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Updated: Jul 30, 2026
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Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9
Published on: January 3, 2015
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Protocol for generating splice isoform-specific mouse mutants using CRISPR-Cas9 and a minigene splicing reporter
Yudong Teng1, Kelsey Arbogast2, Harald Junge3
1The Genetically Engineered Murine Models Core, Department of Immunology & Microbiology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.
STAR Protocols
|January 5, 2025
Summary
This study introduces a CRISPR-Cas9 method to modify specific mRNA variants in mice without altering overall gene expression. This technique generates novel mouse models for advanced genetic research.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Alternative splicing generates diverse mRNA variants from a single gene, crucial for cellular function.
- Existing genetic tools often lack the precision to target specific splice isoforms.
- Understanding splice variant function requires precise genetic manipulation tools.
Purpose of the Study:
- To develop a protocol for precise alteration of alternative mRNA splicing using CRISPR-Cas9 in mice.
- To create splice isoform-specific mouse mutants for detailed genetic analysis.
- To enable the study of gene function beyond traditional knockout or overexpression models.
Main Methods:
- Designing guide RNA (gRNA) to target Cas9 endonuclease to consensus splice sites.
- Generating transgenic mice via pronuclear injection of CRISPR-Cas9 components.
- Screening for desired mutations in mammalian cells using a minigene splicing reporter assay.
Main Results:
- Successful generation of mouse models with altered alternative splicing patterns.
- Demonstration that overall gene expression remains unaffected by the targeted splicing modifications.
- Validation of the minigene reporter system for efficient screening of splice-site mutations.
Conclusions:
- CRISPR-Cas9-mediated genomic editing offers a precise method to control alternative splicing in mice.
- Splice isoform-specific mouse mutants are powerful tools for dissecting gene function and biological pathways.
- This protocol facilitates the creation of novel research models for genetic and molecular studies.
