From Natural Discovery to AI‐Guided Design: A Curated Collection of Compact Enhancers for Crop Engineering

ABSTRACT Precise transgene‐free gene upregulation remains a challenge in crop biotechnology, as conventional enhancers often exceed CRISPR‐mediated knock‐in size constraints and face regulatory hurdles. Here we establish a foundational cross‐species resource of compact transcriptional enhancers developed via STEM‐seq, a high‐throughput screening platform that systematically evaluated 81 475 genomic elements across maize, wheat, tomato, and soybean. This screen identified 6904 natural short transcriptional enhancers (STEs; 60–80 bp) exhibiting a broad range of activation efficiencies, with the most potent elements derived from wheat (up to 46.3‐fold activation). Augmenting this resource, we developed BaseSearch, an AI‐driven design framework, which computationally generated 5000 synthetic STE candidates and achieved a 9.1% success rate (11.4× higher than genome‐wide screening). This set included ten ultra‐potent enhancers outperforming natural counterparts by 2.27‐fold (64.5‐fold vs. 28.4‐fold activation). Notably, the compact size of these STEs aligns with regulatory frameworks that favor endogenous sequence lengths, offering potential pathways for policy‐compatible precision breeding. This integrated platform provides a substantial collection of functionally validated enhancers for crops, supplying the research community with immediately applicable elements for engineering agronomic traits while advancing the fundamental understanding of plant cis ‐regulation.