Isozygous and selectable marker-free MSTN knockout cloned pigs generated by the combined use of CRISPR/Cas9 and Cre/LoxP

Bi, Ye; Hua, Zhen; Liu, Ximei; Wu, Hua Hsi; Ren, Hongyan; Xiao, Hong‐Wei; Zhang, Liping; Li, Li; ZhiRui, Wang; Laible, Götz; Wang, Yan; Dong, Fengxia; Zheng, Xin

doi:10.1038/srep31729

Cited by 75 publications

(64 citation statements)

References 48 publications

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“…In this study, we show that programmable CRISPR complexes can be delivered to skeletal muscle in chicks, where they mediate targeted gene modification and reduce MSTN mRNA expression. Although in vivo CRISPR-mediated MSTN gene editing has been applied in mammals such as goats [15], pigs [32], sheep [33], and rabbits [34], no reports have yet emerged on the use of MSTN-CRISPR in postnatal chicks.…”

Section: Discussionmentioning

confidence: 99%

Effective MSTN Gene Knockout by AdV-Delivered CRISPR/Cas9 in Postnatal Chick Leg Muscle

Han

Zhou

et al. 2020

IJMS

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Muscle growth and development are important aspects of chicken meat production, but the underlying regulatory mechanisms remain unclear and need further exploration. CRISPR has been used for gene editing to study gene function in mice, but less has been done in chick muscles. To verify whether postnatal gene editing could be achieved in chick muscles and determine the transcriptomic changes, we knocked out Myostatin (MSTN), a potential inhibitor of muscle growth and development, in chicks and performed transcriptome analysis on knock-out (KO) muscles and wild-type (WT) muscles at two post-natal days: 3d (3-day-old) and 14d (14-day-old). Large fragment deletions of MSTN (>5 kb) were achieved in all KO muscles, and the MSTN gene expression was significantly downregulated at 14d. The transcriptomic results indicated the presence of 1339 differentially expressed genes (DEGs) between the 3d KO and 3d WT muscles, as well as 597 DEGs between 14d KO and 14d WT muscles. Many DEGs were found to be related to cell differentiation and proliferation, muscle growth and energy metabolism. This method provides a potential means of postnatal gene editing in chicks, and the results presented here could provide a basis for further investigation of the mechanisms involved in muscle growth and development.

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Section: Discussionmentioning

confidence: 99%

Effective MSTN Gene Knockout by AdV-Delivered CRISPR/Cas9 in Postnatal Chick Leg Muscle

Han

Zhou

et al. 2020

IJMS

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“…Myostatin (MSTN) plays an important negative regulatory role in the proliferation, differentiation, and growth of animal skeletal muscles. MSTN gene knockout or reduced expression of MSTN will promote pig muscle growth and increase lean meat proportion [16,18]. Uncoupling protein 1 (UCP1) plays an important role in energy conversion and maintaining body temperature.…”

Section: Discussionmentioning

confidence: 99%

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

Zhu

Wei

Zhan

et al. 2020

Animals

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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...

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“…Genetic modification in pigs has potential for rapidly improving quantity and quality of pork, compared to traditional selection and breeding methods that are timeconsuming when attempting to increase the desired gene frequency for specific traits (Petersen, 2017;Ruan et al, 2017). Genetically modified pigs have been produced with improved production traits, including altered fatty acid composition (Lai et al, 2006;Zhang et al, 2012), decreased fat deposition (Zheng et al, 2017), increased muscle mass and higher growth rate (Bi et al, 2016;Qian et al, 2015;Wang et al, 2015) and resistance to infectious disease (Burkard et al, 2017;Whitworth et al, 2014Whitworth et al, , 2016Yang et al, 2018). In addition, pigs, especially minipigs, also share a number of similarities with human, such as body size, anatomy, physiology, pathology as well as genome, providing an excellent animal model for mimicking human diseases (Nagashima et al, 2012;Niemann & Lucas-Hahn, 2012;Prather et al, 2013;Whyte & Prather, 2011).…”

Section: Discussionmentioning

confidence: 99%

Generation of transgenic‐cloned Huanjiang Xiang pigs systemically expressing enhanced green fluorescent protein

Zhu

Zhong²,

Ge³

et al. 2018

Reprod Domestic Animals

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Huanjiang Xiang pig is a unique native minipig breed originating in Guangxi, China, and has great utility value in agriculture and biomedicine. Reproductive biotechnologies such as somatic cell nuclear transfer (SCNT) and SCNT-mediated genetic modification show great potential value in genetic preservation and utilization of Huanjiang Xiang pigs. Our previous work has successfully produced cloned and transgenic-cloned embryos using somatic cells from a Huanjiang Xiang pig. In this study, we firstly report the generation of transgenic-cloned Huanjiang Xiang pigs carrying an enhanced green fluorescent protein (eGFP) gene. A total of 504 SCNT-derived embryos were transferred to two surrogate recipients, one of which became pregnant and gave birth to three live piglets. Exogenous eGFP transgene had integrated in all of the three Huanjiang Xiang piglets identified by genotyping. Furthermore, expression of eGFP was also detected from in vitro cultured skin fibroblast cells and various organs or tissues from positive transgenic-cloned Huanjiang Xiang pigs. The present work provides a practical method to preserve this unique genetic resource and also lays a foundation for genetic modification of Huanjiang Xiang pigs with improved values in agriculture and biomedicine.

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Isozygous and selectable marker-free MSTN knockout cloned pigs generated by the combined use of CRISPR/Cas9 and Cre/LoxP

Cited by 75 publications

References 48 publications

Effective MSTN Gene Knockout by AdV-Delivered CRISPR/Cas9 in Postnatal Chick Leg Muscle

Effective MSTN Gene Knockout by AdV-Delivered CRISPR/Cas9 in Postnatal Chick Leg Muscle

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

Generation of transgenic‐cloned Huanjiang Xiang pigs systemically expressing enhanced green fluorescent protein

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