Efficient Generation of Myostatin Mutations in Pigs Using the CRISPR/Cas9 System.

Daxin Pang,Chaogang Yao,Hongsheng Ouyang,Huping Jiao,Kankan Wang,Mengjing Li,Nannan Guo,Zicong Xie

doi:10.1038/srep16623

Abstract

Genetically modified pigs are increasingly used for biomedical and agricultural applications. The efficient CRISPR/Cas9 gene editing system holds great promise for the generation of gene-targeting pigs without selection marker genes. In this study, we aimed to disrupt the porcine myostatin (MSTN) gene, which functions as a negative regulator of muscle growth. The transfection efficiency of porcine fetal fibroblasts (PFFs) was improved to facilitate the targeting of Cas9/gRNA. We also demonstrated that Cas9/gRNA can induce non-homologous end-joining (NHEJ), long fragment deletions/inversions and homology-directed repair (HDR) at the MSTN locus of PFFs. Single-cell MSTN knockout colonies were used to generate cloned pigs via somatic cell nuclear transfer (SCNT), which resulted in 8 marker-gene-free cloned pigs with biallelic mutations. Some of the piglets showed obvious intermuscular grooves and enlarged tongues, which are characteristic of the double muscling (DM) phenotype. The protein level of MSTN was decreased in the mutant cloned pigs compared with the wild-type controls, and the mRNA levels of MSTN and related signaling pathway factors were also analyzed. Finally, we carefully assessed off-target mutations in the cloned pigs. The gene editing platform used in this study can efficiently generate genetically modified pigs with biological safety.

Highlights

The development of custom endonuclease techniques promotes the generation of genetically modified animals, which can be used for exploring gene function and generating animal models of human genetic diseases
The transfection efficiency of donor cells has always been a key factor affecting the generation of genetically modified large animals, especially those in which a precise modification is achieved via homology-directed repair
We demonstrated the feasibility of Cas9/gRNA-mediated gene editing at the MSTN locus of porcine fetal fibroblasts

Summary

Introduction

The development of custom endonuclease techniques promotes the generation of genetically modified animals, which can be used for exploring gene function and generating animal models of human genetic diseases. The NHEJ repair process causes gene mutations with small deletions or insertions in the double-stranded break regions[4] Among these three endonuclease techniques, CRISPR/Cas[9] has emerged as a more powerful tool due to the resultant high efficiency[5,6] and rapid assembly[7]. Gene-targeted pigs have been generated either through cytoplasmic injection of Cas[9] mRNA and sgRNA into zygotes[17,18] or via somatic cell nuclear transfer (SCNT) using targeted fibroblasts selected with antibiotics[19]. The Cas9/gRNA-modified fibroblasts without selection marker genes were used to generate gene-targeted pigs via somatic cell nuclear transfer, which resulted in 8 cloned piglets with biallelic mutations in MSTN

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Results

Conclusion