# Engineered dCas9 with reduced toxicity in bacteria: implications for genetic circuit design

Article · Shuyi Zhang, Christopher A. Voigt

- Archive id: doi:10.1093/nar/gky884
- Published: 2018-09-20
- Venue: Nucleic Acids Research
- DOI: https://doi.org/10.1093/nar/gky884
- PMID: 30289463
- PMCID: PMC6237744
- Citations: 166
- Source URL: https://doi.org/10.1093/nar/gky884
- PDF: https://academic.oup.com/nar/article-pdf/46/20/11115/26578084/gky884.pdf
- Provenance: openalex+europepmc

## 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.

_Abstract reproduced under CC-BY (article license as recorded by Europe PMC; abstract reproduced unmodified apart from markup removal)._

Topics: [Synthetic Biology & Genome Engineering](https://space-kitty.com/topics/synthetic-biology)

Cited in dossiers:
- [Synthetic Biology & Genome Engineering: State of the Field (October 2026)](https://space-kitty.com/dossiers/synthetic-biology)

---
Space Kitty Archive · https://space-kitty.com · API: https://space-kitty.com/api/v1 · MCP: https://space-kitty.com/mcp · Premium: $0.20/request via x402 (Base Sepolia (testnet)). Metadata: arXiv (CC0), OpenAlex (CC0), Europe PMC (CC BY/CC0 works), ClinicalTrials.gov.