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Reactive oxygen signaling molecule inducible regulation of CRISPR-Cas9 gene editing

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Abstract

We report development of a controllable gene editing tool that boronated gRNA, simply generated in situ, could regulate binding of gRNA molecules with either Cas9 endonuclease or target genes, thus serving as a modulator that can control CRISPR-Cas9 gene editing. Subsequent treatment with H2O2 facilitates the restoration of gene editing ability of the boronated gRNA to the level of using untreated gRNA. This is one of the few cases using small molecule to regulate CRISPR-Cas9 gene editing, which is a complement to the light approach, displaying great application potential.

Graphical abstract

We develop a controllable gene editing tools based on the CRISPR-Cas9 gene editing system. This tool can be regulated by oxidative small molecule, i.e., H2O2. Compared with the light method, the application scope of our CRISPR-Cas9 systems have been widened with the small-molecule-triggered approaches, preventing the potential damage of cells or organism caused by UV light. In addition, the gain-of-function tools are expanding the gene code expansion for mechanistic studies of target enzymes since it provides a positive route to evaluate the activity of a given enzyme in dynamic and inversible regulation of targeting cellular processes.

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Supplementary material is available free of charge via internet.

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Acknowledgements

We thank Prof. Jason Chin of the MRC Laboratory of Molecular Biology for providing plasmids. This work was supported by the start-up fund of Huazhong University of Science and Technology (to R.W.).

Funding

This study was supported by the startup fund of Huazhong University of Science and Technology (to R.W.)

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J.Z.Z., H.M.H., and J.W.Z. synthesized chemicals, all the reagents used herein; J.Z.Z., H.M.H., and H.L.Z. constructed the plasmids, RNA extraction, fluorescent labeling, transfection, and transcription assays. R.W. and L.W. designed the experiments and wrote the paper.

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Correspondence to Rui Wang.

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Zhao, J., Hu, H., Zhou, H. et al. Reactive oxygen signaling molecule inducible regulation of CRISPR-Cas9 gene editing. Cell Biol Toxicol 39, 2421–2429 (2023). https://doi.org/10.1007/s10565-022-09723-3

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