Simple Protocol for Generating and Genotyping Genome-Edited Mice With CRISPR-Cas9 Reagents

Area of Science:

• Molecular Biology
• Genetics
• Developmental Biology

Background:

• CRISPR-Cas9 gene editing offers powerful tools for creating genetically modified organisms.
• Developing reliable protocols for generating genome-edited mouse models is crucial for research.

Purpose of the Study:

• To provide a comprehensive, easy-to-follow protocol for generating genome-edited mice.
• To detail methods for CRISPR-Cas9 reagent preparation, microinjection, and genotyping.

Main Methods:

• Designing guide RNAs for gene disruption or editing strategies.
• Preparing and validating CRISPR-Cas9 reagents.
• Microinjection and electroporation of CRISPR-Cas9 components into one-cell mouse embryos.
• Genotyping offspring using next-generation sequencing methods.

Main Results:

• Successful generation of genome-edited mice with targeted modifications.
• Efficient preparation and validation of CRISPR-Cas9 reagents.
• Accurate genotyping of resulting offspring, including detection of indel mutations.

Conclusions:

• The described protocol enables reliable generation of genome-edited mice.
• This method facilitates the creation of knockout and knock-in mouse models for research.
• Advanced genotyping ensures the accuracy of gene editing outcomes.