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Updated: Jun 25, 2026
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A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells
Published on: August 25, 2017
Gene modification in embryonic stem cells by single-stranded DNA oligonucleotides.
Marieke Aarts1, Marleen Dekker, Rob Dekker
1Division of Molecular Biology, The Netherlands Cancer Institute, , Amsterdam, The Netherlands.
Methods in Molecular Biology (Clifton, N.J.)
|March 7, 2009
Summary
Gene editing in mouse embryonic stem cells is improved by targeting DNA mismatch repair. Researchers found suppressing MSH2 facilitates base substitutions, while MSH3 deficiency aids insertions for precise genome modification.
Area of Science:
- Molecular Biology
- Genetics
- Gene Editing
Background:
- Oligonucleotide-mediated gene targeting offers a precise method for genome modification in mouse embryonic stem (ES) cells.
- Existing methods are often hindered by the DNA mismatch repair (MMR) system, limiting the efficiency of small sequence alterations.
Purpose of the Study:
- To develop strategies for overcoming MMR-mediated inhibition of oligonucleotide-directed gene targeting in mouse ES cells.
- To enable efficient single or few-nucleotide substitutions, insertions, or deletions in the genome.
Main Methods:
- Investigated the roles of MSH2/MSH6 and MSH2/MSH3 mismatch recognition complexes in MMR.
- Utilized transient suppression of MSH2 protein levels to facilitate base substitutions.
- Employed MSH3-deficient ES cells to enhance the efficiency of base insertions.
Main Results:
- Transient MSH2 suppression effectively enabled oligonucleotide-mediated base substitutions.
- MSH3-deficient ES cells demonstrated high efficiency for oligonucleotide-mediated base insertions.
- Developed a versatile gene targeting approach applicable to any codon of interest.
Conclusions:
- Targeting specific components of the MMR pathway provides a robust strategy to enhance oligonucleotide-mediated gene editing in mouse ES cells.
- This refined gene targeting method allows for precise and efficient genomic modifications, including codon substitutions.
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