
Blog
Programmable promoter editing for precise control of transgene expression

Website design
Domingo, J. et al. Nonlinear transcriptional responses to gradual modulation of transcription factor dosage. Preprint at bioRxiv https://doi.org/10.1101/2024.03.01.582837, peer reviewed by eLife eeb.embo.org/doi/10.1101/2024.03.01.582837 (2025).
Wang, N. B. et al. Proliferation history and transcription factor levels drive direct conversion to motor neurons. Cell Syst. 16, 101205 (2025).
Article
CAS
PubMed
Google Scholar
Kueh, H. Y. et al. Positive feedback between PU.1 and the cell cycle controls myeloid differentiation. Science 341, 670–673 (2013).
Article
CAS
PubMed
PubMed Central
Google Scholar
Desai, R. V. et al. A DNA-repair pathway can affect transcriptional noise to promote cell fate transitions. Science 373, eabc6506 (2021).
Article
CAS
PubMed
PubMed Central
Google Scholar
Galloway, K. E., Franco, E. & Smolke, C. D. Dynamically reshaping signaling networks to program cell fate via genetic controllers. Science 341, 1235005 (2013).
Article
PubMed
PubMed Central
Google Scholar
Chen, J.-Y. et al. Multi-range ERK responses shape the proliferative trajectory of single cells following oncogene induction. Cell Rep. 42, 112252 (2023).
Article
CAS
PubMed
PubMed Central
Google Scholar
Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).
Article
CAS
PubMed
Google Scholar
Hong, M., Clubb, J. D. & Chen, Y. Y. Engineering CAR-T cells for next-generation cancer therapy. Cancer Cell 38, 473–488 (2020).
Article
CAS
PubMed
Google Scholar
Goglia, A. G. et al. A live-cell screen for altered Erk dynamics reveals principles of proliferative control. Cell Syst. 10, 240–253 (2020).
Article
CAS
PubMed
PubMed Central
Google Scholar
Li, H.-S. et al. Multidimensional control of therapeutic human cell function with synthetic gene circuits. Science 378, 1227–1234 (2022).
Article
CAS
PubMed
PubMed Central
Google Scholar
Collins, R. L. et al. A cross-disorder dosage sensitivity map of the human genome. Cell 185, 3041–3055 (2022).
Article
CAS
PubMed
PubMed Central
Google Scholar
Arita, Y. et al. A genome‐scale yeast library with inducible expression of individual genes. Mol. Syst. Biol. 17, e10207 (2021).
Article
CAS
PubMed
PubMed Central
Google Scholar
Chapman, S. A. & Asthagiri, A. R. Quantitative effect of scaffold abundance on signal propagation. Mol. Syst. Biol. 5, 313 (2009).
Article
PubMed
PubMed Central
Google Scholar
Legut, M. et al. A genome-scale screen for synthetic drivers of T cell proliferation. Nature 603, 728–735 (2022).
Article
CAS
PubMed
PubMed Central
Google Scholar
Eguchi, A. et al. Reprogramming cell fate with a genome-scale library of artificial transcription factors. Proc. Natl Acad. Sci. USA 113, E8257–E8266 (2016).
Article
CAS
PubMed
PubMed Central
Google Scholar
Veerapandian, V. et al. Directed evolution of reprogramming factors by cell selection and sequencing. Stem Cell Reports 11, 593–606 (2018).
Article
CAS
PubMed
PubMed Central
Google Scholar
Joung, J. et al. A transcription factor atlas of directed differentiation. Cell 186, 209–229 (2023).
Article
CAS
PubMed
PubMed Central
Google Scholar
Gao, X. J., Chong, L. S., Kim, M. S. & Elowitz, M. B. Programmable protein circuits in living cells. Science 361, 1252–1258 (2018).
Article
CAS
PubMed
PubMed Central
Google Scholar
Zhu, R., del Rio-Salgado, J. M., Garcia-Ojalvo, J. & Elowitz, M. B. Synthetic multistability in mammalian cells. Science 375, eabg9765 (2022).
