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

ABSTRACT 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.