Configuration of adaptable template RNA architectures to unfold the editable space of a nuclease prime editor

The nuclease prime editor (PEn) combines double-strand break (DSB) induction with reverse transcription for editing. Recently, high-activity PEn forms (e.g. uPEn) have been developed via the concomitant application of DNA repair regulator(s). While the standard uPEn introduces edits only downstream of the nuclease-induced DNA break, we seek innovative designs to enable upstream-directed editing by re-configuring guide/template RNAs to drive prime edits into the target strand (TS), instead of the conventional non-TS. We first devise a dual-RNA uPEn strategy by supplementing a cleavage-competent sgRNA with an accessory template RNA for modifying target strand (ActRNA:t). Characterization of the dual-RNA system allows us to next develop a bifunctional target strand-programming pegRNA (tsp-pegRNA). Both the dual- and single-RNA upstream-modifying uPEn forms (versions 3.1/3.2) successfully drive diverse types of accurate edits into a panel of locations refractory to the standard uPEn and the latest nickase PE. Moreover, we provide insights on the role of uPEn's helper module (i.e. i53) in driving TS prime edits. Additional co-administration of a DNA-dependent protein kinase inhibitor with uPEn3.2 leads to further optimization of editing purities. Together, these advances transform uPEn into a highly applicable tool with much-expanded editable space, and lay a strong foundation for future development of PEn/PE platforms.