Article
CAS
PubMed
Google Scholar
Elowitz, M. B. & Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature 403, 335–338 (2000).
Article
CAS
PubMed
Google Scholar
Gardner, T. S., Cantor, C. R. & Collins, J. J. Construction of a genetic toggle switch in Escherichia coli. Nature 403, 339–342 (2000).
Article
CAS
PubMed
Google Scholar
Danino, T., Mondragón-Palomino, O., Tsimring, L. & Hasty, J. A synchronized quorum of genetic clocks. Nature 463, 326–330 (2010).
Article
CAS
PubMed
PubMed Central
Google Scholar
Tabor, J. J. et al. A synthetic genetic edge detection program. Cell 137, 1272–1281 (2009).
Article
PubMed
PubMed Central
Google Scholar
Bashor, C. J., Helman, N. C., Yan, S. & Lim, W. A. Using engineered scaffold interactions to reshape MAP kinase pathway signaling dynamics. Science 319, 1539–1543 (2008).
Article
CAS
PubMed
Google Scholar
Ravindran, P. T., McFann, S., Thornton, R. H. & Toettcher, J. E. A synthetic gene circuit for imaging-free detection of signaling pulses. Cell Syst. 13, 131–142 (2022).
Article
CAS
PubMed
Google Scholar
Jones, R. D. et al. An endoribonuclease-based feedforward controller for decoupling resource-limited genetic modules in mammalian cells. Nat. Commun. 11, 5690 (2020).
Article
CAS
PubMed
PubMed Central
Google Scholar
Qian, Y., Huang, H.-H., Jiménez, J. I. & Del Vecchio, D. Resource competition shapes the response of genetic circuits. ACS Synth. Biol. 6, 1263–1272 (2017).
Article
CAS
PubMed
Google Scholar
Frei, T. et al. Characterization and mitigation of gene expression burden in mammalian cells. Nat. Commun. 11, 4641 (2020).
Article
CAS
PubMed
PubMed Central
Google Scholar
Ceroni, F. et al. Burden-driven feedback control of gene expression. Nat. Methods 15, 387–393 (2018).
Article
CAS
PubMed
Google Scholar
Di Blasi, R. et al. Resource-aware construct design in mammalian cells. Nat. Commun. 14, 3576 (2023).
Article
PubMed
PubMed Central
Google Scholar
Cabrera, A. et al. The sound of silence: transgene silencing in mammalian cell engineering. Cell Syst. 13, 950–973 (2022).
Article
PubMed
Google Scholar
Duran, A. G. et al. Limiting transactivator amounts contribute to transgene mosaicism in Tet-On all-in-one systems. ACS Synth. Biol. 11, 2623–2635 (2022).
Article
CAS
PubMed
Google Scholar
Heinz, N., Hennig, K. & Loew, R. Graded or threshold response of the tet-controlled gene expression: all depends on the concentration of the transactivator. BMC Biotechnol 13, 5 (2013).
Article
CAS
PubMed
PubMed Central
Google Scholar
Khalil, A. S. et al. A synthetic biology framework for programming eukaryotic transcription functions. Cell 150, 647–658 (2012).
Article
CAS
PubMed
PubMed Central
Google Scholar
Donahue, P. S. et al. The COMET toolkit for composing customizable genetic programs in mammalian cells. Nat. Commun. 11, 779 (2020).
Article
CAS
PubMed
PubMed Central
Google Scholar
Wu, M.-R. et al. A high-throughput screening and computation platform for identifying synthetic promoters with enhanced cell-state specificity (SPECS). Nat. Commun. 10, 2880 (2019).
Article
PubMed
PubMed Central
Google Scholar
Bragdon, M. D. J. et al. Cooperative assembly confers regulatory specificity and long-term genetic circuit stability. Cell 186, 3810–3825 (2023).
