Qi Yao, Jin Gao, Keling Wang, Yufeng Liu, Peijin Han et al.
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.
Qi Yao, Jin Gao, Keling Wang, Yufeng Liu, Peijin Han, Hong Pan, Xiaofeng Yang, Qianlan Yin, Dating Zhong, Lu Ye, Qi Deng, Lingling Gao, Xiaoyu Tu, Dequan Wang, Yuming Lu. From Natural Discovery to AI‐Guided Design: A Curated Collection of Compact Enhancers for Crop Engineering. 2026-01-04. https://doi.org/10.1002/advs.202516600
An 80-bp natural short transcriptional enhancer (STE) library was assayed in Solanum lycopersicum (tomato) using the STEM-seq plasmid reporter with the EF1apro context. The table contains the authors’ two-replicate RNA/DNA-normalized activity measurements and their replicate-combined statistics.
An 80-bp natural short transcriptional enhancer (STE) library was assayed in Triticum aestivum (wheat) using the STEM-seq plasmid reporter with the UBIpro context. The table contains the authors’ two-replicate RNA/DNA-normalized activity measurements and their replicate-combined statistics.
An 80-bp natural short transcriptional enhancer (STE) library was assayed in Triticum aestivum (wheat) using the STEM-seq plasmid reporter with the 35Sminipro context. The table contains the authors’ two-replicate RNA/DNA-normalized activity measurements and their replicate-combined statistics.
An 80-bp natural short transcriptional enhancer (STE) library was assayed in Glycine max (soybean) using the STEM-seq plasmid reporter with the EF1apro context. The table contains the authors’ two-replicate RNA/DNA-normalized activity measurements and their replicate-combined statistics.
A 60-bp AI-designed synthetic enhancer library was tested in maize protoplasts with the pSTEM02 35S minimal-promoter STEM-seq reporter. The table contains the measured synthetic records provided in Supplementary Sheet 11, including BaseSearch predictions and two-replicate activity values.
An 80-bp natural short transcriptional enhancer (STE) library was assayed in Solanum lycopersicum (tomato) using the STEM-seq plasmid reporter with the 35Sminipro context. The table contains the authors’ two-replicate RNA/DNA-normalized activity measurements and their replicate-combined statistics.
An 80-bp natural short transcriptional enhancer (STE) library was assayed in Glycine max (soybean) using the STEM-seq plasmid reporter with the 35Sminipro context. The table contains the authors’ two-replicate RNA/DNA-normalized activity measurements and their replicate-combined statistics.
An 80-bp natural short transcriptional enhancer (STE) library was assayed in Zea mays (maize) using the STEM-seq plasmid reporter with the UBIpro context. The table contains the authors’ two-replicate RNA/DNA-normalized activity measurements and their replicate-combined statistics.
An 80-bp natural short transcriptional enhancer (STE) library was assayed in Zea mays (maize) using the STEM-seq plasmid reporter with the 35Sminipro context. The table contains the authors’ two-replicate RNA/DNA-normalized activity measurements and their replicate-combined statistics.
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