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.

Figure 1. Optimization of PFF transfection.

(a) The pEGFP-N1 expression plasmid was transfected into PFFs using the indicated methods. GFP fluorescence was analyzed via fluorescence microscopy and FACS at 24 h post-transfection. (b) Percent of GFP-expressing cells and mean fluorescence intensity of GFP-expressing cells.n = 3. Graphs show the mean ± S.E.M.

Figure 2. Editing of the porcine MSTN locus using the CRISPR/Cas9 system.

(a) Schematic representation of sgRNAs specific to exon 3 of the porcine MSTN locus. SgRNA targeting sequences are highlighted in red, and PAMs are highlighted in blue. (b) Genomic sequences of CRISPR target regions in wild-type PFFs and transfected PFFs, as indicated. PAMs are underlined in red and the cleavage sites are labeled with arrow. (c) Dual sgRNA-induced mutation at the porcine MSTN locus. PCR results for the targeted regions from co-transfected (CT) and un-transfected (Con) PFFs are shown. Both deletion and inversion bands were detected using specific primers. (d) The dual sgRNA-induced deletion and inversion frequency were further determined based on 149 amplicons covering the target sites. (e). Sequence of the ssDNA donor (upper panel). The newly added nucleotides are highlighted in orange. Sequencing of sgRNA#1 and the ssDNA donor induced mutations in the MSTN gene (lower panel). PCR amplicons spanning the target sites were analyzed for indels. The wild-type sequences are placed on the first line, while mutant TA clones are placed below. Target sites are indicated in red, PAM in blue, and inserted or substituted bases are presented in lower case. Clones showing correct HDR are labeled to the right of the sequences.

Figure 3. Generation of gene-targeted piglets via SCNT.

(a) Genotypes of a portion of the MSTN mutant single-cell colonies. The target sites are labeled in red and PAM in blue. C8,C10,C14 and C18 are Landrace PFFs while E4,E8,E15,E17,E21,E23 and E35 are Erhualian PFFs. (b) Newborn piglets harboring MSTN mutations and some of the piglets show visible intermuscular boundaries (upper panel) and enlarged tongues (lower panel) compared with the WT controls.

Figure 4. Identification of MSTN biallelic mutant piglets.

(a) Representative images of H&E-stained sections to compare the total longissimus dorsi muscle cross-sectional area at a × 4 magnification and general morphology at a × 20 magnification. (b) Quantification of number of myofiber nuclei in longissimus dorsi muscle slices of two mutant and two WT piglets. n = 5. Each bar indicates the mean ± S.E.M. (c) Semi-quantitative RT-PCR analysis of MSTN and myogenic regulatory factors in mutant and WT pigs. (d) Western blotting analysis of MSTN levels in mutant and WT piglets.

Figure 5. Detection of sgRNA#1-mediated off-target effects in mutant pigs.

(a) PCR products covering potential off-target site 16 were subjected to T7 endonuclease I (T7E1) assays. Two pigs (asterisk) showed cleavage bands indicating non-specific mutations in OTS-16. (b) Further cloning of the PCR products confirmed the mutations. The target sites and PAM of OTS-16 are labeled in red and blue, respectively.