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Updated: Nov 24, 2025
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CIRCLE-Seq for Interrogation of Off-Target Gene Editing
Published on: November 1, 2024
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Evaluation of Homology-Independent CRISPR-Cas9 Off-Target Assessment Methods
Hemangi G Chaudhari1, Jon Penterman1, Holly J Whitton2
1CRISPR Therapeutics, Inc., Cambridge, Massachusetts, USA; San Francisco, California 94102, USA.
The CRISPR Journal
|December 21, 2020
Summary
CRISPR-Cas gene editing requires accurate off-target detection for therapies. This study compared three methods, finding GUIDE-seq suitable for preclinical assessment due to its low false-positive rate.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- CRISPR-Cas gene editing offers revolutionary potential in medicine and research.
- Therapeutic applications necessitate rigorous preclinical evaluation of off-target editing.
- Existing off-target site nomination assays require comparative analysis to determine efficacy.
Purpose of the Study:
- To compare the performance of three homology-independent off-target nomination methods: cell-based assay, GUIDE-seq, and CIRCLE-seq/SITE-seq.
- To benchmark these methods against homology-nominated sites using sequencing and hybrid capture.
- To provide a comprehensive assessment of off-target activity detection for CRISPR-Cas technologies.
Main Methods:
- HEK293T cells were treated with Streptococcus pyogenes Cas9 and eight guide RNAs.
- Comparison of cell-based assay, GUIDE-seq, and CIRCLE-seq/SITE-seq for off-target nomination.
- Benchmarking via sequencing of 75,000 homology-nominated sites using hybrid capture and high-throughput sequencing.
Main Results:
- All three homology-independent methods performed similarly in nominating sequence-confirmed off-target sites.
- Significant differences were observed in the total number of sites nominated by each method.
- GUIDE-seq demonstrated a low false-positive rate and high correlation with observed editing.
Conclusions:
- When combined with homology-dependent methods, all tested assays provide a comprehensive assessment of CRISPR-Cas off-target activity.
- GUIDE-seq is particularly suitable for nominating off-target sites in ex vivo CRISPR-Cas therapies.
- Accurate off-target detection is crucial for the safe and effective therapeutic application of gene editing.
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