PARP1 Constrains Insertional Prime Editing in a Context-Dependent Manner

Prime editing (PE) enables precise insertions, deletions, and substitutions without double-strand breaks, but editing efficiency remains highly variable. To identify modulators of PE outcomes, we screened 569 compounds targeting DNA damage and repair pathways in K562 cells using targeted next-generation sequencing. PARP inhibitors selectively enhanced PE-mediated insertions at the HEK3 locus by more than 2-fold, without improving substitution or deletion editing. Genetic loss of PARP1 reproduced this phenotype, identifying PARP1 as the principal mediator. PARP1 inhibition enhanced insertion efficiency across multiple PE architectures, insertion sizes, and cellular backgrounds, including primary human T cells, and promoted more complete incorporation of pegRNA-encoded sequence changes without increasing indels or scaffold incorporation. However, this effect was dependent on genomic locus and reverse transcription template configuration. Together, these findings identify PARP1 as a context-dependent constraint on insertional PE.