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Updated: Sep 5, 2025
CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models
Published on: August 24, 2022
Optimized CRISPR-Cas9-based Strategy for Complex Gene Targeting in Murine Embryonic Stem Cells for Germline
Thomas J O'Neill1, Daniel Krappmann1, Andreas Gewies1
1Research Unit Cellular Signal Integration, Institute of Molecular Toxicology and Pharmacology, Molecular Targets and Therapies Center (MTTC), Helmholtz Zentrum München - German Research Center for Environmental Health, 85764 Neuherberg, Germany.
CRISPR-Cas9 gene targeting in mouse embryonic stem cells (ESCs) is improved using flow cytometry for enrichment and qPCR for screening. This method enhances efficiency and reduces animal use for generating genetically defined mouse models.
Area of Science:
- Molecular Biology
- Genetics
- Developmental Biology
Background:
- CRISPR-Cas9 genome editing in mouse zygotes has limitations for complex modifications.
- Targeting and validating genomic modifications in mouse embryonic stem cells (ESCs) before germline transmission is a viable alternative.
- This approach can reduce animal numbers and improve cost-effectiveness, aligning with animal welfare regulations.
Purpose of the Study:
- To present a streamlined protocol for CRISPR-Cas9-directed gene targeting in mouse ESCs.
- To demonstrate an efficient method for enriching CRISPR-Cas9-expressing ESCs and screening for successful genomic modifications.
Main Methods:
- Transfection of homology templates and PX458 CRISPR-Cas9 plasmids into mouse ESCs.
- Enrichment of CRISPR-Cas9-expressing cells using flow cytometry sorting based on EGFP expression.
- Screening of ESC clones for genomic modifications using quantitative PCR (qPCR) followed by PCR and sequencing validation.
Main Results:
- Flow cytometry effectively enriches for CRISPR-Cas9-expressing ESCs, obviating antibiotic selection.
- qPCR provides a faster and more convenient method for screening genomic modifications compared to agarose gel electrophoresis.
- Validated ESC clones are suitable for injection into early-stage mouse embryos to generate chimeric mice with germline transmission.
Conclusions:
- The described protocol offers a simple and efficient method for CRISPR-Cas9 gene targeting in mouse ESCs.
- Utilizing flow cytometry and qPCR streamlines the process, leading to faster generation of genetically defined mouse models.
- This approach supports the 3R principles by potentially reducing the number of animals required.
