Efficient isolation of mouse deletion mutant embryonic stem cells by CRISPR

Area of Science:

• Molecular Biology
• Genetics
• Developmental Biology

Background:

• Understanding gene function is crucial for biological research.
• Mouse embryonic stem (ES) cells are valuable tools for genetic studies and creating mutant mice.
• Efficiently generating genetically modified ES cells is essential for these studies.

Purpose of the Study:

• To develop and describe an efficient method for isolating mouse ES cell clones with targeted gene deletions using CRISPR-Cas9.
• To facilitate the generation of mutant mice for functional genomics research.

Main Methods:

• Co-expression of two single-guide RNAs (sgRNAs) targeting a specific gene along with a puromycin-resistant gene in mouse ES cells.
• Utilizing CRISPR-Cas9 technology for precise gene editing.
• Employing transient puromycin selection to enrich for cells with successful gene deletion.
• Identifying deletion mutations in individual ES cell clones via Polymerase Chain Reaction (PCR).

Main Results:

• Successful and efficient isolation of ES cell clones harboring deletion mutations in target genes.
• Demonstration of a streamlined workflow from gene editing to clone identification.

Conclusions:

• The described CRISPR-Cas9 approach provides an efficient strategy for isolating ES cell clones with desired gene deletions.
• This method can accelerate the study of gene function through the rapid generation of genetically modified mouse models.