Engineering Base Changes and Epitope-Tagged Alleles in Mice Using Cas9 RNA-Guided Nuclease

Area of Science:

• Genetics and Genomics
• Molecular Biology
• Animal Models

Background:

• Patient-derived genetic changes in mice are crucial for understanding disease causality.
• Epitope tags are essential for studying proteins lacking available antibodies.
• These genetically engineered mouse models facilitate detailed research in development, mechanisms, and translation.

Purpose of the Study:

• To outline a comprehensive protocol for generating genetically modified mice using CRISPR-Cas9.
• To enable the creation of mouse models with specific base changes or epitope tags for disease research.

Main Methods:

• Identification of Cas9 protospacer sequences for targeted modifications.
• Design and synthesis of single-stranded oligonucleotide repair templates and single guide RNAs (sgRNAs).
• Microinjection or electroporation of Cas9, sgRNA, and repair template into mouse zygotes, followed by screening of offspring.

Main Results:

• Successful generation of mice carrying precise sequence alterations or epitope tags.
• Implementation of quality control measures to screen for off-target effects and correct template integration.
• Detailed protocols cover sgRNA design, repair template preparation, zygote manipulation, and screening.

Conclusions:

• This methodology provides a robust framework for creating custom mouse models.
• These models are invaluable for advancing mechanistic and translational studies of human diseases.
• The described protocols ensure the generation of high-quality, genetically precise mouse lines.