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CRISPR/Cas9 Genome Editing

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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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CIRCLE-Seq for Interrogation of Off-Target Gene Editing

CIRCLE-Seq for Interrogation of Off-Target Gene Editing

Published on: November 1, 2024

Quantifying on- and off-target genome editing.

Ayal Hendel¹, Eli J Fine², Gang Bao²

¹Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.

Trends in Biotechnology

|January 18, 2015

Summary

This summary is machine-generated.

Researchers need better assays to measure genome editing outcomes. This review covers current methods for on- and off-target analysis and identifies needs for future genome editing technologies.

Keywords:

CRISPR/Cas9 RNA-guided endonucleases TALENs ZFNs gene editing gene targeting homologous recombination nonhomologous end-joining

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Area of Science:

Molecular Biology
Genetics
Biotechnology

Background:

Genome editing technologies, such as engineered nucleases, offer precise control over genomic alterations.
Applications span basic research, biotechnology, and human gene therapy.
Current methods for assessing genome editing outcomes at endogenous loci are not standardized.

Purpose of the Study:

To review existing assays for quantifying on- and off-target genome editing.
To describe the utility of these assays in advancing genome editing technology.
To highlight unmet needs in assay development for comprehensive genome editing outcome assessment.

Main Methods:

Literature review of current genome editing assay methodologies.
Analysis of the strengths and limitations of existing on- and off-target detection techniques.
Identification of critical gaps in current assay capabilities.

Main Results:

A range of assays exist for quantifying genome editing, but standardization is lacking.
Existing assays vary in their ability to accurately measure on- and off-target modifications.
Key areas for improvement include sensitivity, specificity, and comprehensive outcome detection.

Conclusions:

Standardized and comprehensive assay batteries are crucial for the advancement of genome editing.
Addressing unmet assay needs will accelerate the development and application of genome editing technologies.
Improved assay development is essential for reliable translation of genome editing into clinical and biotechnological applications.