Article
CAS
PubMed
PubMed Central
Google Scholar
Randolph, L. N., Bao, X., Zhou, C. & Lian, X. An all-in-one, Tet-On 3G inducible PiggyBac system for human pluripotent stem cells and derivatives. Sci. Rep. 7, 1549 (2017).
Article
PubMed
PubMed Central
Google Scholar
Nevozhay, D., Zal, T. & Balázsi, G. Transferring a synthetic gene circuit from yeast to mammalian cells. Nat. Commun. 4, 1451 (2013).
Article
PubMed
Google Scholar
Beerli, R. R. & Barbas, C. F. Engineering polydactyl zinc-finger transcription factors. Nat. Biotechnol. 20, 135–141 (2002).
Article
CAS
PubMed
Google Scholar
Jamieson, A. C., Kim, S. H. & Wells, J. A. In vitro selection of zinc fingers with altered DNA-binding specificity. Biochemistry 33, 5689–5695 (1994).
Article
CAS
PubMed
Google Scholar
Ede, C., Chen, X., Lin, M.-Y. & Chen, Y. Y. Quantitative analyses of core promoters enable precise engineering of regulated gene expression in mammalian cells. ACS Synth. Biol. 5, 395–404 (2016).
Article
CAS
PubMed
PubMed Central
Google Scholar
Zopf, C. J., Quinn, K., Zeidman, J. & Maheshri, N. Cell-cycle dependence of transcription dominates noise in gene expression. PLoS Comput. Biol. 9, e1003161 (2013).
Article
CAS
PubMed
PubMed Central
Google Scholar
Quarton, T. et al. Uncoupling gene expression noise along the central dogma using genome engineered human cell lines. Nucleic Acids Res. 48, 9406–9413 (2020).
Article
CAS
PubMed
PubMed Central
Google Scholar
Babos, K. N. et al. Mitigating antagonism between transcription and proliferation allows near-deterministic cellular reprogramming. Cell Stem Cell 25, 486–500 (2019).
Article
CAS
PubMed
PubMed Central
Google Scholar
Fitzgerald, M., Livingston, M., Gibbs, C. & Deans, T. L. Rosa26 docking sites for investigating genetic circuit silencing in stem cells. Synth. Biol. 5, ysaa014 (2020).
Article
CAS
Google Scholar
Jones, R. D. et al. Robust and tunable signal processing in mammalian cells via engineered covalent modification cycles. Nat. Commun. 13, 1720 (2022).
Article
CAS
PubMed
PubMed Central
Google Scholar
Frei, T., Chang, C.-H., Filo, M., Arampatzis, A. & Khammash, M. A genetic mammalian proportional–integral feedback control circuit for robust and precise gene regulation. Proc. Natl Acad. Sci. USA 119, e2122132119 (2022).
Article
CAS
PubMed
PubMed Central
Google Scholar
Johnstone, C. P. & Galloway, K. E. Supercoiling-mediated feedback rapidly couples and tunes transcription. Cell Rep. 41, 111492 (2022).
Article
CAS
PubMed
PubMed Central
Google Scholar
Lillacci, G., Benenson, Y. & Khammash, M. Synthetic control systems for high performance gene expression in mammalian cells. Nucleic Acids Res. 46, 9855–9863 (2018).
Article
CAS
PubMed
PubMed Central
Google Scholar
Strovas, T. J., Rosenberg, A. B., Kuypers, B. E., Muscat, R. A. & Seelig, G. MicroRNA-based single-gene circuits buffer protein synthesis rates against perturbations. ACS Synth. Biol. 3, 324–331 (2014).
Article
CAS
PubMed
Google Scholar
Peterman, E. L. et al. High-resolution profiling reveals coupled transcriptional and translational regulation of transgenes. Nucleic Acids Res. 53, gkaf528 (2025).
Article
CAS
PubMed
PubMed Central
Google Scholar
Askary, A. et al. In situ readout of DNA barcodes and single base edits facilitated by in vitro transcription. Nat. Biotechnol. 38, 66–75 (2020).
