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

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.