Sager J. Gosai, Rodrigo I. Castro, Natalia Fuentes, John C. Butts, Kousuke Mouri et al.
Cis-regulatory elements (CREs) control gene expression, orchestrating tissue identity, developmental timing and stimulus responses, which collectively define the thousands of unique cell types in the body. While there is great potential for strategically incorporating CREs in therapeutic or biotechnology applications that require tissue specificity, there is no guarantee that an optimal CRE for these intended purposes has arisen naturally. Here we present a platform to engineer and validate synthetic CREs capable of driving gene expression with programmed cell-type specificity. We take advantage of innovations in deep neural network modelling of CRE activity across three cell types, efficient in silico optimization and massively parallel reporter assays to design and empirically test thousands of CREs. Through large-scale in vitro validation, we show that synthetic sequences are more effective at driving cell-type-specific expression in three cell lines compared with natural sequences from the human genome and achieve specificity in analogous tissues when tested in vivo. Synthetic sequences exhibit distinct motif vocabulary associated with activity in the on-target cell type and a simultaneous reduction in the activity of off-target cells. Together, we provide a generalizable framework to prospectively engineer CREs from massively parallel reporter assay models and demonstrate the required literacy to write fit-for-purpose regulatory code.
Sager J. Gosai, Rodrigo I. Castro, Natalia Fuentes, John C. Butts, Kousuke Mouri, Michael Alasoadura, Susan Kales, Thanh Thanh L. Nguyen, Ramil R. Noche, Arya S. Rao, Mary T. Joy, Pardis C. Sabeti, Steven K. Reilly, Ryan Tewhey. Machine-guided design of cell-type-targeting cis-regulatory elements. 2024-10-23. https://doi.org/10.1038/s41586-024-08070-z
A 200-bp Fast SeqProp-designed CRE was tested in SK-N-SH cells with all three alternate nucleotides at each position, producing 600 single-nucleotide substitutions plus the measured parent sequence. The processed table adds the variant's log2FC change relative to the parent sequence.
The 77,157-oligo CODA library of synthetic candidates, natural human CREs and experimental controls was tested in K562 cells using an episomal GFP reporter. The processed table reports K562 empirical activity and Malinois predictions after the publication-based reproducibility filter.
The 77,157-oligo CODA library of synthetic candidates, natural human CREs and experimental controls was tested in HepG2 cells using an episomal GFP reporter. The processed table reports HepG2 empirical activity and Malinois predictions after the publication-based reproducibility filter.
The 77,157-oligo CODA library of synthetic candidates, natural human CREs and experimental controls was tested in SK-N-SH cells using an episomal GFP reporter. The processed table reports SK-N-SH empirical activity and Malinois predictions after the publication-based reproducibility filter.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
Download all 6 files (ZIP)README.txtsupplementary_information.pdfsupplementary_table_10_saturation_mutagenesis.txtsupplementary_table_12_coda_mpra_results.txtsupplementary_table_2_malinois_training_mpra.txtsupplementary_table_4_coda_library_breakdown.xlsx