Article
CAS
PubMed
Google Scholar
Kim, T., Weinberg, B., Wong, W. & Lu, T. K. Scalable recombinase-based gene expression cascades. Nat. Commun. 12, 2711 (2021).
Article
CAS
PubMed
PubMed Central
Google Scholar
Loveless, T. B. et al. Lineage tracing and analog recording in mammalian cells by single-site DNA writing. Nat. Chem. Biol. 17, 739–747 (2021).
Article
CAS
PubMed
PubMed Central
Google Scholar
Sheth, R. U. & Wang, H. H. DNA-based memory devices for recording cellular events. Nat. Rev. Genet. 19, 718–732 (2018).
Article
CAS
PubMed
PubMed Central
Google Scholar
Shi, D., Toyonaga, S. & Anderson, D. G. In vivo RNA delivery to hematopoietic stem and progenitor cells via targeted lipid nanoparticles. Nano Lett. 23, 2938–2944 (2023).
Article
CAS
PubMed
PubMed Central
Google Scholar
Sago, C. D. et al. High-throughput in vivo screen of functional mRNA delivery identifies nanoparticles for endothelial cell gene editing. Proc. Natl Acad. Sci. USA 115, E9944–E9952 (2018).
Article
CAS
PubMed
PubMed Central
Google Scholar
Blanch-Asensio, A. et al. STRAIGHT-IN enables high-throughput targeting of large DNA payloads in human pluripotent stem cells. Cell Rep. Methods 2, 100300 (2022).
Article
CAS
PubMed
PubMed Central
Google Scholar
Weinberg, B. H. et al. High-performance chemical- and light-inducible recombinases in mammalian cells and mice. Nat. Commun. 10, 4845 (2019).
Article
PubMed
PubMed Central
Google Scholar
Lende-Dorn, B. A., Atkinson, J. C., Bae, Y. & Galloway, K. E. Chemogenetic tuning reveals optimal MAPK signaling for cell-fate programming. Cell Rep. 44, 116226 (2025).
Article
CAS
PubMed
Google Scholar
Ilia, K. et al. Synthetic genetic circuits to uncover the OCT4 trajectories of successful reprogramming of human fibroblasts. Sci. Adv. 9, eadg8495 (2023).
Article
CAS
PubMed
PubMed Central
Google Scholar
Velychko, S. et al. Excluding Oct4 from Yamanaka cocktail unleashes the developmental potential of iPSCs. Cell Stem Cell 25, 737–753 (2019).
Article
CAS
PubMed
PubMed Central
Google Scholar
Hu, X. et al. Reprogramming progressive cells display low CAG promoter activity. Stem Cells 39, 43–54 (2021).
Article
CAS
PubMed
Google Scholar
Okada, M. & Yoneda, Y. The timing of retroviral silencing correlates with the quality of induced pluripotent stem cell lines. Biochim. Biophys. Acta 1810, 226–235 (2011).
Article
CAS
PubMed
Google Scholar
Wang, N. B. et al. Compact transcription factor cassettes generate functional, engraftable motor neurons by direct conversion. Cell Syst. 16, 101206 (2025).
Article
CAS
PubMed
Google Scholar
Beitz, A. et al. Cells transit through a quiescent-like state to convert to neurons at high rates. Preprint at bioRxiv https://doi.org/10.1101/2024.11.22.624928 (2024).
Du, R., Flynn, M. J., Honsa, M., Jungmann, R. & Elowitz, M. B. miRNA circuit modules for precise, tunable control of gene expression. Preprint at bioRxiv https://doi.org/10.1101/2024.03.12.583048 (2024).
Flynn, M. J., Mayfield, A. M., Du, R., Gradinaru, V. & Elowitz, M. B. Synthetic dosage-compensating miRNA circuits for quantitative gene therapy. Preprint at bioRxiv https://doi.org/10.1101/2024.03.13.584179 (2024).
Bashor, C. J., Hilton, I. B., Bandukwala, H., Smith, D. M. & Veiseh, O. Engineering the next generation of cell-based therapeutics. Nat. Rev. Drug Discov. 21, 655–675 (2022).
