Riboregulated toehold-gated gRNA for programmable CRISPR–Cas9 function

Abstract

Predictable control over gene expression is essential to elicit desired synthetic cellular phenotypes. Although CRISPR–Cas9 offers a simple RNA-guided method for targeted transcriptional control, it lacks the ability to integrate endogenous cellular information for efficient signal processing. Here, we present a new class of riboregulators termed toehold-gated gRNA (thgRNA) by integrating toehold riboswitches into sgRNA scaffolds, and demonstrate their programmability for multiplexed regulation in Escherichia coli with minimal cross-talks.

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Fig. 1: Design and screening of toehold-gated guide RNAs (thgRNAs).
Fig. 2: thgRNAs can be selectively activated intracellularly by induced expression of cognate trigger RNAs.
Fig. 3: thgRNA can be activated specifically by endogenous RNAs.

Data availability

Sequences of all thgRNAs and trigger strands studied are included in the Supplementary Information. Additional data that support the findings of this study are available from the authors on reasonable request.

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Acknowledgements

This work was supported by grants to W.C. from the National Science Foundation (MCB1615731 and MCB1817675). We thank D. Liu (Harvard University), T. Pederson (University of Massachusetts Medical School), and M. Koffas (Rensselaer Polytechnic Institute) for their generous gifts of plasmids as noted in the manuscript.

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K-H.S. and W.C. conceived the project. K-H.S. designed experiments, performed the experiments, analyzed the data, and wrote the manuscript. W.C. designed experiments, analyzed the data, and wrote the manuscript. Both authors discussed the results and commented on the manuscript.

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Correspondence to Wilfred Chen.

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The authors declare no competing interests.

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Supplementary Figures 1–10, Supplementary Tables 1–2

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Siu, K., Chen, W. Riboregulated toehold-gated gRNA for programmable CRISPR–Cas9 function. Nat Chem Biol 15, 217–220 (2019). https://doi.org/10.1038/s41589-018-0186-1

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