The Impact of Chromatin Structure on the Formation of Double Strand Breaks by CRISPR‐CAS9 in the Yeast Genome

Abstract

DNA is persistently at risk for being damaged by endogenous and exogenous factors. One type of DNA damage is a double‐strand break (DSB), which occurs when both strands of the helix become severed. DSBs are important because upon repair, they could lead to mutations or genomic alteration. The packaging of the DNA into chromatin may affect the susceptibility of an area to DSBs and thus could make the area more prone to mutation. The purpose of this study is to look at how the chromatin environment affects the susceptibility of genes to DSBs in Saccharomyces cerevisiae. A new system, tetracycline‐inducible CRISPR‐Cas9, was used in this experiment to induce DSBs at different sites in the yeast genome Histone modification levels were measured at each site to correlate these chromatin marks with DSB formation frequency. DSB frequency at each genomic locus was measured using quantitative PCR and directly compared to those same sites in unchromatinized DNA to see how chromatin environment and the surrounding histone modifications affects susceptibility to DSBs.To measure the frequency of DSBs at different genomic sites using qPCR, the break site was amplified. DSBs would be indicated through a decrease in product when compared to an uncut control site NFS1 on Chromosome III. While correlation analysis of histone modifications and DSB frequency is still ongoing, preliminary data suggests that repressed genes have higher levels of cutting than active genes after three hours of DSB induction. Of the repressed genes investigated, PPE1 had 82.06% DSB formation and RRT8 had 66.67% after three hours of induction. Active genes RPL22A, TDH2, and TDH3 had on average about 15%, 9.5%, and 56% DSB formation after three hours of DSB induction, respectively. The level of DSB formation in these sites will be directly compared to DSB formation of the same sites on an unchromatinized plasmid, which is currently underway.This research is important because it shows which areas of the genome may be at higher risk for DSBs and whether or that is affected by chromatin structure. Understanding this could help deepen the knowledge of how the genome evolves through spontaneous DSB formation and repair.