Investigating the Effect of Chromatin Environment on Susceptibility to Double Strand Breaks and Choice of Repair Mechanism

Double strand breaks are an extremely dangerous type of DNA damage. When a cell detects them they undergo apoptosis if it isn't repaired. If repaired improperly, mutations resulting in disease states can accumulate, so understanding the susceptibility and mechanism of DSB repair is important. While it is known how DSBs are repaired, it is unknown how the surrounding chromatin environment affects the DSB susceptibility and the choice of repair mechanism. The goal of this study is to compare DSB frequency and repair in regions with different chromatin environments around the genome. To induce DSBs, we first compared Galactose‐inducible and Tetracycline (tet)‐inducible CRISPR systems for cutting frequency, and found that the tet‐inducible system produces DSBs at a higher frequency in the time course examined. In our study, we will use this tet‐inducible system to measure DSB frequency at different genome sites as well as probe repair pathway choice, first comparing three cut sites near active genes and three near repressed genes. We first measured DSB frequency using quantitative PCR across break sites. Preliminary data shows that the repressed genes have higher levels of DSBs after 3 h of DSB induction. This suggests that the specific chromatin environment that surrounds the cut site that may contribute to the increased susceptibility to DSBs, and correlation to histone modifications is underway. Alternatively, the increased amount of DSBs may be due to a reduced repair efficiency in the repressed gene regions compared to the active gene regions, thus more double strand breaks were present at each time point. This a topic of ongoing investigation. The survivor analysis tests the ability of cells to become resistant to continuous DSB induction due to imprecise repair of the cut site and destruction of the Cas9‐guide RNA recognition site. Calculating percentage of survivor colonies can give an estimate of the mutation frequency of a genomic locus. Preliminary results suggest that active chromatin locations might generally have fewer survivors, but more data and genomic loci must be examined. Interestingly, a few active loci appear to be hypermutable. The TDH2 gene, for example, showed a multitude of inaccuracies when repairing after cutting, one to two orders of magnitude higher than other active genes, including its paralog, TDH3 . Sequencing of the imprecisely joined regions is underway to investigate this further. In addition, we will be determining if there is a correlation between specific histone modifications and mutation frequency. This work will help us better understand which locations in the genome are more susceptible to DSBs and to imprecise repair, and subsequent mutation, thus advancing our understanding of how DSB repair can drive genomic change. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .