Systematic optimization of prime editing for enhanced efficiency and versatility in genome engineering across diverse cell types

• DOI: 10.3389/fcell.2025.1589034
• Publication Date: 2025-04-29T05:25:39Z
• Authors: Huiling Mu; Yeyi Liu; Yijia Chi; Fei Wang; Shuting Meng; Yi Zhang; Xunting Wang; Dongxin Zhao
• Keywords: Genome Engineering

Prime editing offers remarkable versatility in genome editing, but its efficiency remains a major bottleneck. While continuous optimization of the prime enzymes and guide RNAs (pegRNAs) has improved outcomes, method delivery also plays crucial role overall performance. To maximize efficiency, we implemented series systematic optimizations, achieving up to 80% across multiple loci cell lines. Beyond integrating latest advancements our approach combined stable genomic integration editors via piggyBac transposon system, selection integrated single clones, use an enhanced promoter, lentiviral pegRNAs, ensuring robust, ubiquitous, sustained expression both pegRNAs. further assess efficacy challenging types, validated optimized system human pluripotent stem cells (hPSCs) primed naïve states, substantial efficiencies 50%. Collectively, strategy provides highly efficient versatile framework for engineering vitro, serving as roadmap refining technologies expanding their applications genetic research therapeutic development.