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Updated: Jul 5, 2026
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Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter
Published on: March 31, 2022
iMUT-seq mapping of DSB-induced mutations with high sensitivity at single-nucleotide resolution.
Aldo S Bader1,2, Martin Bushell3,4
1Cancer Research UK Scotland Institute, Glasgow, UK. ab2510@cam.ac.uk.
Nature Protocols
|July 3, 2026
Summary
We developed iMUT-seq, a high-sensitivity DNA sequencing method to precisely detect mutations caused by DNA double-strand breaks (DSBs). This technique offers new insights into disease-related mutagenesis and DNA repair mechanisms.
Area of Science:
- Molecular Biology
- Genetics
- Genomics
Background:
- DNA double-strand breaks (DSBs) are significant contributors to human diseases.
- Current methods for quantifying DSB-induced mutations lack sensitivity.
- Accurate profiling of these mutations is crucial for understanding disease pathogenesis.
Purpose of the Study:
- To introduce iMUT-seq, a novel, high-sensitivity technique for profiling mutations induced by DSBs.
- To enable the detection of mutations at very low incidence rates around endogenous DSBs.
- To facilitate the identification of novel mutagenic outcomes and mechanisms.
Main Methods:
- iMUT-seq couples high-depth sequencing with restriction enzyme-inducible DSBs in human cell lines.
- Genomic DNA is extracted, followed by multiplexed PCR and library preparation for Illumina sequencing.
- A specialized analytical pipeline is employed for per-nucleotide data analysis.
Main Results:
- iMUT-seq achieves high sensitivity, detecting mutations at rates as low as 1 in 500,000.
- The technique provides single-nucleotide resolution around DSBs.
- It allows for the characterization of previously unknown mutagenesis mechanisms.
Conclusions:
- iMUT-seq is a powerful tool for advancing the study of DSB mutagenesis.
- It offers potential for discovering novel DNA repair components.
- The method is adaptable to other organisms and DNA damage types.