Article
CAS
PubMed
PubMed Central
Google Scholar
Rai, K. et al. Ultra-high throughput mapping of genetic design space. Preprint at bioRxiv https://doi.org/10.1101/2023.03.16.532704 (2025).
Tycko, J. et al. High-throughput discovery and characterization of human transcriptional effectors. Cell 183, 2020–2035.e16 (2020).
Article
CAS
PubMed
PubMed Central
Google Scholar
Ravindran, P. T., Wilson, M. Z., Jena, S. G. & Toettcher, J. E. Engineering combinatorial and dynamic decoders using synthetic immediate-early genes. Commun. Biol. 3, 436 (2020).
Article
CAS
PubMed
PubMed Central
Google Scholar
Hong, C. K. Y. & Cohen, B. A. Genomic environments scale the activities of diverse core promoters. Genome Res. 32, 85–96 (2022).
Article
CAS
PubMed
PubMed Central
Google Scholar
Johnstone, C. P., Wang, N. B., Sevier, S. A. & Galloway, K. E. Understanding and engineering chromatin as a dynamical system across length and timescales. Cell Syst. 11, 424–448 (2020).
Article
CAS
PubMed
Google Scholar
Bartman, C. R., Hsu, S. C., Hsiung, C. C.-S., Raj, A. & Blobel, G. A. Enhancer regulation of transcriptional bursting parameters revealed by forced chromatin looping. Mol. Cell 62, 237–247 (2016).
Article
CAS
PubMed
PubMed Central
Google Scholar
Popp, A. P., Hettich, J. & Gebhardt, J. C. M. Altering transcription factor binding reveals comprehensive transcriptional kinetics of a basic gene. Nucleic Acids Res. 49, 6249–6266 (2021).
Article
CAS
PubMed
PubMed Central
Google Scholar
Weinberg, B. H. et al. Large-scale design of robust genetic circuits with multiple inputs and outputs for mammalian cells. Nat. Biotechnol. 35, 453–462 (2017).
Article
CAS
PubMed
PubMed Central
Google Scholar
Short, A. E., Kim, D., Milner, P. T. & Wilson, C. J. Next generation synthetic memory via intercepting recombinase function. Nat. Commun. 14, 5255 (2023).
Article
CAS
PubMed
PubMed Central
Google Scholar
Meinke, G., Bohm, A., Hauber, J., Pisabarro, M. T. & Buchholz, F. Cre recombinase and other tyrosine recombinases. Chem. Rev. 116, 12785–12820 (2016).
Article
CAS
PubMed
Google Scholar
Shen, Y. et al. SCRaMbLE generates designed combinatorial stochastic diversity in synthetic chromosomes. Genome Res. 26, 36–49 (2016).
Article
CAS
PubMed
PubMed Central
Google Scholar
Liu, W. et al. Rapid pathway prototyping and engineering using in vitro and in vivo synthetic genome SCRaMbLE-in methods. Nat. Commun. 9, 1936 (2018).
Article
PubMed
PubMed Central
Google Scholar
Koeppel, J. et al. Randomizing the human genome by engineering recombination between repeat elements. Science 387, eado3979 (2025).
Article
CAS
PubMed
Google Scholar
Pinglay, S. et al. Multiplex generation and single-cell analysis of structural variants in mammalian genomes. Science 387, eado5978 (2025).
Article
CAS
PubMed
PubMed Central
Google Scholar
Cautereels, C. et al. Combinatorial optimization of gene expression through recombinase-mediated promoter and terminator shuffling in yeast. Nat. Commun. 15, 1112 (2024).
Article
CAS
PubMed
PubMed Central
Google Scholar
Chakravarti, D., Caraballo, L. D., Weinberg, B. H. & Wong, W. W. Inducible gene switches with memory in human T cells for cellular immunotherapy. ACS Synth. Biol. 8, 1744–1754 (2019).
