Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance

In this project we used a combination of TRIP and CRISPR/Cas9 to study the effect of chromatin on DNA double strand break repair. A barcoded piggyBac transposon carrying the target cutting site for Cas9 was randomly integrated in the genome. We derived two pools of cells with integrated pathway reporters (IPRs) as well as multiple clones. The integration sites were mapped using two techniques, inverted PCR (Akhtar et al. 2014) and Tagmentation based mapping (Stern et al. 2017). The mutations arising from the Cas9 cuts were our readout and were amplified using a simple PCR encompassing the target site as well as a barcode that is unique for a specific integration. The timepoints of collection for the experiments were either 64 or 72 hours after Cas9 induction for the pool and clonal experiments in various conditions all with at least 2 replicates. We have also done timeseries of the accumulation of indels from 0 to 69 hours (24 time points) in a clone with two different conditions and two replicates.