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

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. Overall design: Bulk RNA sequencing of K562 cells (wild-type) and cells treated with CRISPRoff. For the CRISPRoff experiment, cells were transiently transfected with CRISPRoff-v2.1 and paired gRNA targeting gene promoters. After FACS sorting and expansion of the transfected cells, cells' total RNAs were extracted and used for bulk RNA sequencing. Differential gene expression analyses were performed between promoter-targeting gRNA (USP7, METTL2A, and LRRC8C) treated cells versus cells treated with non-targeting control gRNAs.