Advances in prime editing: Molecular innovations, Large-fragment engineering, and AI-driven design.

Prime editing has rapidly emerged as a versatile and precise genome engineering technology capable of introducing targeted substitutions, insertions, and deletions without requiring double-strand breaks or donor DNA templates. Since its initial development, substantial progress has been achieved through iterative innovations in core components, including Cas protein variants, reverse transcriptase engineering, and prime editing guide RNA (pegRNA) design. These advancements have significantly improved editing efficiency, fidelity, and target accessibility across diverse biological systems. In parallel, the development of large-fragment editing platforms such as TwinPE, PASTE, and PrimeRoot has expanded the scope of prime editing from small sequence modifications to kilobase-scale genome rewriting. Furthermore, the integration of artificial intelligence-driven design tools has transformed pegRNA optimisation and system selection, enabling more predictable and efficient editing outcomes. This review provides a comprehensive overview of recent molecular innovations, large-fragment editing strategies, and computational approaches shaping the evolution of prime editing. Collectively, these developments position prime editing as a highly programmable and scalable platform with broad applications in medicine, agriculture, and synthetic biology.