Baeza Centurión, Pablo, 1989-Miñana Gómez, BelénFaure, Andre J.Thompson, MikeBonnal, SophieQuarantani, GioiaClarke, JosephLehner, Ben, 1978-Valcárcel, J. (Juan)2025-10-152025-10-152025Baeza-Centurión P, Miñana B, Faure AJ, Thompson M, Bonnal S, Quarantani G, et al. Deep indel mutagenesis reveals the regulatory and modulatory architecture of alternative exon splicing. Nat Commun. 2025 Aug 30;16(1):8117. DOI: 10.1038/s41467-025-62957-72041-1723http://hdl.handle.net/10230/71517While altered pre-mRNA splicing is a frequent mechanism by which genetic variants cause disease, the regulatory architecture of human exons remains poorly understood. Antisense oligonucleotides (AONs) that target pre-mRNA splicing have been approved as therapeutics for various pathologies including patient-customised treatments for rare diseases, but AON discovery is currently slow and expensive, limiting the wider adoption of the approach. Here we show that deep indel mutagenesis (DIM) -which can be made experimentally at very low cost - provides an efficient strategy to chart the regulatory landscape of human exons and rapidly identify candidate splicing-modulating oligonucleotides. DIM reveals autonomous effects of insertions, while systematic deletion scans delineate the checkerboard architecture of sequential enhancers and silencers in a model alternative exon. The results also suggest a mechanism for repression of transmembrane domain-encoding exons and for the generation of microexons. Leveraging deep learning tools, we provide a resource, DANGO, that predicts the splicing regulatory landscape of all human exons and can help to identify effective splicing-modulating antisense oligonucleotides.application/pdfeng© The Author(s) 2025. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.info:eu-repo/semantics/articlehttp://dx.doi.org/10.1038/s41467-025-62957-7GeneticsRNA splicinginfo:eu-repo/semantics/openAccess