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CRISPR

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Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...

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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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Related Experiment Video

Updated: Dec 23, 2025

CIRCLE-Seq for Interrogation of Off-Target Gene Editing

CIRCLE-Seq for Interrogation of Off-Target Gene Editing

Published on: November 1, 2024

CRISPR off-target detection with DISCOVER-seq.

Beeke Wienert^1,2, Stacia K Wyman³, Charles D Yeh⁴

¹Innovative Genomics Institute, University of California, Berkeley, Berkeley, California, USA. beeke.wienert@gladstone.ucsf.edu.

Nature Protocols

|April 22, 2020

Summary

This summary is machine-generated.

DISCOVER-seq offers unbiased CRISPR-Cas off-target identification by tracking DNA repair factors at double-strand breaks (DSBs). This method provides low false-positive rates for accurate genome editing verification in various systems.

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Area of Science:

Molecular Biology
Genomics
Biotechnology

Background:

CRISPR-Cas genome editing necessitates accurate identification of off-target mutations.
Existing methods for off-target detection can suffer from high false-positive rates or limited applicability.
Unbiased and sensitive detection of CRISPR-Cas induced off-target events is crucial for therapeutic development.

Purpose of the Study:

To present DISCOVER-seq, a novel method for unbiased, in situ identification and verification of CRISPR-Cas off-target events.
To provide a detailed protocol and analysis pipeline for performing DISCOVER-seq.
To demonstrate the broad applicability of DISCOVER-seq in various biological systems.

Main Methods:

DISCOVER-seq utilizes chromatin immunoprecipitation (ChIP) to track MRE11 recruitment to CRISPR-Cas induced double-strand breaks (DSBs).
Next-generation sequencing (NGS) is employed to identify the genomic locations of these DSBs.
A custom bioinformatics pipeline, BLENDER, analyzes sequencing data to identify off-target sequences genome-wide.

Main Results:

DISCOVER-seq successfully identifies and quantifies off-target mutations in both cell cultures and in situ.
The method exhibits low false-positive rates due to the requirement of DNA repair factor binding.
DISCOVER-seq is applicable to diverse systems, including primary cells, patient-derived cells, and animal models.

Conclusions:

DISCOVER-seq is a broadly applicable and robust approach for unbiased CRISPR-Cas off-target identification.
The method offers high sensitivity and specificity, reducing concerns about unintended genomic alterations.
The entire DISCOVER-seq protocol, including data analysis, can be completed within two weeks, facilitating rapid assessment of genome editing outcomes.