Title: Leveraging DNA Damage Response Pathways to Enhance the Precision of CRISPR-Mediated Genome Editing
Description: The ability to efficiently and precisely modify the genome of living cells forms the basis of genetic studies and offers great potential to research and therapy. With its unprecedented ease of use and efficiency, CRISPR-Cas9 has revolutionized genome editing at a stunning pace. Functioning like a pair of molecular scissors, the RNA-guided endonuclease Cas9 can cleave genomic DNA to generate double-stranded breaks (DSBs). DSBs trigger the DNA damage response (DDR), that sets into motion multiple cellular processes that attempt to repair these lesions. One such cellular pathway, named homology-directed repair (HDR), enables researchers to make desirable changes precisely to genomic DNA sequences. HDR facilitates nearly any genomic DNA change, from the replacement of a single nucleotide to the insertion of several thousands of nucleotides. Thus, the precision, as well as versatility at modifying genomic DNA, make HDR a particularly promising repair pathway for genome editing. However, competition with other error-prone DSB repair pathways reduces the efficiency of HDR and results in the generation of an excess of undesirable mutations.

In this thesis, I address these two challenges associated with CRISPR-Cas9 genome editing: i) low efficiency of HDR and ii) large deletion mutations generated upon repair of Cas9-induced DSBs. The first part of the thesis describes our study to identify genetic factors that stimulate HDR at Cas9 induced DSBs. Towards this goal, we individually express in human cells 204 open reading frames involved in the DDR and determine their impact on CRISPR-mediated HDR. From these studies, we identify RAD18 as a stimulator of CRISPR-mediated HDR. By defining the RAD18 domains required to promote HDR, we derive an enhanced RAD18 variant (e18) that stimulates HDR induced by CRISPR-Cas9 in multiple human cell types, including embryonic stem cells. Mechanistically, e18 suppresses the localization of the HDR-inhibiting factor 53BP1 to DSBs. Through this suppression of 53BP1, e18 promotes HDR at the expense of insertion and deletion mutations introduced by error-prone DSB repair pathways. Altogether, this study identifies e18 as an enhancer of CRISPR-mediated HDR and highlights the promise of engineering DDR factors to augment the efficiency of precision genome editing.

In the second part of the thesis I describe our study of the genetic mechanisms regulating large deletions that are generated upon repair of Cas9-induced DSBs. We perform a pooled CRISPR screen to interrogate the effect of knocking out 610 DDR genes on the frequency of CRISPR-mediated long deletions. The screen identifies genes that consistently affect the frequency of long deletions when knocked-out in different experimental conditions. Thus, our study lays the foundations for uncovering the mechanisms regulating CRISPR-mediated long deletions and has the potential to aid in the development of new strategies to limit their generation.