Article
CAS
PubMed
PubMed Central
Google Scholar
Durrant, M. G. et al. Systematic discovery of recombinases for efficient integration of large DNA sequences into the human genome. Nat. Biotechnol. 41, 488–499 (2023).
Article
CAS
PubMed
Google Scholar
Jelicic, M. et al. Discovery and characterization of novel Cre-type tyrosine site-specific recombinases for advanced genome engineering. Nucleic Acids Res. 51, 5285–5297 (2023).
Article
CAS
PubMed
PubMed Central
Google Scholar
Kretzschmar, K. & Watt, F. M. Lineage tracing. Cell 148, 33–45 (2012).
Article
CAS
PubMed
Google Scholar
Yang, D. et al. Lineage tracing reveals the phylodynamics, plasticity, and paths of tumor evolution. Cell 185, 1905–1923 (2022).
Article
CAS
PubMed
PubMed Central
Google Scholar
Anderson, T. et al. Ligament injury in adult zebrafish triggers ECM remodeling and cell dedifferentiation for scar-free regeneration. NPJ Regen. Med. 8, 51 (2023).
Article
CAS
PubMed
PubMed Central
Google Scholar
Nowak, J. A., Polak, L., Pasolli, H. A. & Fuchs, E. Hair follicle stem cells are specified and function in early skin morphogenesis. Cell Stem Cell 3, 33–43 (2008).
Article
CAS
PubMed
PubMed Central
Google Scholar
Le Bin, G. C. et al. Oct4 is required for lineage priming in the developing inner cell mass of the mouse blastocyst. Development 141, 1001–1010 (2014).
Article
PubMed
PubMed Central
Google Scholar
Meador, K. et al. Achieving tight control of a photoactivatable Cre recombinase gene switch: new design strategies and functional characterization in mammalian cells and rodent. Nucleic Acids Res. 47, e97 (2019).
Article
PubMed
PubMed Central
Google Scholar
Bleris, L. et al. Synthetic incoherent feedforward circuits show adaptation to the amount of their genetic template. Mol. Syst. Biol. 7, 519 (2011).
Article
PubMed
PubMed Central
Google Scholar
Yang, J. et al. A synthetic circuit for buffering gene dosage variation between individual mammalian cells. Nat. Commun. 12, 4132 (2021).
Article
CAS
PubMed
PubMed Central
Google Scholar
Love, K. S. et al. Model-guided design of microRNA-based gene circuits supports precise dosage of transgenic cargoes into diverse primary cells. Cell Syst. 16, 101269 (2025).
Article
CAS
PubMed
Google Scholar
An, M. et al. Engineered virus-like particles for transient delivery of prime editor ribonucleoprotein complexes in vivo. Nat. Biotechnol. 42, 1526–1537 (2024).
Article
CAS
PubMed
PubMed Central
Google Scholar
Kreitz, J. et al. Programmable protein delivery with a bacterial contractile injection system. Nature 616, 357–364 (2023).
Article
CAS
PubMed
PubMed Central
Google Scholar
Choi, H. M. T. et al. Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust. Development 145, dev165753 (2018).
Article
PubMed
PubMed Central
Google Scholar
Jia, Z., Dong, Y., Xu, H. & Wang, F. Optimizing the hybridization chain reaction-fluorescence in situ hybridization (HCR-FISH) protocol for detection of microbes in sediments. Mar. Life Sci. Technol. 3, 529–541 (2021).
Article
CAS
PubMed
PubMed Central
Google Scholar
Kabaria, S. & Galloway, K. E. Data for Kabaria et al “Programmable Promoter Editing for Precise Control of Transgene Expression”. Zenodo https://doi.org/10.5281/zenodo.17014280 (2025).
Kabaria, S. R. et al. Programmable promoter editing for precise control of transgene expression. Source code. GitHub https://github.com/GallowayLabMIT/Kabaria_Promoter_Editing_DIAL (2025).
Kabaria, S. R. et al. Programmable promoter editing for precise control of transgene expression. Zenodo https://doi.org/10.5281/zenodo.17042201 (2025).