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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

CIRCLE-Seq for Interrogation of Off-Target Gene Editing.

Jeffrey Inen¹, Chann Makara Han¹, David M Farrel²

¹Department of Dermatology, University of Colorado School of Medicine, Anschutz Medical Campus; Gates Institute, University of Colorado School of Medicine, Anschutz Medical Campus.

Journal of Visualized Experiments : Jove

|November 18, 2024

Summary

Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq) identifies unintended CRISPR-Cas9 DNA cleavage sites. This novel method offers sensitive, efficient, and impartial detection of off-target effects for genomic research.

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Area of Science:

Molecular Biology
Genomics
Biotechnology

Background:

CRISPR-Cas9 gene editing technology offers precise DNA modification.
Off-target cleavage by CRISPR-Cas9 can lead to unintended mutations.
Accurate identification of unintended cleavage sites is crucial for safe and effective gene editing applications.

Purpose of the Study:

To present a detailed protocol for Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq).
To demonstrate the utility of CIRCLE-seq for impartial identification of CRISPR-Cas9 off-target cleavage sites.
To validate the CIRCLE-seq protocol using genome-wide analysis of CRISPR-Cas9 activity at the AAVS1 locus.

Main Methods:

Genomic DNA (gDNA) is circularized.
Circularized gDNA is treated with Cas9 protein and a specific guide RNA (gRNA).
Cleaved DNA is purified, prepared as an Illumina sequencing library, and sequenced.

Main Results:

CIRCLE-seq enables sensitive detection of both intended and unintended Cas9 cleavage events.
The method requires minimal sequencing depth and exhibits low background noise.
CIRCLE-seq demonstrated high enrichment for Cas9-cleaved gDNA, facilitating accurate site mapping.

Conclusions:

CIRCLE-seq is a robust and efficient method for unbiased assessment of CRISPR-Cas9 off-target activity.
The protocol is comprehensive, validated, and can be completed within two weeks.
CIRCLE-seq streamlines the analysis of CRISPR-Cas9 cleavage sites, supporting safer gene editing research.