Riboregulated toehold-gated gRNA for programmable CRISPR–Cas9 function

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. Engineering of toehold-gated guide RNA (thgRNA) by tethering toehold riboswitches to sgRNAs enables the activation of CRISPR–Cas9 genome editing or transcriptional regulation in response to complementary synthetic or endogenous cellular RNAs.