Protocol: A Multiplexed Reporter Assay to Study Effects of Chromatin Context on DNA Double-Strand Break Repair

Area of Science:

• Molecular Biology
• Genetics
• Genomics

Background:

• DNA double-strand breaks (DSBs) are critical DNA lesions repaired by diverse pathways.
• Understanding DSB repair pathway regulation is vital for cancer research and gene editing.
• The influence of the local chromatin environment on DSB repair pathway choice remains poorly understood.

Purpose of the Study:

• To present a detailed protocol for DSB-TRIP (DNA double-strand break repair pathway) analysis.
• To enable the study of DSB repair pathway usage across the genome.
• To investigate the relationship between chromatin state and DSB repair pathway selection.

Main Methods:

• DSB-TRIP involves random integration of a repair reporter into multiple genomic locations.
• DSBs are induced within the reporter, generating unique DNA scars upon repair.
• Multiplexed sequencing of scars quantifies repair pathway usage at each integration site.

Main Results:

• The study provides a step-by-step protocol for DSB-TRIP in K562 cells.
• A dedicated computational pipeline is presented for data analysis.
• The technique allows linking repair pathway balance to the local chromatin state.

Conclusions:

• DSB-TRIP is a powerful technique for studying genome-wide DSB repair pathway usage.
• The method facilitates understanding the impact of chromatin environment on DNA repair.
• Potential applications include further research into DNA repair mechanisms and optimization of gene editing tools.