Abstract
RNA molecules perform various crucial roles in diverse cellular processes, from translating genetic information to decoding the genome, regulating gene expression and catalyzing chemical reactions. RNA-binding proteins (RBPs) play an essential role in regulating the diverse behaviors and functions of RNA in live cells, but techniques for the spatiotemporal control of RBP activities and RNA functions are rarely reported yet highly desirable. We recently reported the development of LicV, a synthetic photoswitchable RBP that can bind to a specific RNA sequence in response to blue light irradiation. LicV has been used successfully for the optogenetic control of RNA localization, splicing, translation and stability, as well as for the photoswitchable regulation of transcription and genomic locus labeling. Compared to classical genetic or pharmacologic perturbations, LicV-based light-switchable effectors have the advantages of large dynamic range between dark and light conditions and submicron and millisecond spatiotemporal resolutions. In this protocol, we provide an easy, efficient and generalizable strategy for engineering photoswitchable RBPs for the spatiotemporal control of RNA metabolism. We also provide a detailed protocol for the conversion of a CRISPR–Cas system to optogenetic control. The protocols typically take 2–3 d, including transfection and results analysis. Most of this protocol is applicable to the development of novel LicV-based photoswitchable effectors for the optogenetic control of other RNA metabolisms and CRISPR–Cas functions.
Key points
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This protocol provides an efficient and generalizable strategy for engineering photoswitchable RNA-binding proteins (RBPs) for the spatiotemporal control of RNA activity. It uses LicV, a synthetic RBP that can bind to a specific RNA sequence in response to blue light irradiation.
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This optogenetic method circumvents the limitations of previous strategies by enabling the activity of functional RNAs or effectors to be more precisely controlled because they can be switched on and off by using light.
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Data availability
The main data discussed in this protocol are available in the supporting primary research paper12. The raw datasets are too large to be publicly shared but are available for research purposes from the corresponding authors upon reasonable request. Source data are provided with this paper.
References
Cech, T. R. & Steitz, J. A. The noncoding RNA revolution—trashing old rules to forge new ones. Cell 157, 77–94 (2014).
Battich, N., Stoeger, T. & Pelkmans, L. Image-based transcriptomics in thousands of single human cells at single-molecule resolution. Nat. Methods 10, 1127–1133 (2013).
Wang, K. C. & Chang, H. Y. Molecular mechanisms of long noncoding RNAs. Mol. Cell 43, 904–914 (2011).
Buxbaum, A. R., Haimovich, G. & Singer, R. H. In the right place at the right time: visualizing and understanding mRNA localization. Nat. Rev. Mol. Cell Biol. 16, 95–109 (2015).
Pichon, X. et al. RNA binding protein/RNA element interactions and the control of translation. Curr. Protein Pept. Sci. 13, 294–304 (2012).
Abil, Z., Denard, C. A. & Zhao, H. Modular assembly of designer PUF proteins for specific post-transcriptional regulation of endogenous RNA. J. Biol. Eng. 8, 7 (2014).
Tischer, D. & Weiner, O. D. Illuminating cell signalling with optogenetic tools. Nat. Rev. Mol. Cell Biol.y 15, 551–558 (2014).
Kawano, F., Shi, F. & Yazawa, M. Optogenetics: switching with red and blue. Nat. Chem. Biol. 13, 573–574 (2017).
Weber, A. M. et al. A blue light receptor that mediates RNA binding and translational regulation. Nat. Chem. Biol.y 15, 1085–1092 (2019).
Pilsl, S., Morgan, C., Choukeife, M., Moglich, A. & Mayer, G. Optoribogenetic control of regulatory RNA molecules. Nat. Commun. 11, 4825 (2020).
Renzl, C., Kakoti, A. & Mayer, G. Aptamer-mediated reversible transactivation of gene expression by light. Angew. Chem. Int. Ed. Engl. 59, 22414–22418 (2020).
