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

Abstract

DNA double-strand breaks (DSBs) can be repaired through various pathways. Understanding how these pathways are regulated is of great interest for cancer research and optimization of gene editing. The local chromatin environment can affect the balance between repair pathways, but this is still poorly understood. Here we provide a detailed protocol for DSB-TRIP, a technique that utilizes the specific DNA scars left by DSB repair pathways to study pathway usage throughout the genome. DSB-TRIP randomly integrates a repair reporter into many genomic locations, followed by the induction of DSBs in the reporter. Multiplexed sequencing of the resulting scars at all integration sites then reveals the balance between several repair pathways, which can be linked to the local chromatin state of the integration sites. Here we present a step-by-step protocol to perform DSB-TRIP in K562 cells and to analyse the data by a dedicated computational pipeline. We discuss strengths and limitations of the technique, as well as potential additional applications to study DNA repair.

Widespread chromatin context-dependencies of DNA double-strand break repair proteins

“…7B ). We ran the CCD analysis using publicly available 10 chromatin tracks of RPE-1 cells (Supplementary Data 3 ) processed as described in 58 . A list of integration coordinates and chromatin features of all the IPRs assayed in RPE-1 cells can be found in Supplementary Data 4 .…”

Widespread chromatin context-dependencies of DNA double-strand break repair proteins

“…Scoring of indels and linking to their IPR barcodes in the sequence reads was done using a previously reported computational pipeline ( 46 ). In short, for each sequence the barcode was extracted and the indel state was classified.…”

Chromatin context-dependent effects of epigenetic drugs on CRISPR-Cas9 editing

“…All experiments were performed on the earlier established K562#17, which is a clonal cell line of the female K562 cells (ATCC) stably expressing DD-Cas9 (20,39). In this study, we used a heterogenous DSB-TRIP cell-line, harboring thousands of DSB reporters, and for the screening we selected a clone (clone #5) carrying 19 reporters (15,58). Both the clone and cell pools were cultured in tissue culture flasks in RPMI 1640 (GIBCO) supplemented with 10% fetal bovine serum (FBS, HyClone) and 1% penicillin/streptomycin.…”

“…For both screens, three separate cryovials of clone 5 cells were cultured separately for two weeks, one for each replicate. The TRIP cell pools were kept in culture for less than two weeks to avoid reporter drifting (58).…”

“…Analysis of the raw demultiplexed sequencing data was done using our previously reported computational pipeline (58). Briefly, indel size counts were generated for each integration of each well.…”