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Updated: Jun 16, 2026
08:23
CIRCLE-Seq for Interrogation of Off-Target Gene Editing
Published on: November 1, 2024
Short DNA sequences inserted for gene targeting can accidentally interfere with off-target gene expression
Ingo D Meier1, Christian Bernreuther, Thomas Tilling
1Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Summary
Gene targeting in mice using neo cassettes or loxP/FRT sites can affect neighboring gene expression. Researchers found that while neo insertions impacted mRNA levels, loxP/FRT sites also caused off-target effects when near regulatory elements, necessitating careful analysis of mouse models.
Area of Science:
- Genetics
- Genomics
- Molecular Biology
Background:
- Gene targeting in mice is crucial for studying development and disease.
- Commonly used targeting methods, such as neo cassettes and loxP/FRT sites, can be inserted into the mouse genome.
- While neo insertions are known to affect gene expression, the impact of loxP sites has been less studied.
Purpose of the Study:
- To investigate the potential for loxP/FRT sites, similar to neo cassettes, to influence the expression of neighboring genes in mice.
- To analyze off-target effects of gene targeting strategies on mRNA levels.
- To assess the impact of inserted DNA sequences on gene regulation in mouse models.
Main Methods:
- Microarray analyses were performed on mice with targeted Ncam or Tnr genes.
- Mice were engineered with either neo cassettes or loxP/FRT sites inserted into specific genes.
- Gene expression levels were compared between different targeting strategies and control conditions.
Main Results:
- Neo insertion near the Ncam gene resulted in a 2-fold reduction in mRNA levels of three neighboring genes.
- LoxP/FRT insertion into the Tnr gene introns led to a 2.5- to 4-fold reduction in Gas5 gene transcript levels, located 1.1 Mb away.
- These off-target effects were attributed to the disruption of regulatory elements by the inserted DNA sequences.
Conclusions:
- Short DNA sequences like loxP/FRT can inadvertently affect off-target mRNA levels by interfering with regulatory elements.
- Conditional knockout mice require thorough analysis for unintended genomic positional effects that could influence observed phenotypes.
- These findings highlight the importance of considering the impact of targeting strategies on the broader genomic landscape.
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