PhD defence R.J.A. (Ruben) Schep

Summary:

This thesis aimed to investigate the impact of chromatin on DNA repair pathway balance and CRISPR-Cas9 editing efficiency. To achieve this, a reporter sequence consisting of a target sequence for double strand break (DSB) formation with CRISPR-Cas9 and a unique barcode was used. These reporter sequences were randomly inserted into the genome of human cells, and each reporter was mapped to its corresponding genomic coordinate. By integrating publicly available chromatin data, we established associations between the chromatin environment and the reporters. The formation of DSBs allowed us to quantify both the overall mutations and the mutations specific to non-homologous end joining (NHEJ) and microhomology mediated end joining (MMEJ) repair pathways, the two primary DSB repair mechanisms. Analyzing the data in the context of chromatin revealed a shift towards MMEJ in heterochromatin, an inactive form of chromatin. Furthermore, we confirmed previous findings by demonstrating a reduction in total mutation frequency within heterochromatin. Additionally, we conducted an extensive drug screening to explore the interplay between drugs and chromatin in the context of DNA repair and CRISPR-Cas9 editing. Numerous drugs showed susceptibility to various chromatin states, and we identified a few potential candidates that could enhance Cas9 editing. Lastly, we investigated the kinetics of the two main repair pathways and Cas9, which revealed slower processes within heterochromatin.