Synthetic multistability in mammalian cells
In multicellular organisms, gene regulatory circuits generate thousands of molecularly distinct, mitotically heritable states through the property of multistability. Designing synthetic multistable circuits would provide insight into natural cell fate control circuit architectures and would allow engineering of multicellular programs that require interactions among distinct cell types. We created MultiFate, a naturally inspired, synthetic circuit that supports long-term, controllable, and expandable multistability in mammalian cells. MultiFate uses engineered zinc finger transcription factors that transcriptionally self-activate as homodimers and mutually inhibit one another through heterodimerization. Using a model-based design, we engineered MultiFate circuits that generate as many as seven states, each stable for at least 18 days. MultiFate permits controlled state switching and modulation of state stability through external inputs and can be expanded with additional transcription factors. These results provide a foundation for engineering multicellular behaviors in mammalian cells.
Additional Information© 2022 American Association for the Advancement of Science. Received 10 February 2021; accepted 29 November 2021. We thank M. Budde for suggestions on MultiFate circuit design; J. Tijerina at Caltech Flow Cytometry Facility for help with cell sorting; X. Wang and F. Horns for timely help with experiments during COVID and lab move; S. Xie for help with MultiFate-2 monoclone screening; S. Xie and S. Satia for advice on coding; J. Bois for teaching and sharing Caltech BE150 course materials for mathematical modeling; A. Khalil for suggestions on the choice of zinc fingers; R. Kuintzle, F. Horns, L. Chong, Z. Chen, M. Flynn, H. Klumpe, M. Budde, B. Gu, J. Gregrowicz, and E. Mun for critical feedback; and other members of the Elowitz lab for scientific input and support. Supported by DARPA (HR0011-17-2-0008, M.B.E.); the Allen Discovery Center program, a Paul G. Allen Frontiers Group advised program of the Paul G. Allen Family Foundation (UWSC10142, M.B.E.); the Spanish Ministry of Science and Innovation and FEDER (PGC2018-101251-B-I00, J.G.-O.); "Maria de Maeztu" Programme for Units of Excellence in R&D (CEX2018-000792-M, J.G.-O.); and the Generalitat de Catalunya (ICREA Academia program, J.G.-O.). M.B.E. is a Howard Hughes Medical Institute investigator. Author contributions: R.Z. and M.B.E. conceived of the project. R.Z. and M.B.E. designed experiments. R.Z. performed experiments. R.Z. and M.B.E. analyzed data. R.Z., J.M.d.R.-S., J.G.-O., and M.B.E. did mathematical modeling. R.Z. and M.B.E. wrote the manuscript with input from all authors. Competing interests: R.Z. and M.B.E. are inventors on a US provisional patent application related to this work. Data and materials availability: All DNA constructs (table S2) and cell lines (table S3) are available from M.B.E. or through the Addgene repository under a material agreement with California Institute of Technology. All data generated and all the computational and data analysis and modeling code used in the current study are available at data.caltech.edu/records/1882.
Submitted - 2021.02.10.430659v1.full.pdf
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