Agro-STARR-seq enables in planta genome-wide enhancer discovery and AI-empowerd enhancer design

Bao, Y.; Zhang, X.; Song, Y.; Li, H.; Qi, J.; Cui, H.; Li, N.; Chen, Y.; Zheng, W.; Xu, L.; Zhang, Y.; Chen, C.; Zhu, F.; Zhou, S.; Li, Z.; Liu, Y.

doi:10.1101/2025.10.10.681754

bioRxiv · 10.1101/2025.10.10.681754

Agro-STARR-seq enables in planta genome-wide enhancer discovery and AI-empowerd enhancer design

Bao, Y.·Zhang, X.·Song, Y.·Li, H.·Qi, J.·Cui, H.·Li, N.·Chen, Y.·Zheng, W.·Xu, L.·Zhang, Y.·Chen, C.·Zhu, F.·Zhou, S.·Li, Z.·Liu, Y.

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Abstract

Comprehensive enhancer screening is essential for dissecting gene regulatory networks. However, methods for such screening remain limited in plants. We present Agro-STARR-seq, an in planta enhancer screening platform using Agrobacterium-mediated delivery of STARR-seq libraries into Nicotiana benthamiana leaves. In the Arabidopsis genome, we identified 14,715 sequences with enhancer activity, of which 91.59% were located outside of open chromatin regions. This largest in planta enhancer dataset to date enabled AI-assisted design of highly active synthetic enhancers. For the large N. benthamiana genome, we pre-processed the genomic DNA with an Assay for Transposase Accessible Chromatin (ATAC) procedure before the Agro-STARR-seq protocol,and found that only one-third of open chromatin exhibited enhancer activity, 76.00% of which showed no canonical H3K27ac histone marks. Footprinting analysis further enables systematic linkage of transcription factor (TF) binding to enhancer function. Overall, Agro-STARR-seq provides a novel platform for dissecting and engineering plant enhancers.

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Bao, Y., Zhang, X., Song, Y., Li, H., Qi, J., Cui, H., Li, N., Chen, Y., Zheng, W., Xu, L., Zhang, Y., Chen, C., Zhu, F., Zhou, S., Li, Z., Liu, Y.. 2025-10-11. Agro-STARR-seq enables in planta genome-wide enhancer discovery and AI-empowerd enhancer design. https://doi.org/10.1101/2025.10.10.681754

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