Tuning Rice Gene Expression via In Situ Promoter Truncations Using a Prime Editing Library

Promoter engineering holds immense potential for fine-tuning gene expression and optimising agronomic traits, yet conventional genome-editing tools face limitations in precision, scalability, and risk mitigation. Here, we develop Prime Editing-mediated Promoter Engineering (PEPE), a DSB-free platform integrating bidirectional Protospacer adjacent motif (PAM) recognition (NGG/CCN) with combinatorial duo-pegRNA strategies to achieve tiled, overlapping deletions across entire plant promoters. Applying PEPE to the 1.8-kb rice D53 promoter, we generated a mutant library with stepwise deletions. Edited alleles showed stable inheritance, and dual-method validation confirmed the precision at junctions. Quantitative profiling revealed functional modularity: core-region deletions suppressed D53 expression by 70%-85%, while a distal deletion (D53-G9C10) paradoxically upregulated transcription 2.2-fold, uncovering a cryptic repressor element. Phenotypic variation corresponded with transcriptional changes, establishing a direct link between cis-regulatory diversity and agronomic traits. By circumventing DSBs and enabling kilobase-scale CRE manipulation, PEPE establishes a robust framework for decoding promoter logic and accelerating trait pyramiding in crops. This study advances plant genome editing by merging precision with scalability, offering transformative potential for breeding climate-resilient, high-yield cultivars.