CRISPR/Cas9-based genome editing in mice by single plasmid injection

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR/Cas genome modification is crucial for gene function research.
• Generating gene knockout mice traditionally involves injecting mRNA and guide RNA (sgRNA), which requires complex preparation and storage.

Purpose of the Study:

• To simplify the delivery method for CRISPR/Cas-mediated gene editing in mice.
• To develop a rapid, easy, and reproducible strategy for targeted mutagenesis.

Main Methods:

• Injection of circular pX330 plasmids expressing humanized Cas9 (hCas9) and sgRNA into fertilized eggs.
• Development of a pCAG-EGxxFP reporter plasmid to evaluate sgRNA activity via EGFP fluorescence in HEK293T cells.
• Generation of targeted gene mutant mice using the optimized protocol.

Main Results:

• Circular plasmid injection achieved efficient generation of mutant mice, comparable to RNA injection.
• Circular plasmids reduced the likelihood of integration into the host genome compared to linearized plasmids.
• The developed strategy enables the generation of targeted gene mutant mice within one month.

Conclusions:

• The use of circular plasmids offers a simplified and efficient method for CRISPR/Cas-mediated gene knockout mouse generation.
• This approach enhances reproducibility and reduces technical challenges in creating genetically modified mice.
• The combined techniques provide a valuable tool for accelerating genetic research and gene function elucidation.