Production Of IRX3 Gene Knockout Bama Miniature Pig By CRISPR/Cas9 Technology

Bama minipigs are one of local pig breeds that is unique to China and is of high development and utilization value.However,its high fat content,low feed utilization rate,and slow growth rate limit the popularization and utilization of this swine species.Compared with the long breeding cycle and high cost of traditional genetic breeding of pigs,CRISPR/Cas9-mediated gene editing can cost-effectively implement targeted editing in animal genomes,thereby providing a powerful application tool for rapid improvement of the production traits of pigs.Iroquois homeobox3（IRX3）has been implicated in human obesity.Mouse experiments have shown that knocking out IRX3significantly enhances basal metabolism,reduces the fat content,and controls body mass and composition.The primary purpose of this study is to improve the production and application value of Bama minipigs,to knock out IRX3 using the CRISPR/Cas9gene editing method to breed Bama minipigs with significantly reduced fat content and significantly improved production traits.So as to promote the popularization and application of Bama minipigsIn this study,CRISPR/Cas9 gene editing technology was successfully used to obtain Cas9-IRX3-sg RNA vector with high cutting efficiency and targeting the first exon of IRX3 gene of Bama minipigs.The vector were transfected into Bama minipigs kidney fibroblasts by liposome transfection,and 8 single cell clones were obtained.7of them had double allele mutation,the editing efficiency was 87.5%.Through PCR and gene sequencing,IRX3^-/-cells without plasmid integration and off-targets were selected as nuclear donors.Nuclear transfer combined with embryo transfer,a total of1000 gene editing cloned embryos were transferred to 4 surrogate sows,of which 2were pregnant.Three cloned pigs were successfully produced.Live born IRX3^-/-pigs have poor postnatal viability and all die shortly after birth.The ear tissues of three cloned piglets were collected for genotyping and protein expression.It shows that IRX3 gene is completely knockout.According to production data,compared with the production of the same period of wild-type somatic clone pig,IRX3^-/-pig’s birth weight decreased significantly（0.7±0.2kg;P<0.05）,the average litter size decreased significantly（7.6±2.4 vs 1.5±0.7,P<0.01）,while the average litter size increased significantly（1.4±1.4 vs 6.5±0.7,P<0.01）.Therefore,this study suggests that IRX3 gene may play an important role in early growth and development of pigs,and it is not suitable to be an early regulatory target gene for reducing fat content in Bama minipigs.So far,there has not been any research on directly targeting pig adipose tissue through gene editing technology to reduce pig obesity.In this study,CRISPR/Cas9editing technology was utilized to knock out IRX3 gene of Bama minipigs and establish IRX3 gene knockout genetic molecular breeding technology.The significance of IRX3is to explore the mechanism of IRX3 gene on Bama minipigs.Secondly,if pigs with IRX3 gene deletion can reduce fat content,which is expected to be used as breeding material in the future,to promote the popularization and application of Bama minipigs.So it has high production and application value.