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

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.

Applications of CRISPR Genome Engineering in Cell Biology

“…While mouse models have been widely used, the CRISPR/Cas9 gene editing approach has been established in many other animal models including worm [84], fly [85], fish [86, 87], rat [88], rabbit [89, 90], goat [91], sheep [92], dog [93], pig [94], and monkeys [95]. The expansion of transgenic animal models beyond mouse is advantageous to biomedical research that accelerates the development of new therapeutic strategies.…”

Genome Editing in Large Animals

“…Prather’s group has created pigs with substantial immunity to PRRSv virus by knocking out the PRRSv receptor with random NHEJ [33]. Others have, for instance, knocked out myostatin in pigs [34]. The Roslin institute, the original developers of cloning and one of the great pioneers in this field, have in one of the first examples of use of homologous recombination in large animals, changed a pig allele to a warthog allele to induce resistance to African Swine Fever virus [35].…”

Partial loss of interleukin 2 receptor gamma function in pigs provides mechanistic insights for the study of human immunodeficiency syndrome

“…For example, designed mega-nucleases can induce double-stranded breaks (DSBs) at specific sites in the genome, resulting in random modifications through non-homologous end joining (NHEJ) [11–13]. Recent achievements in the use of mega-nucleases such as Zinc-finger nucleases (ZFNs) [11, 12], transcription activator-like effector nucleases (TALENs) [14–16], and CRISPR/Cas9 [17] have also suggested that KO pigs can be efficiently generated. Recently, we and other groups have described X-linked or autosomal SCID-like pigs that were created by disrupting interleukin 2 receptor gamma (IL2RG) [18, 19], autosomal Rag-1/2 [20], and RAG-2 SCID [21] or by natural breeding [22].…”