ArticleNucleic acids research2018
Engineered dCas9 with reduced toxicity in bacteria: implications for genetic circuit design.
Article in Nucleic acids research, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 111 papers.
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Who cites it
111 citing papers in PubMed.
- 3D Genome Engineering Using CRISPR/dCas Systems.International journal of molecular sciences · 2026Review
- Quantitative Measurement of Synthetic Repression Curves Reveals Design Challenges for Genetic Circuit Engineering under Growth Arrest.ACS synthetic biology · 2026Article
- A portable Cas6f-based system for multiplex translational repression in bacteria.Nature communications · 2026Article
- Inducible CRISPRi enables efficient and high-fidelity genome editing in Streptomyces.Nucleic acids research · 2026Article
- Programmable, multiplexed and orthogonal gene control in bacteria with attenuated Cas13d systems.Nature biotechnology · 2026Article
- Cross-Strain Transferability of CRISPRi Systems and Design Rules from Laboratory to ClinicalACS synthetic biology · 2026Article
- Associate toxin-antitoxin with CRISPR-Cas to harness (ATTACH) engineered microbes.Nucleic acids research · 2026Article
- Cross-strain transferability of CRISPRi systems and design rules from laboratory to clinicalbioRxiv : the preprint server for biology · 2026Article
- Synthetic Biology of Plants and Microbes for Agriculture, Environment, and Future Applications.Chemical reviews · 2026Review
- Mobile-CRISPRi as a tool for genetic manipulation in the intracellular pathogenApplied and environmental microbiology · 2026Article
- A Simple Programmable Cas12a/crRNA Induced Walking System for Sensitive Methicillin-ResistantJournal of microbiology and biotechnology · 2026Article
- CRISPR-Cas based plasmid design for multidrug-resistant Klebsiella pneumoniae isolates.FEMS microbiology letters · 2026Article
- Intracellularly coupled oscillators for synthetic biology.Nature communications · 2025Review
- CRISPRi-Linked Multimodule Negative Feedback Loops to Address Winner-Take-All Resource Competition.ACS synthetic biology · 2025Article
- A quorum sensing-controlled type I CRISPRi toolkit for dynamically regulating metabolic flux.Nucleic acids research · 2025Article
- Modular and signal-responsive transcriptional regulation using CRISPRi-aided genetic switches in Escherichia coli.Journal of biological engineering · 2025Article
- Codon usage modulates the relationship between the burden and yield of protein overexpression.Journal of biological engineering · 2025Article
- CRISPRi-Linked Multi-Module Negative Feedback Loops to Address Winner-Take-All Resource Competition.bioRxiv : the preprint server for biology · 2025Article
- Highly efficient CRISPR-Cas9 base editing inApplied and environmental microbiology · 2025Article
- Engineering intercellular communication using M13 phagemid and CRISPR-based gene regulation for multicellular computing in Escherichia coli.Nature communications · 2025Article
51 more citing papers are in PubMed but not listed here.
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Authors and funding
2 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Large synthetic genetic circuits require the simultaneous expression of many regulators. Deactivated Cas9 (dCas9) can serve as a repressor by having a small guide RNA (sgRNA) direct it to bind a promoter. The programmability and specificity of RNA:DNA basepairing simplifies the generation of many orthogonal sgRNAs that, in theory, could serve as a large set of regulators in a circuit. However, dCas9 is toxic in many bacteria, thus limiting how high it can be expressed, and low concentrations are quickly sequestered by multiple sgRNAs. Here, we construct a non-toxic version of dCas9 by eliminating PAM (protospacer adjacent motif) binding with a R1335K mutation (dCas9*) and recovering DNA binding by fusing it to the PhlF repressor (dCas9*_PhlF). Both the 30 bp PhlF operator and 20 bp sgRNA binding site are required to repress a promoter. The larger region required for recognition mitigates toxicity in Escherichia coli, allowing up to 9600 ± 800 molecules of dCas9*_PhlF per cell before growth or morphology are impacted, as compared to 530 ± 40 molecules of dCas9. Further, PhlF multimerization leads to an increase in average cooperativity from n = 0.9 (dCas9) to 1.6 (dCas9*_PhlF). A set of 30 orthogonal sgRNA-promoter pairs are characterized as NOT gates; however, the simultaneous use of multiple sgRNAs leads to a monotonic decline in repression and after 15 are co-expressed the dynamic range is <10-fold. This work introduces a non-toxic variant of dCas9, critical for its use in applications in metabolic engineering and synthetic biology, and exposes a limitation in the number of regulators that can be used in one cell when they rely on a shared resource.
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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.