Efficient and Specific Generation of MSTN-Edited Hu Sheep Using C-CRISPR

Area of Science:

• Animal Genetics
• Gene Editing Technologies
• Livestock Improvement

Background:

• Hu sheep, a Chinese indigenous breed, possess high fecundity but require enhanced growth and carcass traits.
• Myostatin (MSTN) is a key negative regulator of muscle development; its inactivation leads to muscularity.
• The C-CRISPR system enables efficient, one-step gene knockout using multiple guide RNAs.

Purpose of the Study:

• To generate MSTN-knockout (KO) Hu sheep using the C-CRISPR system.
• To investigate the resulting double-muscled (DM) phenotype and assess gene editing efficiency and specificity.
• To evaluate the potential of C-CRISPR for farm animal breeding.

Main Methods:

• Cas9 mRNA and four single-guide RNAs targeting sheep MSTN exon 3 were microinjected into Hu sheep embryos.
• Embryos were transferred to recipient ewes, and resulting lambs were analyzed for MSTN gene editing.
• Phenotypic characterization and molecular analyses (AKT/ERK signaling) were performed on edited sheep.

Main Results:

• Nine out of ten lambs born exhibited complete MSTN KO with various mutations, demonstrating high editing efficiency.
• No off-target mutations were detected, indicating high specificity of the C-CRISPR system.
• MSTN-KO sheep displayed a pronounced double-muscled phenotype, increased body weight, and altered muscle signaling pathways.

Conclusions:

• The C-CRISPR system efficiently and specifically generated MSTN-complete KO Hu sheep with a double-muscled phenotype.
• This study validates C-CRISPR as a powerful tool for genetic modification in farm animals.
• The developed MSTN-KO Hu sheep model offers potential for advancing livestock breeding programs.