Generation of site-specific mutant mice using the CRISPR/Cas9 system

Area of Science:

• Genetics
• Molecular Biology
• Animal Models

Background:

• CRISPR/Cas9 is a powerful genome editing tool for research.
• Generating precise mutations in animal models is crucial for studying gene function.

Purpose of the Study:

• To establish a CRISPR/Cas9-based system for generating site-specific mutant mice.
• To utilize both homologous recombination-dependent and -independent DNA repair mechanisms for mutation induction.

Main Methods:

• Co-microinjection of Cas9 mRNA and single-guide RNA (sgRNA) targeting specific genes (Kdm2b, Fmo3).
• Utilizing DNA double-strand breaks (DSBs) to trigger repair pathways.
• Employing single-strand oligonucleotides (ssODN) as donor templates for precise editing.

Main Results:

• Simultaneous generation of frame-shifted Kdm2b null mutant and enzyme activity disrupted mouse strains.
• Successful generation of frame-shifted Fmo3 knockout and site-specific Fmo3 knock-in mice with single base substitution.
• Demonstrated stable transmission of generated mutations to the next generation.

Conclusions:

• The CRISPR/Cas9 system effectively generates site-specific mutations in mice.
• Both homologous recombination-dependent and -independent DSB repair pathways can be utilized for genome editing.
• This method provides a robust platform for creating genetically modified mouse models for research.