Liu, R. et al. Optogenetic control of RNA function and metabolism using engineered light-switchable RNA-binding proteins. Nat. Biotechnol. 40, 779–786 (2022).
Ranzani, A. T. et al. Light-dependent control of bacterial expression at the mRNA Level. ACS Synth. Biol. 11, 3482–3492 (2022).
Bubeck, F. et al. Engineered anti-CRISPR proteins for optogenetic control of CRISPR-Cas9. Nat. Methods 15, 924–927 (2018).
Knott, G. J. & Doudna, J. A. CRISPR-Cas guides the future of genetic engineering. Science 361, 866–869 (2018).
Liu, K. I. et al. A chemical-inducible CRISPR-Cas9 system for rapid control of genome editing. Nat. Chem. Biol. 12, 980–987 (2016).
Setten, R. L., Rossi, J. J. & Han, S. P. The current state and future directions of RNAi-based therapeutics. Nat. Rev. Drug Discov. 18, 421–446 (2019).
Chen, Y. & Varani, G. Engineering RNA-binding proteins for biology. FEBS J. 280, 3734–3754 (2013).
Choudhury, R., Tsai, Y. S., Dominguez, D., Wang, Y. & Wang, Z. Engineering RNA endonucleases with customized sequence specificities. Nat. Commun. 3, 1147 (2012).
Cooke, A., Prigge, A., Opperman, L. & Wickens, M. Targeted translational regulation using the PUF protein family scaffold. Proc. Natl. Acad. Sci. USA 108, 15870–15875 (2011).
Abudayyeh, O. O. et al. RNA targeting with CRISPR-Cas13. Nature 550, 280–284 (2017).
Batra, R. et al. Elimination of toxic microsatellite repeat expansion RNA by RNA-targeting Cas9. Cell 170, 899–912.e10 (2017).
Cox, D. B. T. et al. RNA editing with CRISPR-Cas13. Science 358, 1019–1027 (2017).
Chaulk, S. G. & MacMillan, A. M. Caged RNA: photo-control of a ribozyme reaction. Nucleic Acids Res. 26, 3173–3178 (1998).
Zetsche, B., Volz, S. E. & Zhang, F. A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat. Biotechnol. 33, 139–142 (2015).
Nihongaki, Y., Otabe, T., Ueda, Y. & Sato, M. A split CRISPR-Cpf1 platform for inducible genome editing and gene activation. Nat. Chem. Biol. 15, 882–888 (2019).
Nihongaki, Y., Kawano, F., Nakajima, T. & Sato, M. Photoactivatable CRISPR-Cas9 for optogenetic genome editing. Nat. Biotechnol. 33, 755–760 (2015).
Nihongaki, Y. et al. CRISPR-Cas9-based photoactivatable transcription systems to induce neuronal differentiation. Nat. Methods 14, 963–966 (2017).
Richter, F. et al. Engineering of temperature- and light-switchable Cas9 variants. Nucleic Acids Res. 44, 10003–10014 (2016).
Zhou, X. X. et al. A single-chain photoswitchable CRISPR-Cas9 architecture for light-inducible gene editing and transcription. ACS Chem. Biol. 13, 443–448 (2018).
Ma, H. et al. CRISPR-Sirius: RNA scaffolds for signal amplification in genome imaging. Nat. Methods 15, 928–931 (2018).
Qin, P. et al. Live cell imaging of low- and non-repetitive chromosome loci using CRISPR-Cas9. Nat. Commun. 8, 14725 (2017).
Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517, 583–588 (2015).
Shao, S. et al. Long-term dual-color tracking of genomic loci by modified sgRNAs of the CRISPR/Cas9 system. Nucleic Acids Res. 44, e86 (2016).
Ma, H. et al. Multiplexed labeling of genomic loci with dCas9 and engineered sgRNAs using CRISPRainbow. Nat. Biotechnol. 34, 528–530 (2016).
Tanenbaum, M. E., Gilbert, L. A., Qi, L. S., Weissman, J. S. & Vale, R. D. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159, 635–646 (2014).
