A new class of toehold-gated gRNAs (thgRNAs) has been created to provide conditional gene regulation via RNA-mediated activation. However, the detailed design principles remain elusive. Here, we presented an investigation into the design rules for conditional gRNAs by systematically varying the toehold, stem, and flexible loop regions of thgRNA for optimal gene activation in HeLa cells. We determined that nonspecific interactions between the toehold region and the flexible loop are the main driver for the background leak observed in the OFF state. By trimming the toehold length from 15 to 5 nt, the improved thgNT-F design led to a 38-fold increase in the activated ON state with no observable background leak. The same design rule was successfully adapted to target two different regions on the mCherry mRNA with the same impressive fold change. Using the thgRNA to direct conditional protein degradation, we showed up to 8-fold knockdown of a reporter protein through activating expression of a bifunctional ubiquibody GS2-IpaH9.8. This new strategy may find many new applications for cell culture control or cell therapy by removing unwanted proteins in an RNA-responsive manner.

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Using a newly created AND-gate toehold-gated guide RNA (thgRNA), presence of specific miRNA and mRNA allows conditional gene activation and protein degradation.