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CRISPRi-Linked Multimodule Negative Feedback Loops to Address Winner-Take-All Resource Competition.

Sadikshya Rijal1, Kylie Standage-Beier1, Rong Zhang1

  • 1School of Biological and Health Systems Engineering, Arizona State University, Tempe, Arizona 85281, United States.

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|September 10, 2025
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Summary
This summary is machine-generated.

Synthetic gene circuits often suffer from resource competition, leading to Winner-Takes-All (WTA) behavior. A novel Negatively Competitive Regulatory (NCR) controller using CRISPR interference (CRISPRi) effectively suppressed WTA, enhancing synthetic biology circuit modularity.

Keywords:
dCas9 competitionmodularitymodule coactivationmulti-module controlresource decoupling

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Area of Science:

  • Synthetic biology
  • Genetic engineering
  • CRISPR technology

Background:

  • Cellular resource limitations cause unintended module interactions in synthetic gene circuits.
  • Positive feedback loops in circuits exacerbate resource competition, leading to Winner-Takes-All (WTA) behavior.
  • Compromised circuit modularity hinders predictable synthetic gene circuit design and application.

Purpose of the Study:

  • To experimentally implement and evaluate a Negatively Competitive Regulatory (NCR) controller.
  • To assess the NCR controller's efficacy in mitigating WTA behavior in synthetic gene circuits.
  • To improve the modularity and resource allocation of synthetic gene circuits.

Main Methods:

  • Implementation of an NCR controller utilizing CRISPR interference (CRISPRi) with a tunable dCas9 gene.
  • Chromosomal integration of dCas9 and design of module-specific guide RNAs (gRNAs) for self-repression.
  • Evaluation in dual self-activation and cascading bistable switch gene circuits, comparing with a control group.

Main Results:

  • The NCR controller significantly suppressed Winner-Takes-All (WTA) behavior in tested gene circuits.
  • Module coactivation was substantially increased by the NCR controller compared to controls.
  • Quantitative analysis confirmed balanced module activation and improved resource reallocation.

Conclusions:

  • The Negatively Competitive Regulatory (NCR) controller is an effective strategy for managing resource competition.
  • NCR enhances the modularity of synthetic gene circuits by mitigating WTA dynamics.
  • This approach offers a robust method for improving the predictability and performance of synthetic gene circuits.