Abstract

Precise and efficient replacement of large genomic DNA segments without inducing double-strand breaks (DSBs) remains a central challenge in genome engineering. Traditional homologous recombination relies on DSBs and long homologous arms, yet it remains inefficient, while recombinase or integrase systems suffer from residual sequences at integration sites. Prime editing (PE), limited by the processivity of reverse transcriptase, struggles to integrate large fragments (>100 bp). To address this challenge, we introduce Prime Editing–Microhomology-Enabled Replacement (PREMIER), a DSB-free platform by installing single-stranded microhomology arms at donor and genomic junctions via PE. In cell lines, PREMIER achieved a mean efficiency of 63.4% (median 65.2%) in diverse target sites, with peak efficiencies reaching 85.9%, exceeding homology-directed repair by 10–20-fold and reducing off-target integrations by over 100-fold compared to nonhomologous end joining. It bypasses the need for long homology arms, simplifies donor preparation, achieves targeted replacement of sequences up to 10.3 kb. In vivo , PREMIER integrates a 6.2-kb oncogene cassette into the mouse liver. Additionally, PREMIER replaces murine  Trp53 with human TP53 CDS, generating functional humanized mice. Altogether, PREMIER provides a precise, high-efficiency, and DSB-free strategy for large-scale genome rewriting, offering a powerful tool for complex modeling and therapeutic genome editing.