Boersma, S. et al. Multi-color single-molecule imaging uncovers extensive heterogeneity in mRNA decoding. Cell 178, 458–472.e19 (2019).
Nowak, C. M., Lawson, S., Zerez, M. & Bleris, L. Guide RNA engineering for versatile Cas9 functionality. Nucleic Acids Res. 44, 9555–9564 (2016).
Terenin, I. M., Smirnova, V. V., Andreev, D. E., Dmitriev, S. E. & Shatsky, I. N. A researcher’s guide to the galaxy of IRESs. Cell. Mol. Life Sci. 74, 1431–1455 (2017).
Wang, W. et al. Impact of different promoters, promoter mutation, and an enhancer on recombinant protein expression in CHO cells. Sci. Rep. 7, 10416 (2017).
Kulaberoglu, Y. et al. RNA polymerase III, ageing and longevity. Front. Genet. 12, 705122 (2021).
Roelz, R., Pilz, I. H., Mutschler, M. & Pahl, H. L. Of mice and men: human RNA polymerase III promoter U6 is more efficient than its murine homologue for shRNA expression from a lentiviral vector in both human and murine progenitor cells. Exp. Hematol. 38, 792–797 (2010).
Buckle, A. M., Schreiber, G. & Fersht, A. R. Protein-protein recognition: crystal structural analysis of a barnase-barstar complex at 2.0-A resolution. Biochemistry 33, 8878–8889 (1994).
Wang, X., Chen, X. & Yang, Y. Spatiotemporal control of gene expression by a light-switchable transgene system. Nat. Methods 9, 266–269 (2012).
Li, T. et al. A synthetic BRET-based optogenetic device for pulsatile transgene expression enabling glucose homeostasis in mice. Nat. Commun. 12, 615 (2021).
Chen, X., Zaro, J. L. & Shen, W. C. Fusion protein linkers: property, design and functionality. Adv. Drug Deliv. Rev. 65, 1357–1369 (2013).
Schwartz, M. A., Schaller, M. D. & Ginsberg, M. H. Integrins: emerging paradigms of signal transduction. Annu. Rev. Cell Dev. Biol. 11, 549–599 (1995).
Zou, Y. et al. Analysis of redox landscapes and dynamics in living cells and in vivo using genetically encoded fluorescent sensors. Nat. Protoc. 13, 2362–2386 (2018).
Chen, X. et al. Visualizing RNA dynamics in live cells with bright and stable fluorescent RNAs. Nat. Biotechnol. 37, 1287–1293 (2019).
Chen, X., Wang, X., Du, Z., Ma, Z. & Yang, Y. Spatiotemporal control of gene expression in mammalian cells and in mice using the LightOn system. Curr. Protoc. Chem. Biol. 5, 111–129 (2013).
Luft, C. et al. Application of Gaussia luciferase in bicistronic and non-conventional secretion reporter constructs. BMC Biochem. 15, 14 (2014).
Tannous, B. A., Kim, D. E., Fernandez, J. L., Weissleder, R. & Breakefield, X. O. Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo. Mol. Ther. 11, 435–443 (2005).
Chavez, A. et al. Highly efficient Cas9-mediated transcriptional programming. Nat. Methods 12, 326–328 (2015).
Chen, B. et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155, 1479–1491 (2013).
Abil, Z. & Zhao, H. Engineering reprogrammable RNA-binding proteins for study and manipulation of the transcriptome. Mol. Biosyst. 11, 2658–2665 (2015).
Zhang, L., Chen, C., Fan, X. & Tang, X. Photomodulating gene expression by using caged siRNAs with single-aptamer modification. Chembiochem 19, 1259–1263 (2018).
Zhang, L. et al. Caged circular siRNAs for photomodulation of gene expression in cells and mice. Chem. Sci. 9, 44–51 (2018).
Wu, L. et al. Caged circular antisense oligonucleotides for photomodulation of RNA digestion and gene expression in cells. Nucleic Acids Res. 41, 677–686 (2013).
