Chromatin context-dependent regulation and modulation of prime editing [sci-RNA-seq3]

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. A pooled DNA damage response (DDR) gene-focused shRNA screen in single cells was carried out to analyze the interaction between the cis-chromatin and trans-acting regulatory factors of prime editing. Briefly, K562 PE2-Puro cells with multiple randomly integrated synHEK3 reporters were transduced with a lentiviral shRNA library (driven by a tetracycline-controlled promoter) containing 304 hairpins targeting 76 DDR-related genes. We treated the cells with Doxycycline for 2 days to fully induce knockdown effects. We then transfected pegRNAs introducing random 6-bp insertions to the synHEK3 reporters. After 3-4 days, cells were profiled using a modified version of sci-RNA-seq3 (Martin et al., 2022). In particular, a T7 in situ transcription step was used to drive expression of the synHEK3 reporter in fixed, permeabilized nuclei, such that the cellular transcriptome, the perturbation molecules (shRNAs), and prime editing outcomes recorded in synHEK3 reporters can be co-profiled. The nuclei underwent three levels of split-pool barcoding and three types of libraries were generated: the sci-RNA-seq3 transcriptome library, the synHEK3 capture library, and the shRNA capture library.