CRISPR-Cas9-Based Knockout of Myostatin Gene in Ovine Somatic Cells

A study was aimed to develop a workflow for CRISPR-Cas9-mediated editing of ovine myostatin (MSTN) gene in primary fibroblast cells. Multiple single-guide RNAs (sgRNAs) targeting exonic regions of the ovine MSTN gene were designed using in silico tools and screened through in vitro cleavage assays to identify functionally active guides. Primary fibroblast cultures at passage 4 were established from ear pinna tissue of Malpura sheep and transfected with Cas9- sgRNA ribonucleoprotein (RNP) complexes. Post-transfection, cell morphology and viability were maintained, with 96% viable cells. Genome editing outcomes were evaluated through PCR amplification of the target locus followed by Sanger sequencing. Indel formation quantified using inference of CRISPR edits (ICE) and tracking of indels by decomposition (TIDE) analysis revealed that population-level editing efficiencies ranged from 15 to 20% for the selected sgRNA. On-target CRISPR activity was confirmed through consistent cut-site-centred sequence divergence and indel distributions. Overall, the study establishes a reliable cell-based platform for MSTN gene editing in sheep fibroblasts and also provides a foundational insight for subsequent clonal isolation and assisted reproductive applications in ovine genome editing programme.