Chromatin context-dependent regulation and modulation of prime editing [amplicon]

Prime editing is a powerful means of introducing precise changes to specific locations in mammalian genomes. However, the widely varying efficiency of prime editing across target sites of interest has limited its adoption in the context of both basic research and clinical settings. Here, we set out to exhaustively characterize the impact of the cis-chromatin environment on prime editing efficiency. Utilizing a newly developed and highly sensitive method for mapping the genomic locations of a randomly integrated “sensor”, we identify specific epigenetic features that strongly correlate with the highly variable efficiency of prime editing across different genomic locations. Next, to assess the interaction of trans-acting factors with the cis-chromatin environment, we develop and apply a pooled genetic screening approach with which the impact of knocking down various DNA repair factors on prime editing efficiency can be stratified by cis-chromatin context. Finally, we demonstrate that we can dramatically modulate the efficiency of prime editing through epigenome editing, i.e. enhancing (or restricting) local chromatin accessibility in order to increase (or decrease) the efficiency of prime editing at a target site. Looking forward, we envision that the insights and tools described here will broaden the range of both basic research and therapeutic contexts in which prime editing is useful. We randomly integrated a complex library of the synHEK3 reporters into a monoclonal K562 cell line constitutively expressing the PE2 prime editor. We further bottlenecked the pool to ~500 clones, each containing a unique combination of randomly inserted reporters, and performed T7-assisted reporter mapping on this population (T7-500-1). We further performed amplicon sequencing of synHEK3 reporters to measure the outcomes of prime editing (K562-HEK3-insCTT) as well as Cas9 editing (K562-HEK3-Cas9) in this pool of synHEK3 reporters. A few monoclonal lines were also derived from the 500-clone population. Files named "K562-SC1-5-OFF*" contain prime editing efficiencies measured in synHEK3 reporters in one of the monoclonal lines (Clone 5) in the settings of targeted gene silencing by CRISPRoffv2.1. "K562-HEK3-SC*" experiments measured the effects of various shRNAs on prime editing efficiencies in synHEK3 reporters in two monoclonal lines. Finally, the rest files are amplicon sequencing results of endogenous prime editing targets in the presense and absense of CRISPRa.