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

Area of Science:

• Animal genetics
• Gene editing technologies
• Biotechnology

Background:

• Genetically modified pigs are crucial for biomedical research and agriculture.
• CRISPR/Cas9 offers efficient gene targeting without selection markers.
• Myostatin (MSTN) is a key regulator of muscle growth in pigs.

Purpose of the Study:

• To disrupt the porcine myostatin (MSTN) gene using CRISPR/Cas9.
• To generate marker-gene-free cloned pigs with targeted MSTN mutations.
• To assess the efficiency, outcomes, and safety of MSTN gene editing in pigs.

Main Methods:

• Optimized CRISPR/Cas9/gRNA delivery into porcine fetal fibroblasts (PFFs).
• Induced various DNA repair outcomes (NHEJ, HDR, deletions/inversions) at the MSTN locus.
• Generated cloned pigs via somatic cell nuclear transfer (SCNT) from MSTN knockout PFFs.

Main Results:

• Successfully generated 8 marker-gene-free cloned pigs with biallelic MSTN mutations.
• Observed double muscling (DM) phenotype characteristics in some piglets.
• Confirmed reduced MSTN protein and altered mRNA levels, with thorough off-target analysis.

Conclusions:

• The CRISPR/Cas9 platform efficiently generates genetically modified pigs.
• Marker-gene-free gene editing is feasible for creating pigs with desired traits.
• The study demonstrates the biological safety of the gene editing approach for pigs.