Using CRISPR/Cas9 for Gene Knockout in Immunodeficient NSG Mice.

Area of Science:

• Immunology
• Genetics
• Molecular Biology

Background:

• NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mice are crucial for human cell xenografts but have a complex genetic background hindering further modifications.
• Traditional methods for genetic modification in mice are labor-intensive and require extensive cross-breeding.

Purpose of the Study:

• To describe a protocol for genetically modifying NSG mice using CRISPR/Cas9 technology.
• To demonstrate the efficiency of CRISPR/Cas9 for creating knockout mutations in NSG mice.

Main Methods:

• Design and synthesis of two single-guide RNAs (sgRNAs) targeting exons 1 and 3 of the Cybb gene.
• Microinjection of sgRNAs and Cas9 mRNA into fertilized NSG mouse eggs.
• Transfer of injected embryos into pseudopregnant surrogate mothers and subsequent genotyping of offspring.

Main Results:

• Successful knockout of the Cybb gene in NSG mice offspring.
• Genotyping confirmed the genetic modification through PCR and DNA sequencing.
• Demonstrated high efficiency of CRISPR/Cas9 for genetic engineering in NSG mice.

Conclusions:

• CRISPR/Cas9 technology provides an efficient and streamlined approach for genetically modifying NSG mice.
• This method simplifies the generation of congenic strains with desired genetic traits in the NSG background.
• Facilitates the development of advanced mouse models for biomedical research.