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Optogenetic control of RNA function and metabolism using engineered light-switchable RNA-binding proteins

Nature Biotechnology volume 40, pages 779–786 (2022)Cite this article

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Abstract

RNA-binding proteins (RBPs) play an essential role in regulating the function of RNAs in a cellular context, but our ability to control RBP activity in time and space is limited. Here, we describe the engineering of LicV, a photoswitchable RBP that binds to a specific RNA sequence in response to blue light irradiation. When fused to various RNA effectors, LicV allows for optogenetic control of RNA localization, splicing, translation and stability in cell culture. Furthermore, LicV-assisted CRISPR–Cas systems allow for efficient and tunable photoswitchable regulation of transcription and genomic locus labeling. These data demonstrate that the photoswitchable RBP LicV can serve as a programmable scaffold for the spatiotemporal control of synthetic RNA effectors.

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Fig. 1: Engineered photoswitchable RBP.
Fig. 2: Synthetic photoswitchable effectors for the optogenetic control of RNA metabolic processes.
Fig. 3: Highly efficient and tunable activation of transcription by the LA-CRISPR system.
Fig. 4: Optogenetic control of genomic locus labeling using the LA-CRISPR system.

Data availability

The data supporting the findings of this study are available within the paper and its Supplementary Information files.

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Acknowledgements

We thank Z. Wang and M. Mao at CAS Key Lab for Computational Biology for technical advice and sharing the splicing reporter and PUF-based SFs. We thank Z. Xie at Tsinghua University for sharing dAsCpf1–VPR and dSaCas9–VPR constructs. We thank C. Wang at the State Key Laboratory of Bioreactor Engineering for technical advice. This research was supported by the National Key Research and Development Program of China (2019YFA0904800 and 2017YFA050400 to Y.Y.), NSFC (91857202, 32121005, 21937004, 31225008 and 31470833 to Y.Y. and 31600688 and 31971349 to X.C.), the Shanghai Science and Technology Commission (18JC1411900, 14XD1401400 and 16430723100 to Y.Y. and 19ZR1472800 to X.C.), the Shanghai Municipal Education Commission-Frontier Science Research Base of Optogenetic Techniques for Cell Metabolism (2021 Sci & Tech 03-28) to Y.Y., X.C. and Y.Z., the Shanghai Rising-Star Program to X.C., the Young Elite Scientists Sponsorship Program by CAST to X.C., the State Key Laboratory of Bioreactor Engineering to Y.Y. and the Fundamental Research Funds for the Central Universities to Y.Y. and X.C.

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Author notes

  1. These authors contributed equally: Renmei Liu, Jing Yang.

Authors and Affiliations

  1. Optogenetics & Synthetic Biology Interdisciplinary Research Center, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China

    Renmei Liu, Jing Yang, Jing Yao, Zhou Zhao, Wei He, Ni Su, Zeyi Zhang, Chenxia Zhang, Zhuo Zhang, Haibo Cai, Linyong Zhu, Yuzheng Zhao, Shu Quan, Xianjun Chen & Yi Yang

  2. School of Pharmacy, East China University of Science and Technology, Shanghai, China

    Renmei Liu, Jing Yang, Jing Yao, Zhou Zhao, Ni Su, Zeyi Zhang, Chenxia Zhang, Yuzheng Zhao, Xianjun Chen & Yi Yang

  3. School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai, China

    Linyong Zhu

  4. CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, China

    Xianjun Chen & Yi Yang

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  1. Renmei Liu
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Contributions

Concepts were conceived by Y.Y. and X.C. Y.Y., X.C., R.L. and J. Yang designed the experiments and analyzed the data. R.L. and Z. Zhao performed screening to obtain the light-switchable RBP. R.L., J. Yang, J. Yao and Z. Zhao performed plasmid construction and live-cell experiments. R.L. performed imaging experiments. J. Yang, Z. Zhao and W.H. performed LicV protein purification and in vitro characterization. N.S., Y.Z., S.Q., C.Z., Zeyi Zhang, Zhuo Zhang, H.C. and L.Z. gave technical support and conceptual advice. Y.Y. and X.C. wrote the manuscript.

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Correspondence to Xianjun Chen or Yi Yang.

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Competing interests

Y.Y., X.C., R.L. and J. Yang are named inventors of patent application number 202111024451.5. The remaining authors declare no competing interests.

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Peer review information Nature Biotechnology thanks Robert Campbell, Andres Jäschke and Barbara Di Ventura for their contribution to the peer review of this work.

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Liu, R., Yang, J., Yao, J. et al. Optogenetic control of RNA function and metabolism using engineered light-switchable RNA-binding proteins. Nat Biotechnol 40, 779–786 (2022). https://doi.org/10.1038/s41587-021-01112-1

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