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Updated: Jun 3, 2026
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Mouse Genome Engineering Using Designer Nucleases
Published on: April 2, 2014
Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes
Ting Li1, Sheng Huang, Xuefeng Zhao
1Department of Genetics, Development and Cell Biology, Laurence H Baker Center for Bioinformatics and Biological Statistics, University of Nebraska, Lincoln, NE 68588, USA.
Nucleic Acids Research
|April 5, 2011
Summary
Researchers developed a modular assembly method for creating designer TALENs (Transcription Activator-Like Effector Nucleases). These engineered TALENs efficiently disrupt genes and promote gene replacement in yeast with minimal off-target mutations.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Transcription Activator-Like Effectors (TALEs) fused with FokI nuclease domains create TALENs.
- TALENs induce DNA double-strand breaks, activating cellular repair pathways like non-homologous end-joining and homologous recombination.
Purpose of the Study:
- To develop a modular assembly method for rapid production of designer TALENs (dTALENs).
- To engineer dTALENs for targeting specific loci in yeast chromosomal genes.
- To evaluate the efficiency and specificity of dTALENs in a eukaryotic organism.
Main Methods:
- Exploited TAL repeat DNA recognition cipher to create a modular assembly method.
- Engineered 10 dTALENs targeting unique DNA sequences up to 23 bases.
- Applied dTALENs to native yeast chromosomal genes.
Main Results:
- All engineered dTALENs achieved high rates of site-specific gene disruption.
- dTALENs induced significant gene replacement via homologous recombination (up to 34%).
- No detectable cytotoxicity or significant off-target mutations were observed in treated yeast.
Conclusions:
- Modular assembly provides a rapid and dependable method for dTALEN production.
- dTALENs are effective tools for gene modification in intact eukaryotic organisms like yeast.
- dTALENs hold promise for applications in human and animal health, agriculture, and industry.
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