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Updated: Aug 4, 2025
CIRCLE-Seq for Interrogation of Off-Target Gene Editing
Published on: November 1, 2024
Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq
Roger S Zou1,2, Yang Liu2,3, Oscar E Reyes Gaido4
1Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Abstract:
Discovery of off-target CRISPR-Cas activity in patient-derived cells and animal models is crucial for genome editing applications, but currently exhibits low sensitivity. We demonstrate that inhibition of DNA-dependent protein kinase catalytic subunit accumulates the repair protein MRE11 at CRISPR-Cas-targeted sites, enabling high-sensitivity mapping of off-target sites to positions of MRE11 binding using chromatin immunoprecipitation followed by sequencing. This technique, termed DISCOVER-Seq+, discovered up to fivefold more CRISPR off-target sites in immortalized cell lines, primary human cells and mice compared with previous methods. We demonstrate applicability to ex vivo knock-in of a cancer-directed transgenic T cell receptor in primary human T cells and in vivo adenovirus knock-out of cardiovascular risk gene PCSK9 in mice. Thus, DISCOVER-Seq+ is, to our knowledge, the most sensitive method to-date for discovering off-target genome editing in vivo.
Insights
We developed DISCOVER-Seq+, a highly sensitive method to detect unintended DNA changes from CRISPR-Cas genome editing. This new technique identifies significantly more off-target sites in cells and animal models than previous approaches.
Area of Science:
- Genetics
- Molecular Biology
- Biotechnology
Background:
- CRISPR-Cas genome editing offers therapeutic potential but requires sensitive detection of off-target effects.
- Current methods for identifying unintended DNA modifications by CRISPR-Cas systems lack sufficient sensitivity.
Purpose of the Study:
- To develop a highly sensitive method for discovering off-target CRISPR-Cas activity in various biological systems.
- To improve the safety assessment of genome editing technologies.
Main Methods:
- Inhibition of DNA-dependent protein kinase catalytic subunit to enrich MRE11 repair protein at targeted sites.
- Chromatin immunoprecipitation followed by sequencing (ChIP-seq) to map MRE11 binding sites, termed DISCOVER-Seq+.
- Application in immortalized cell lines, primary human cells, and animal models.
Main Results:
- DISCOVER-Seq+ identified up to fivefold more CRISPR off-target sites compared to existing methods.
- Demonstrated successful application in ex vivo T cell receptor knock-in and in vivo gene knockout of PCSK9.
- Validated the technique's sensitivity across diverse cell types and in vivo models.
Conclusions:
- DISCOVER-Seq+ represents a significant advancement in detecting off-target genome editing.
- The method enhances the safety evaluation of CRISPR-Cas applications in preclinical and clinical settings.
- This technique is the most sensitive method to date for discovering in vivo off-target genome editing.
