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.
Highlights
The double-strand break (DSB) repair machinery consists of multiple pathways, including nonhomologous end-joining (NHEJ), homologous recombination (HR) and microhomology-mediated end-joining (MMEJ) (McVey and Lee, 2008; Iliakis et al, 2015; Chang et al, 2017; Scully et al, 2019)
double-strand breaks (DSBs)-TRIP works by random integration of a specially designed DSB repair pathway reporter into hundreds or thousands of genomic locations in a pool of cells, by means of a transposon vector
A key feature of DSB-TRIP is that an identical reporter sequence is integrated into many different chromatin environments
Summary
The double-strand break (DSB) repair machinery consists of multiple pathways, including nonhomologous end-joining (NHEJ), homologous recombination (HR) and microhomology-mediated end-joining (MMEJ) (McVey and Lee, 2008; Iliakis et al, 2015; Chang et al, 2017; Scully et al, 2019). These studies used methods ranging from using single imprinted endogenous loci (Kallimasioti-Pazi et al, 2018), single transgenic loci (Lemaitre et al, 2014), hundreds of endogenous loci (Iacovoni et al, 2010; Massip et al, 2010; Aymard et al, 2014) to thousands of integrated reporters as presented here (Gisler et al, 2019; Pokusaeva et al, 2021; Schep et al, 2021). The enzyme is fused to a ligand-inducible domain for controlled nuclear localization and can reliably create ∼150 endogenous breaks in U2OS cells in an inducible manner This method can accurately measure differences in repair pathway choice between transcribed and non-transcribed regions. The design of DSB-TRIP effectively rules out confounding effects of surrounding DNA sequences
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
