CRISPR-Cas9-Mediated Gene Editing in Mouse Spermatogonial Stem Cells

Area of Science:

• Genetics
• Molecular Biology
• Reproductive Biology

Background:

• Precise genome editing aids gene function analysis but is challenging in spermatogonial stem cells (SSCs).
• Existing gene editing techniques for SSCs often suffer from low efficiency and complexity.
• The bacterial CRISPR-Cas9 system offers high efficiency and specificity for genome modification.

Purpose of the Study:

• To describe a protocol for CRISPR-Cas9-mediated genome editing in SSCs.
• To enable precise gene modification in SSCs using nonhomologous end joining (NHEJ) or homology-directed repair (HDR).
• To facilitate the production of gene-modified mice from edited SSCs.

Main Methods:

• Derivation of SSCs.
• Nucleofection of SSCs with the CRISPR-Cas9 system.
• Transplantation of gene-modified SSCs into recipient testes.
• Production of mice from transplanted SSC-derived round spermatids.

Main Results:

• Successful CRISPR-Cas9-mediated gene editing in SSCs via NHEJ and HDR pathways.
• Demonstration of a complete protocol from SSC derivation to gene-modified mouse production.
• Establishment of an efficient method for SSC genome modification.

Conclusions:

• CRISPR-Cas9 technology significantly improves genome editing efficiency and specificity in SSCs.
• This protocol provides a robust framework for genetic modification of SSCs.
• The method allows for the generation of genetically engineered mice through SSC transplantation.