Engineering RNA export for measurement and manipulation of living cells
A system for programmable export of RNA molecules from living cells would enable both non-destructive monitoring of cell dynamics and engineering of cells capable of delivering executable RNA programs to other cells. We developed genetically encoded cellular RNA exporters, inspired by viruses, that efficiently and selectively package and secrete target RNA molecules from mammalian cells within protective nanoparticles. Exporting and sequencing RNA barcodes enabled non-destructive monitoring of cell population dynamics with clonal resolution. Further, by incorporating fusogens into the nanoparticles, we demonstrated delivery, expression, and functional activity of exported mRNA in recipient cells. We term these systems COURIER (Controlled Output and Uptake of RNA for Interrogation, Expression, and Regulation). COURIER enables measurement of cell dynamics and establishes a foundation for hybrid cell and gene therapies based on cell-to-cell delivery of RNA.
We thank M. Blanco, M. Guttman, J. Keeffe, Y. Garcia-Flores, S. Mazmanian, and S. Chen for access to equipment; A. Hori for sharing cells; H. Larsen for mRNA production; I.M. Strazhnik for illustrations; D. Chadly, M. Tran, B. Emert, A. Askary, and R. Zhu for scientific input; and S. Xia, R. Du, D. Li, and M. Sui for critical feedback. Electron microscopy was performed in the Beckman Institute Resource Center for Transmission Electron Microscopy at Caltech. Sequencing was performed at the Single-Cell Profiling Center in the Beckman Institute at Caltech. The research was supported by the National Institutes of Health R01MH116508 (to M.B.E.), the Allen Discovery Center, a Paul G. Allen Frontiers Group advised program of the Paul G. Allen Family Foundation (award number UWSC10142 to M.B.E.), and the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health R01EB030015 (to M.B.E.). M.B.E. is a Howard Hughes Medical Institute investigator. F.H. is a Howard Hughes Medical Institute Fellow of the Helen Hay Whitney Foundation.
F.H. conceived of RNA export and its use for reporting. F.H. and M.B.E. conceived of cell-based RNA delivery. F.H., J.A.M., V.T., C.L., and M.B.E. designed experiments. F.H., J.A.M., C.F., M.H., J.M.L., V.T., L.S., and C.L. performed experiments. F.H., J.A.M., A.O.M., and M.B.E. analyzed data. P.J.B. provided instrumentation and support. F.H. and M.B.E. wrote the manuscript, with input from all authors.
Conflict of Interest
Patent applications have been filed by the California Institute of Technology related to this work (US application numbers 17/820,232 and 17/820,235). M.B.E. is a scientific advisory board member or consultant at TeraCyte, Primordium, and Spatial Genomics
- Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
- All original code has been deposited at Github (https://github.com/felixhorns/RNA-export-2023) and is publicly available as of the date of publication. DOIs are listed in the key resources table.
- Raw DNA and RNA sequencing data have been deposited at the NCBI Sequence Read Archive and are publicly available as of the date of publication. Preprocessed data have been deposited at CaltechDATA and are publicly available as of the date of publication. Accession numbers are listed in the key resources table.
Copyright and License
© 2023 The Authors. Published by Elsevier Under a Creative Commons license - Attribution 4.0 International (CC BY 4.0)