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Related Concept Videos

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To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.
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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
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Updated: Mar 20, 2026

CIRCLE-Seq for Interrogation of Off-Target Gene Editing
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Off-target effects of engineered nucleases.

Jiing-Kuan Yee1

  • 1Department of Diabetes and Metabolic Diseases Research, City of Hope National Medical Center, Duarte, CA, USA.

The FEBS Journal
|May 22, 2016
PubMed
Summary

Engineered nucleases enable precise genome editing but can cause unintended mutations. New methods detect these off-target effects, and protein engineering enhances nuclease specificity for safer gene editing applications.

Keywords:
CRISPR/Cas9engineered nucleasegene editinghomology-directed repairmeganucleasenonhomologous end-joiningoff-target effecttranscription activator-like effector nucleasezinc finger nuclease

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Area of Science:

  • Genomics
  • Molecular Biology
  • Biotechnology

Background:

  • Engineered nucleases offer powerful genome manipulation for research and therapy.
  • Off-target cleavage at homologous sites is a major complication, leading to mutations.
  • Detecting these unintended genomic alterations is challenging due to genome complexity.

Purpose of the Study:

  • To review methods for detecting nuclease-generated off-target sites.
  • To highlight advancements in improving nuclease targeting specificity.
  • To assess the safety and broader applicability of enhanced gene editing tools.

Main Methods:

  • Development of five unbiased, genome-wide strategies for off-target cleavage detection.
  • Utilizing deep sequencing for high-sensitivity detection of off-target effects.
  • Protein engineering approaches to modify nuclease structure and target interaction.

Main Results:

  • Several detection strategies achieve high sensitivity and precision for assessing off-target effects.
  • Protein engineering has successfully boosted nuclease targeting specificity.
  • Modified nucleases have demonstrated elimination of off-target effects in studied cases.

Conclusions:

  • Advanced detection methods enable objective assessment of engineered nuclease fidelity.
  • Protein engineering significantly enhances the specificity of gene editing nucleases.
  • These improvements pave the way for safer and broader applications of gene editing in research and medicine.