Targeted mutagenesis in mouse cells and embryos using an enhanced prime editor

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Prime editing is a novel genome-editing technology utilizing CRISPR-Cas9 nickase and reverse transcriptase for targeted mutagenesis.
• Current prime editing methods require optimization to enhance their efficiency and broaden their applicability.

Purpose of the Study:

• To develop and validate strategies for improving prime editing efficiency.
• To generate Igf2 mutant mice using enhanced prime editing and assess its efficacy and safety.

Main Methods:

• Implementation of two novel strategies: proximal dead sgRNA and chromatin-modulating peptides to boost prime editing efficiency.
• Application of enhanced prime editing for the generation of Igf2 mutant mice.
• Analysis of editing frequencies, off-target effects, and phenotypic outcomes, including germline transmission.

Main Results:

• Achieved high editing frequencies, up to 47%, in Igf2 mutant mice using the enhanced prime editing system.
• Demonstrated successful germline transmission of the edited gene.
• Confirmed the absence of detectable off-target mutations.
• Observed a distinct dwarf phenotype in the generated mutant mice.

Conclusions:

• The developed enhanced prime editing strategies significantly improve editing efficiency.
• This method is effective for generating genetically modified mouse models with high precision and efficiency.
• The approach holds promise for applications in cell research and the creation of disease models.