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

Area of Science:

• Synthetic biology
• Genetic engineering
• Systems biology

Background:

• Cellular resource limitations cause unintended interactions between synthetic gene circuit modules, reducing modularity.
• Positive feedback in gene circuits can exacerbate resource competition, leading to Winner-Takes-All (WTA) behavior where one module dominates.

Purpose of the Study:

• To experimentally implement and evaluate a Negatively Competitive Regulatory (NCR) controller using CRISPR interference (CRISPRi).
• To assess the NCR controller's effectiveness in mitigating WTA behavior in dual self-activation and cascading bistable switch synthetic gene circuits.

Main Methods:

• Chromosomal integration of a tunable dCas9 gene.
• Design of module-specific guide RNAs (gRNAs) for self-repression via competition for limited dCas9.
• Implementation of the NCR controller in dual self-activation and cascading bistable switch circuits.

Main Results:

• The NCR controller significantly increased module coactivation compared to control circuits without dCas9-mediated repression.
• WTA behavior was effectively suppressed by the NCR controller, promoting balanced module activation.
• Quantitative results demonstrated successful resource reallocation from more active to less active modules.

Conclusions:

• The Negatively Competitive Regulatory (NCR) controller is an effective strategy for managing resource competition in synthetic gene circuits.
• NCR enhances the modularity of synthetic gene circuits by mitigating unintended interactions and promoting balanced module function.
• CRISPRi-based NCR provides a tunable and robust method for improving synthetic gene circuit performance.