Editing the CYP19 Gene in Goat Embryos Using CRISPR/Cas9 and Somatic Cell Nuclear Transfer Techniques

Area of Science:

• Genetics and Genomics
• Biotechnology
• Animal Science

Background:

• The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) system offers revolutionary potential for genome engineering in livestock.
• The CYP19 gene, encoding aromatase, is vital for steroid metabolism, influencing traits relevant to livestock production and health.
• Genetic modification of livestock holds promise for improving traits and treating diseases.

Purpose of the Study:

• To investigate the efficacy of CRISPR/Cas9 for targeted modification of the CYP19 gene in goat fibroblast cells and subsequent embryo production.
• To assess the efficiency of CYP19 gene knockout in goat embryos produced via somatic cell nuclear transfer (SCNT).
• To evaluate the developmental competence of gene-edited goat embryos.

Main Methods:

• Design and cloning of guide RNAs (gRNAs) targeting the CYP19 gene into the pX459 vector.
• CRISPR/Cas9-mediated gene editing of Lori-Bakhtiari buck fibroblast cells via electrotransfection.
• Somatic cell nuclear transfer (SCNT) using edited cells to produce cloned goat embryos.
• Evaluation of blastocyst development and knockout efficiency in edited cells and embryos.

Main Results:

• Successful generation of CYP19 gene-edited goat embryos using CRISPR/Cas9 technology combined with SCNT.
• Achieved a knockout efficiency of 78.4% in edited cells and 68.9% in resulting goat blastocysts.
• Demonstrated no significant difference in blastocyst development rates between gene-edited and unedited embryos.

Conclusions:

• The study successfully established a method for generating CYP19 gene-edited goat embryos with high efficiency.
• The combined approach of cell electrotransfection and SCNT preserves normal embryonic development.
• These gene-edited embryos provide a valuable resource for future research into the role of CYP19 in goat fertility and production traits.