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However, there are few dynamic CRISPR transcriptional programs, which limit its usage in the creation of living biosensors, self-regulating microbial factories, or conditional therapeutics. Here, we address this limitation by embedding a molecular switch directly into a guide RNA to create a combined sensor\u2013actuator called a metabolite-responsive scaffold RNA (MR-scRNA). We demonstrate the regulatory potential for MR-scRNAs by conditionally activating genes in three different kingdoms of life. We create MR-scRNAs responsive to two distinct metabolites, theophylline and tryptophan, by swapping the molecular switch used. MR-scRNAs respond quickly in a dose-dependent manner specifically to their target metabolite and enhance biochemical production when used as a dynamic regulator of pathway enzyme expression. The broad functionality and ease of design of the MR-scRNAs offer a promising tool for dynamic cellular regulation.<\/jats:p>","DOI":"10.1093\/nar\/gkaf1290","type":"journal-article","created":{"date-parts":[[2025,11,24]],"date-time":"2025-11-24T10:46:47Z","timestamp":1763981207000},"source":"Crossref","is-referenced-by-count":5,"title":["Metabolite-responsive scaffold RNAs for dynamic CRISPR transcriptional regulation"],"prefix":"10.1093","volume":"53","author":[{"given":"Anthony M","family":"Stohr","sequence":"first","affiliation":[{"name":"University of Delaware, Newark Department of Chemical and Biomolecular Engineering, , DE 19716 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Helena","family":"Hansen","sequence":"additional","affiliation":[{"name":"University of Delaware, Newark Department of Chemical and Biomolecular Engineering, , DE 19716 ,","place":["United 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