ArticleNucleic acids research2016
Rapid generation of CRISPR/dCas9-regulated, orthogonally repressible hybrid T7-lac promoters for modular, tuneable control of metabolic pathway fluxes in Escherichia coli.
Article in Nucleic acids research, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 43 papers.
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Who cites it
43 citing papers in PubMed, 87 citations in OpenAlex.
- Metabolic engineering of Escherichia coli for methanol-assisted resveratrol biosynthesis.Bioprocess and biosystems engineering · 2026Article
- A T7 RNAP regulatory toolbox for cell-free network engineering and biosensing applications.Nature communications · 2026Article
- Metabolite-responsive scaffold RNAs for dynamic CRISPR transcriptional regulation.Nucleic acids research · 2025Article
- Multichassis Expression of Cyanobacterial and Other Bacterial Biosynthetic Gene Clusters.ACS synthetic biology · 2025Article
- Innovations, Challenges and Future Directions of T7RNA Polymerase in Microbial Cell Factories.ACS synthetic biology · 2025Review
- Customizable gene sensing and response without altering endogenous coding sequences.Nature chemical biology · 2025Article
- Architecting a transcriptional repressor-based genetic inverter for tryptophan derived pathway regulation in Escherichia coli.Metabolic engineering · 2024Article
- CRISPR in Nucleic Acids Research: the sequel.Nucleic acids research · 2024Article
- Simultaneous glucose and xylose utilization by anApplied and environmental microbiology · 2024Article
- Manipulating Microbial Cell Morphology for the Sustainable Production of Biopolymers.Polymers · 2024Review
- Article
- Engineering activatable promoters for scalable and multi-input CRISPRa/i circuits.Proceedings of the National Academy of Sciences of the United States of America · 2023Article
- Genome analysis of a halophilicFrontiers in microbiology · 2023Article
- Parallel screening and cheminformatics modeling of flavonoid activated aptasensors.Synthetic and systems biotechnology · 2022Article
- Overcoming Leak Sensitivity in CRISPRi Circuits Using Antisense RNA Sequestration and Regulatory Feedback.ACS synthetic biology · 2022Article
- Detection of environmental pollutant cadmium in water using a visual bacterial biosensor.Scientific reports · 2022Article
- Synthetic fused sRNA for the simultaneous repression of multiple genes.Applied microbiology and biotechnology · 2022Article
- Hepatic stellate cell reprogramming via exosome-mediated CRISPR/dCas9-VP64 delivery.Drug delivery · 2021Article
- Nanotargeting of Resistant Infections with a Special Emphasis on the Biofilm Landscape.Bioconjugate chemistry · 2021Review
- A plasmid toolbox for controlled gene expression across the Proteobacteria.Nucleic acids research · 2021Article
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Authors and funding
8 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Robust gene circuit construction requires use of promoters exhibiting low crosstalk. Orthogonal promoters have been engineered utilizing an assortment of natural and synthetic transcription factors, but design of large orthogonal promoter-repressor sets is complicated, labor-intensive, and often results in unanticipated crosstalk. The specificity and ease of targeting the RNA-guided DNA-binding protein dCas9 to any 20 bp user-defined DNA sequence makes it a promising candidate for orthogonal promoter regulation. Here, we rapidly construct orthogonal variants of the classic T7-lac promoter using site-directed mutagenesis, generating a panel of inducible hybrid promoters regulated by both LacI and dCas9. Remarkably, orthogonality is mediated by only two to three nucleotide mismatches in a narrow window of the RNA:DNA hybrid, neighboring the protospacer adjacent motif. We demonstrate that, contrary to many reports, one PAM-proximal mismatch is insufficient to abolish dCas9-mediated repression, and we show for the first time that mismatch tolerance is a function of target copy number. Finally, these promoters were incorporated into the branched violacein biosynthetic pathway as dCas9-dependent switches capable of throttling and selectively redirecting carbon flux in Escherichia coli We anticipate this strategy is relevant for any promoter and will be adopted for many applications at the interface of synthetic biology and metabolic engineering.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.