Ando, H., Furuta, T., Tsien, R. Y. & Okamoto, H. Photo-mediated gene activation using caged RNA/DNA in zebrafish embryos. Nat. Genet. 28, 317–325 (2001).
Ando, H., Furuta, T. & Okamoto, H. Photo-mediated gene activation by using caged mRNA in zebrafish embryos. Methods Cell Biol. 77, 159–171 (2004).
Wang, P. et al. Mapping spatial transcriptome with light-activated proximity-dependent RNA labeling. Nat. Chem. Biol. 15, 1110–1119 (2019).
Benhalevy, D., Anastasakis, D. G. & Hafner, M. Proximity-CLIP provides a snapshot of protein-occupied RNA elements in subcellular compartments. Nat. Methods 15, 1074–1082 (2018).
Nihongaki, Y., Yamamoto, S., Kawano, F., Suzuki, H. & Sato, M. CRISPR-Cas9-based photoactivatable transcription system. Chem. Biol. 22, 169–174 (2015).
Polstein, L. R. & Gersbach, C. A. A light-inducible CRISPR-Cas9 system for control of endogenous gene activation. Nat. Chem. Biol. 11, 198–200 (2015).
Ho, S. C., Mariati, Yeo, J. H., Fang, S. G. & Yang, Y. Impact of using different promoters and matrix attachment regions on recombinant protein expression level and stability in stably transfected CHO cells. Mol. Biotechnol. 57, 138–144 (2015).
Schwerdtfeger, C. & Linden, H. VIVID is a flavoprotein and serves as a fungal blue light photoreceptor for photoadaptation. EMBO J. 22, 4846–4855 (2003).
Zoltowski, B. D. & Crane, B. R. Light activation of the LOV protein vivid generates a rapidly exchanging dimer. Biochemistry 47, 7012–7019 (2008).
Acknowledgements
This research was supported by the National Key Research and Development Program of China (2022YFC3400100 to Y.Y and X.C. and 2019YFA0904800 to Y.Y.), NSFC (32121005, 32150028, 21937004 and 91857202 to Y.Y, 31600688 to X.C. and 32001026 to R.L.), the Shanghai Municipal Education Commission (2021 Sci & Tech 03-28 to Y.Y. and X.C.), the ALS Project from Shanghai Jiao Tong University School of Medicine Affiliated Sixth People’s Hospital (to Y.Y.), the Shanghai Rising-Star Program (to X.C.), the State Key Laboratory of Bioreactor Engineering (to Y.Y. and X.C.) and the Fundamental Research Funds for the Central Universities (to Y.Y. and X.C.).
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Concepts were conceived by Y.Y. and X.C. Y.Y., X.C., R.L., S.Z. and J. Yang. designed the experiments and analyzed the data. R.L., J. Yao, J. Yang and S.Z. performed plasmid construction and live-cell experiments. Y. Zhang performed mice experiments. S.Z. performed zebrafish experiments. J. Yang performed LicV protein purification and in vitro characterization. X.Y., L.L., Y. B. Zhang and Y. Zhuang gave technical support and conceptual advice. Y.Y., X.C. and R.L. wrote the manuscript.
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Related links
Key references using this protocol:
Liu, R. et al. Nat. Biotechnol. 40, 779–786 (2022): https://doi.org/10.1038/s41587-021-01112-1
Li, T. et al. Nat. Commun. 12, 615 (2021): https://doi.org/10.1038/s41467-021-20913-1
Wang, X. et al. Nat. Methods 9, 266–269 (2012): https://doi.org/10.1038/nmeth.1892
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Liu, R., Yao, J., Zhou, S. et al. Spatiotemporal control of RNA metabolism and CRISPR–Cas functions using engineered photoswitchable RNA-binding proteins. Nat Protoc 19, 374–405 (2024). https://doi.org/10.1038/s41596-023-00920-w
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DOI: https://doi.org/10.1038/s41596-023-00920-w
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