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Multistable and dynamic CRISPRi-based synthetic circuits.

Javier Santos-Moreno1, Eve Tasiudi2, Joerg Stelling2

  • 1Department of Fundamental Microbiology, University of Lausanne, Biophore Building, 1015, Lausanne, Switzerland.

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This summary is machine-generated.

CRISPR interference (CRISPRi) enables robust synthetic gene circuits like oscillators and toggle switches in E. coli. Unspecific binding is key for multistability in these novel CRISPR-based synthetic networks.

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Area of Science:

  • Synthetic biology
  • Molecular biology
  • Microbial engineering

Background:

  • CRISPR interference (CRISPRi) is a powerful gene expression tool for prokaryotes and eukaryotes.
  • Lack of cooperativity in CRISPRi has limited dynamic and multistable synthetic circuit construction.
  • Developing predictable, orthogonal, and low-burden synthetic circuits is crucial.

Purpose of the Study:

  • To engineer novel synthetic gene circuits using CRISPRi technology.
  • To demonstrate the construction of an oscillator, bistable network, and feed-forward loop.
  • To investigate the role of unspecific binding in CRISPRi-mediated multistability.

Main Methods:

  • Utilized CRISPR interference (CRISPRi) for gene expression control.
  • Designed and constructed synthetic oscillator (CRISPRlator), toggle switch, and incoherent feed-forward loop circuits.
  • Performed experiments in Escherichia coli populations and employed mathematical modeling.

Main Results:

  • Successfully built and demonstrated robust synthetic oscillator, bistable network, and IFFL circuits.
  • Achieved high predictability, orthogonality, low metabolic burden, and context-dependency in circuit designs.
  • Mathematical modeling indicated unspecific binding is essential for establishing multistability.

Conclusions:

  • CRISPRi is broadly applicable for engineering diverse synthetic gene circuits.
  • The developed circuits exhibit robust behaviors in E. coli.
  • This work advances the potential for engineering complex synthetic networks using CRISPR technology.