Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos

Area of Science:

• Genetics
• Molecular Biology
• Developmental Biology

Background:

• Generating knock-in mice with large targeted insertions is challenging.
• Existing methods often lack efficiency and precision for complex genetic modifications.

Purpose of the Study:

• To develop a highly efficient method for generating knock-in mice with large targeted insertions.
• To improve the precision and efficiency of genome editing in mouse embryos.

Main Methods:

• Utilized CRISPR reagents at the two-cell embryo stage for homologous recombination (2C-HR-CRISPR).
• Employed a modified biotin-streptavidin system to localize repair templates to target DNA sites.
• Applied the method to target 20 endogenous genes for fluorescent reporter insertion.

Main Results:

• Achieved a >10-fold increase in knock-in efficiency, reaching up to 95%.
• Successfully generated reporter mouse lines by targeting endogenous genes in blastocysts.
• Created triple-color blastocysts and embryos with inducible protein degradation systems.

Conclusions:

• 2C-HR-CRISPR offers superior efficiency and precision compared to random transgenesis or standard genome editing.
• This method enables highly efficient insertions at endogenous loci and safe harbor sites.
• Facilitates the generation of advanced mouse models for genetic research and functional studies.