Targeted Germline Modifications in Rats Using CRISPR/Cas9 and Spermatogonial Stem Cells

Area of Science:

• Genetics
• Reproductive Biology
• Molecular Biology

Background:

• CRISPR/Cas9 gene editing enables targeted genomic modifications in various organisms.
• Efficient germline editing is crucial for genetic research and disease modeling.

Purpose of the Study:

• To establish a method for targeted germline gene editing in rats using CRISPR/Cas9 in spermatogonial stem cells.
• To generate rats with specific mutations (Epsti1, Erbb3) for studying spermatogenesis and heritability.

Main Methods:

• Cultured rat spermatogonial stem cells were modified using CRISPR/Cas9.
• Modified cells were transplanted into rat testes to assess regeneration of spermatogenesis.
• Mutant progeny were analyzed for transmission of targeted genomic alterations.

Main Results:

• CRISPR/Cas9-modified spermatogonia regenerated spermatogenesis and maintained long-term sperm-forming potential.
• Targeted mutations in Epsti1 and Erbb3 were vertically transmitted, producing pure, non-mosaic mutant offspring.
• Recessive spermatogenesis defects were unmasked in Erbb3 mutant rats, demonstrating the utility of the platform.

Conclusions:

• Spermatogonial gene editing with CRISPR/Cas9 is an effective platform for generating targeted germline mutations in rats.
• This approach facilitates the study of spermatogenesis and the generation of genetically defined animal models.
• The method allows for the production of pure mutant progeny, simplifying genetic analysis and breeding.