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A robust CRISPR-Cas9-based fluorescent reporter assay for the detection and quantification of DNA double-strand break
Rebeka Eki1,2,3, Jane She1, Mahmut Parlak1
1Department of Radiation Oncology, University of Virginia, Charlottesville, VA 22908, USA.
Abstract:
DNA double-strand breaks (DSBs) are highly cytotoxic lesions that can lead to chromosome rearrangements, genomic instability and cell death. Consequently, cells have evolved multiple mechanisms to efficiently repair DSBs to preserve genomic integrity. We have developed a DSB repair assay system, designated CDDR (CRISPR-Cas9-based Dual-fluorescent DSB Repair), that enables the detection and quantification of DSB repair outcomes in mammalian cells with high precision. CDDR is based on the introduction and subsequent resolution of one or two DSB(s) in an intrachromosomal fluorescent reporter following the expression of Cas9 and sgRNAs targeting the reporter. CDDR can discriminate between high-fidelity (HF) and error-prone non-homologous end-joining (NHEJ), as well as between proximal and distal NHEJ repair. Furthermore, CDDR can detect homology-directed repair (HDR) with high sensitivity. Using CDDR, we found HF-NHEJ to be strictly dependent on DNA Ligase IV, XRCC4 and XLF, members of the canonical branch of NHEJ pathway (c-NHEJ). Loss of these genes also stimulated HDR, and promoted error-prone distal end-joining. Deletion of the DNA repair kinase ATM, on the other hand, stimulated HF-NHEJ and suppressed HDR. These findings demonstrate the utility of CDDR in characterizing the effect of repair factors and in elucidating the balance between competing DSB repair pathways.
Insights
A new assay, CRISPR-Cas9-based Dual-fluorescent DSB Repair (CDDR), precisely measures DNA double-strand break (DSB) repair. CDDR reveals that canonical NHEJ factors regulate repair pathway choice and balance.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- DNA double-strand breaks (DSBs) are critical DNA lesions that threaten genomic integrity.
- Cellular mechanisms have evolved to efficiently repair DSBs, preventing mutations and cell death.
- Understanding these repair pathways is crucial for comprehending genome stability.
Purpose of the Study:
- To develop a precise assay for detecting and quantifying DNA double-strand break repair outcomes in mammalian cells.
- To characterize the roles of key DNA repair factors in modulating DSB repair pathway choice.
- To elucidate the interplay between high-fidelity non-homologous end-joining (HF-NHEJ) and homology-directed repair (HDR).
Main Methods:
- Development of the CRISPR-Cas9-based Dual-fluorescent DSB Repair (CDDR) assay system.
- Introduction of DSBs into an intrachromosomal fluorescent reporter in mammalian cells.
- Quantification of DSB repair outcomes, distinguishing between HF-NHEJ, error-prone NHEJ, and HDR.
Main Results:
- CDDR accurately detects and quantifies various DSB repair pathways, including HF-NHEJ and HDR.
- HF-NHEJ is strictly dependent on canonical NHEJ factors (DNA Ligase IV, XRCC4, XLF).
- Loss of c-NHEJ factors promotes HDR and error-prone NHEJ, while ATM deletion stimulates HF-NHEJ and suppresses HDR.
Conclusions:
- The CDDR assay is a powerful tool for dissecting DSB repair mechanisms.
- Canonical NHEJ factors play a critical role in directing repair pathway preference.
- ATM kinase influences the balance between HF-NHEJ and HDR, highlighting pathway crosstalk.
