off-targets occurred in base-edited cell colonies (Table 3).…”

“…Bama minipigs have played an important role in local economic growth and agriculture, and thereby in poverty reduction. In addition to food consumption, because of its small size, Bama minipigs have become a well-known local specialty throughout the country, with a high value in the area's economic development (Zhu et al, 2020a;b). Characteristic traits of the breed include early sexual maturity, large litters, and a high degree of inbreeding.…”

“…The pigs also have the potential to be used as medical disease models (Zhu et al, 2018). However, similar to other indigenous pig breeds in China, Bama minipigs have unfavorable characteristics such as low lean meat percentage, high fat content, slow growth rate, and low feed conversion ratio, hindering the breed's application and promotion (Zhu et al, 2020a;b).…”