MAPPING THE CELLULAR DETERMINANTS OF GENOME EDITING

Abstract

Engineering of CRISPR-Cas systems has produced a suite of genome editing tools including programmable nucleases, base editors and prime editors. During my Ph.D., I studied how cellular processes regulate these tools and affect editing outcomes. Chapter 2 focuses on CRISPR nucleases capable of generating DNA double-strand breaks (DSBs) at target sequences. The DSBs are repaired by a complex multi-pathway repair network, leading to heterogenous outcomes. To systematically profile how DNA repair regulates these outcomes, we developed a CRISPR screening platform called Repair-seq; my part in this work was to characterize outcomes uncaptured by Repair-seq including large deletions. I discovered that Cas9-induced DSBs on lentiviral vectors can lead to large deletions through single-strand annealing. Chapter 3 focuses on prime editors which enable precise modifications of genomes through reverse transcription of template sequences appended to the 3′ ends of CRISPR–Cas guide RNAs. To identify cellular determinants of prime editing, I performed Repair-seq screens in K562 cells and identified DNA mismatch repair (MMR) as a major suppressor of small prime edits in those cells. Together with our collaborators, we then developed and validated a dominant negative MLH1 (MLH1dn) whose transient expression enhances prime editing by inhibiting MMR. Chapter 4 features genome-scale CRISPRi screens to identify additional prime editing determinants. A single factor emerged as the strongest mediator: the small RNA-binding exonuclease protection factor La. Further investigation revealed that La promotes prime editing across approaches, edit types, endogenous loci and cell types but has no consistent effect on genome-editing approaches that rely on standard, unextended guide RNAs. I found that La functionally interacts with and stabilizes the 3′ ends of polyuridylated prime editing guide RNAs (pegRNAs). Guided by these results, we developed a prime editor protein (PE7) fused to the RNA-binding, N-terminal domain of La. This editor improved prime editing with expressed pegRNAs and engineered pegRNAs (epegRNAs), as well as with synthetic pegRNAs optimized for La binding. Together, these studies provide key insights into how cellular environment regulates genome editing and suggest useful strategies for improvement.