Rewriting regulatory DNA to dissect and reprogram gene expression

Gabriella E Martyn¹, Michael T Montgomery¹, Hank Jones¹, Katherine Guo¹, Benjamin R Doughty², Johannes Linder³, Deepa Bisht⁴, Fan Xia¹, Xiangmeng S Cai⁵, Ziwei Chen⁶, Kelly Cochran⁶, Kathryn A Lawrence², Glen Munson⁷, Anusri Pampari⁶, Charles P Fulco⁸, Nidhi Sahni⁹, David R Kelley³, Eric S Lander¹⁰, Anshul Kundaje¹¹, Jesse M Engreitz¹²

Affiliations

¹ Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA; Basic Science and Engineering Initiative, Stanford Children's Health, Betty Irene Moore Children's Heart Center, Stanford, CA 94305, USA.
² Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA.
³ Calico Life Sciences LLC, South San Francisco, CA 94080, USA.
⁴ Department of Genitourinary Medical Oncology, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Houston, TX 77230, USA.
⁵ Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA; Basic Science and Engineering Initiative, Stanford Children's Health, Betty Irene Moore Children's Heart Center, Stanford, CA 94305, USA; Department of Bioengineering, Stanford University, Stanford, CA 94305, USA.
⁶ Department of Computer Science, Stanford University, Stanford, CA 94305, USA.
⁷ Novo Nordisk Foundation Center for Genomic Mechanisms of Disease, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Gene Regulation Observatory, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
⁸ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
⁹ Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Houston, TX 77230, USA; Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77230, USA; Quantitative and Computational Biosciences Program, Baylor College of Medicine, Houston, TX 77030, USA.
¹⁰ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Biology, MIT, Cambridge, MA 02139, USA; Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
¹¹ Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Computer Science, Stanford University, Stanford, CA 94305, USA.
¹² Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA; Basic Science and Engineering Initiative, Stanford Children's Health, Betty Irene Moore Children's Heart Center, Stanford, CA 94305, USA; Novo Nordisk Foundation Center for Genomic Mechanisms of Disease, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Gene Regulation Observatory, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Stanford Cardiovascular Institute, Stanford University, Stanford, CA 94305, USA. Electronic address: engreitz@stanford.edu.

PMID: 40245860
PMCID: PMC12167154 (available on 2026-06-12)
DOI: 10.1016/j.cell.2025.03.034

Rewriting regulatory DNA to dissect and reprogram gene expression

Gabriella E Martyn et al. Cell. 2025.

. 2025 Jun 12;188(12):3349-3366.e23.

doi: 10.1016/j.cell.2025.03.034. Epub 2025 Apr 16.

Authors

Affiliations

¹ Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA; Basic Science and Engineering Initiative, Stanford Children's Health, Betty Irene Moore Children's Heart Center, Stanford, CA 94305, USA.
² Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA.
³ Calico Life Sciences LLC, South San Francisco, CA 94080, USA.
⁴ Department of Genitourinary Medical Oncology, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Houston, TX 77230, USA.
⁵ Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA; Basic Science and Engineering Initiative, Stanford Children's Health, Betty Irene Moore Children's Heart Center, Stanford, CA 94305, USA; Department of Bioengineering, Stanford University, Stanford, CA 94305, USA.
⁶ Department of Computer Science, Stanford University, Stanford, CA 94305, USA.
⁷ Novo Nordisk Foundation Center for Genomic Mechanisms of Disease, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Gene Regulation Observatory, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
⁸ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
⁹ Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Houston, TX 77230, USA; Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77230, USA; Quantitative and Computational Biosciences Program, Baylor College of Medicine, Houston, TX 77030, USA.
¹⁰ Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Biology, MIT, Cambridge, MA 02139, USA; Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
¹¹ Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Computer Science, Stanford University, Stanford, CA 94305, USA.
¹² Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA; Basic Science and Engineering Initiative, Stanford Children's Health, Betty Irene Moore Children's Heart Center, Stanford, CA 94305, USA; Novo Nordisk Foundation Center for Genomic Mechanisms of Disease, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Gene Regulation Observatory, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Stanford Cardiovascular Institute, Stanford University, Stanford, CA 94305, USA. Electronic address: engreitz@stanford.edu.

PMID: 40245860
PMCID: PMC12167154 (available on 2026-06-12)
DOI: 10.1016/j.cell.2025.03.034

Abstract

Regulatory DNA provides a platform for transcription factor binding to encode cell-type-specific patterns of gene expression. However, the effects and programmability of regulatory DNA sequences remain difficult to map or predict. Here, we develop variant effects from flow-sorting experiments with CRISPR targeting screens (Variant-EFFECTS) to introduce hundreds of designed edits to endogenous regulatory DNA and quantify their effects on gene expression. We systematically dissect and reprogram 3 regulatory elements for 2 genes in 2 cell types. These data reveal endogenous binding sites with effects specific to genomic context, transcription factor motifs with cell-type-specific activities, and limitations of computational models for predicting the effect sizes of variants. We identify small edits that can tune gene expression over a large dynamic range, suggesting new possibilities for prime-editing-based therapeutics targeting regulatory DNA. Variant-EFFECTS provides a generalizable tool to dissect regulatory DNA and to identify genome editing reagents that tune gene expression in an endogenous context.

Keywords: CRISPR; RNA FlowFISH; enhancers; gene regulation; high-throughput screening; non-coding variants; predictive models; prime editing; sequence design; transcription factors.

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Conflict of interest statement

Declaration of interests J.M.E. is a consultant and equity holder in Martingale Labs, Inc.; has received materials from 10× Genomics unrelated to this study; and has received speaking honoraria from GSK plc and Roche Genentech. J.L. and D.R.K. are employed by Calico Life Sciences LLC. C.P.F. is employed by Sanofi. A.K. is on the scientific advisory board of PatchBio, SerImmune, and OpenTargets; was a consultant with Illumina; and owns shares in DeepGenomics, ImmunAI, and Freenome. M.T.M., G.E.M., B.R.D., H.J., K.G., and J.M.E. are inventors on a provisional patent application related to this work.

Update of

Rewriting regulatory DNA to dissect and reprogram gene expression.
Martyn GE, Montgomery MT, Jones H, Guo K, Doughty BR, Linder J, Chen Z, Cochran K, Lawrence KA, Munson G, Pampari A, Fulco CP, Kelley DR, Lander ES, Kundaje A, Engreitz JM. Martyn GE, et al. bioRxiv [Preprint]. 2023 Dec 21:2023.12.20.572268. doi: 10.1101/2023.12.20.572268. bioRxiv. 2023. Update in: Cell. 2025 Jun 12;188(12):3349-3366.e23. doi: 10.1016/j.cell.2025.03.034. PMID: 38187584 Free PMC article. Updated. Preprint.

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Rewriting regulatory DNA to dissect and reprogram gene expression

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Rewriting regulatory DNA to dissect and reprogram gene expression

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