Prime editing in mice with an engineered pegRNA.

Area of Science:

• Molecular Biology
• Genetics
• Gene Editing

Background:

• CRISPR gene editing causes DNA double-strand breaks, leading to indels and potential embryonic lethality.
• Prime editing (PE) offers precise nucleotide substitutions but can be limited by conventional guide RNAs.

Purpose of the Study:

• To evaluate the efficacy of engineered pegRNAs (epegRNAs) for precise gene editing in vivo.
• To demonstrate germline transmission of precise edits using epegRNAs in a mouse model.

Main Methods:

• Utilized engineered pegRNAs (epegRNAs) with a 3' extension for enhanced stability.
• Attempted precise nucleotide substitution in the Capn2 gene using CRISPR, conventional PE, and epegRNA.
• Conducted genotyping, long-read sequencing, Western blotting, and histological analysis.

Main Results:

• Conventional CRISPR and PE failed to introduce the desired Capn2 substitution.
• epegRNA successfully installed the precise Capn2 edit in founder mice with high sequence fidelity.
• Reduced CAPN2 phosphorylation and alleviated lung inflammation in treated mice.

Conclusions:

• epegRNAs enable precise germline editing in animals, overcoming limitations of previous methods.
• This technology provides a solution for editing essential developmental genes previously difficult to target.