Generating CRISPR/Cas9-Derived Mutant Mice by Zygote Cytoplasmic Injection Using an Automatic Microinjector

Area of Science:

• Genetics and Genomics
• Developmental Biology
• Molecular Biology

Background:

• CRISPR/Cas systems offer efficient gene editing for creating mutant animals.
• Traditional methods like embryonic stem cells (ESCs) can be time-consuming and complex.
• Simultaneous targeting of multiple genes is desirable for functional genomics.

Purpose of the Study:

• To describe a detailed protocol for generating CRISPR mutant animals via cytoplasmic injection.
• To provide a streamlined and efficient method for CRISPR-based mutagenesis.
• To highlight the advantages of this technique over existing microinjection methodologies.

Main Methods:

• Step-by-step protocol for CRISPR mutant generation.
• Preparation of reagents and materials for injection.
• Cytoplasmic microinjection into zygotes and subsequent validation.

Main Results:

• The described protocol allows for CRISPR mutant generation within 2-4 hours.
• High survival and developmental rates of injected zygotes were observed.
• The method offers significant advantages over pronuclear and other microinjection techniques.

Conclusions:

• Cytoplasmic injection provides a rapid, efficient, and high-throughput method for generating CRISPR mutant animals.
• This protocol facilitates the elucidation of gene function in development and disease.
• The technique simplifies the production of genetically modified organisms.