A tunable orthogonal coiled-coil interaction toolbox for engineering mammalian cells

Abstract

Protein interactions guide most cellular processes. Orthogonal hetero-specific protein–protein interaction domains may facilitate better control of engineered biological systems. Here, we report a tunable de novo designed set of orthogonal coiled-coil (CC) peptide heterodimers (called the NICP set) and its application for the regulation of diverse cellular processes, from cellular localization to transcriptional regulation. We demonstrate the application of CC pairs for multiplex localization in single cells and exploit the interaction strength and variable stoichiometry of CC peptides for tuning of gene transcription strength. A concatenated CC peptide tag (CCC-tag) was used to construct highly potent CRISPR–dCas9-based transcriptional activators and to amplify the response of light and small molecule-inducible transcription in cell culture as well as in vivo. The NICP set and its implementations represent a valuable toolbox of minimally disruptive modules for the recruitment of versatile functional domains and regulation of cellular processes for synthetic biology.

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Fig. 1: Orthogonality of the designed NICP peptide set in HEK293T cells.
Fig. 2: CC-directed localization of multiple proteins.
Fig. 3: Tunability of the interaction strength within the NICP set toolbox.
Fig. 4: Highly potent designed CRISPR–CCC transcriptional activation platform.
Fig. 5: Enhancement of conditionally regulated transcription with CRISPR–CCC.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

This research was supported by grants from the Slovenian Research Agency (nos. P4-0176, J1-9173, J3-7034 and N4-0080), ERC grant MaCChines to R.J, Horizon2020 CSA Bioroboost and ERANET project MediSurf. T.L. is partially supported by the UNESCO-L’OREAL national fellowship ‘For Women in Science’. We thank H. Gradišar for providing the sequences of CC peptides and for valuable advice.

Author information

T.L. and E.M. prepared the plasmid constructs and performed the experiments on cell culture. D.L. performed the experiments on mice. J.A. performed the bioinformatics analysis. T.L. and R.J. designed and analyzed the experiments and wrote the manuscript. R.J. conceived the study.

Correspondence to Roman Jerala.

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Lebar, T., Lainšček, D., Merljak, E. et al. A tunable orthogonal coiled-coil interaction toolbox for engineering mammalian cells. Nat Chem Biol (2020). https://doi.org/10.1038/s41589-019-0443-y

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