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Multilevel characterization of genome editor nuclease activity with BreakTag
Gabriel M C Longo1,2,3,4, Sergi Sayols5, Vassilis Roukos6,7
1Institute of Molecular Biology, Mainz, Germany. gmellodacunhalongo@mgh.harvard.edu.
Nature Protocols
|November 19, 2025
Summary
BreakTag is a novel sequencing method for unbiased characterization of gene editing nucleases and guide RNAs. It enables precise assessment of nuclease activity, specificity, and DNA break profiles for improved CRISPR applications.
Area of Science:
- Molecular Biology
- Genomics
- Bioinformatics
Background:
- CRISPR-Cas gene editing requires precise characterization of nuclease activity and specificity.
- Existing methods may lack the scalability or unbiased nature needed for comprehensive analysis.
- Understanding DNA double-strand break profiles is crucial for predicting gene editing outcomes.
Purpose of the Study:
- To introduce BreakTag, a scalable next-generation sequencing-based method for unbiased characterization of programmable nucleases and guide RNAs.
- To enable high-throughput assessment of nuclease activity, specificity, and scission profiles.
- To develop computational tools (BreakInspectoR and XGScission) for data analysis and prediction of cleavage patterns.
Main Methods:
- BreakTag involves Cas9/guide RNA digestion of genomic DNA, enrichment of double-strand breaks, and next-generation sequencing.
- BreakInspectoR software facilitates high-throughput analysis of nuclease activity, specificity, and scission profiles.
- XGScission, a machine learning model, predicts double-strand break types (blunt/staggered) and aids in target sequence preselection.
- HiPlex enables pooled generation of thousands of single guide RNAs for robust BreakTag datasets.
Main Results:
- BreakTag provides unbiased, multi-level characterization of programmable nucleases and guide RNAs.
- The method accurately assesses nuclease activity, off-target effects, and detailed scission profiles.
- BreakInspectoR and XGScission enable efficient data analysis and prediction of cleavage outcomes.
- The entire BreakTag workflow, including analysis, can be completed in approximately 3 days.
Conclusions:
- BreakTag is a powerful, scalable tool for comprehensive characterization of gene editing nucleases.
- The associated computational tools enhance the utility of BreakTag for CRISPR research and development.
- BreakTag facilitates the optimization of gene editing strategies by providing mechanistic insights into nuclease function and DNA repair outcomes.
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