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

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.