miRNA circuit modules for precise, tunable control of gene expression
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1.
California Institute of Technology
Abstract
The ability to express transgenes at specified levels is critical for understanding cellular behaviors, and for applications in gene and cell therapy. Transfection, viral vectors, and other gene delivery methods produce varying protein expression levels, with limited quantitative control, while targeted knock-in and stable selection are inefficient and slow. Active compensation mechanisms can improve precision, but the need for additional proteins or lack of tunability have prevented their widespread use. Here, we introduce a toolkit of compact, synthetic miRNA-based circuit modules that provide precise, tunable control of transgenes across diverse cell types. These circuits, termed DIMMERs (Dosage-Invariant miRNA-Mediated Expression Regulators) use multivalent miRNA regulatory interactions within an incoherent feed-forward loop architecture to achieve nearly uniform protein expression over more than two orders of magnitude variation in underlying gene dosages or transcription rates. They also allow coarse and fine control of expression, and are portable, functioning across diverse cell types. In addition, a heuristic miRNA design algorithm enables the creation of orthogonal circuit variants that independently control multiple genes in the same cell. These circuits allowed dramatically improved CRISPR imaging, and super-resolution imaging of EGFR receptors with transient transfections. The toolbox provided here should allow precise, tunable, dosage-invariant expression for research, gene therapy, and other biotechnology applications.
Copyright and License
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
Acknowledgement
We thank Phillip Zamore (UMass), Viviana Gradinaru, Acacia Mayfield, James Linton, Dongyang Li, Bo Gu, Kaiwen Luo, Martin Tran, Duncan Chadly, Felix Horns, Lucy Chong, Leah Santat, Sheng Wang, Shiyu Xia, Yodai Takei for discussion and technical support; Inna-Marie Strazhnik (Caltech) for graphical design; Fei Chen (Broad Institute), Jacob Parres-Gold, Evan Mun, Judy Shon, and other members of the Elowitz lab for critical feedback, and administrative support. This work is supported by the National Institute Of Biomedical Imaging And Bioengineering of the National Institutes of Health under Award Number R01EB030015. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. M.B.E. is a Howard Hughes Medical Institute Investigator. We apologize for incomplete citations due to space limits.
Funding
This work is supported by the National Institute Of Biomedical Imaging And Bioengineering of the National Institutes of Health under Award Number R01EB030015. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. M.B.E. is a Howard Hughes Medical Institute Investigator.
Contributions
R.D., M.F., and M.B.E. conceived and designed the study. M.B.E. directed and supervised the study. R.D., M.F., and M.H. performed or assisted with experiments and analyzed data. R.D. and M.B.E. wrote the manuscript with input from all authors.
Data Availability
Data needed to evaluate the conclusions are available in the paper or the Supplementary Materials. Raw data and computer codes used for data analysis are available from the corresponding author.
Conflict of Interest
Patent applications related to this work have been filed by the California Institute of Technology (US application numbers CIT-9070-P/CIT-9070-P2). M.B.E is a scientific advisory board member or consultant at TeraCyte, Primordium, and Spatial Genomics.
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Additional details
- PMCID
- PMC10979901
- R01EB030015
- National Institutes of Health
- Howard Hughes Medical Institute
- Caltech groups
- Division of Biology and Biological Engineering