Methods in molecular biology (Clifton, N.J.)Jul 8, 2026

Using CRISPRi Competition to Build Multi-Part Controllers for Managing Resources in Genetic Circuits

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Jul 9, 2026

Abstract

A Negatively Competitive Regulatory (NCR) controller based on CRISPR interference was experimentally implemented in dual self-activation circuits.

Resource competition can lead to imbalances in engineered genetic circuits due to Winner-Takes-All dynamics.
The NCR controller introduces negative feedback to more active modules while reallocating resources to less active ones.
Self-repression is achieved through competition for limiting dCas9 using module-specific guide RNAs.
Guidelines for quantitatively assessing resource competition regulation in DSA modules using the NCR strategy are provided.
Implementation of the NCR strategy may enhance the modularity and predictability of gene circuits.

Simplified

Cellular resource limitations give rise to resource competition, undermining the modularity and predictability of engineered genetic circuits. In systems containing positive feedback, such competition can drive Winner-Takes-All (WTA) dynamics, resulting in severe imbalances in resource allocation across circuit modules. In this chapter, we present an experimental implementation of a Negatively Competitive Regulatory (NCR) controller based on CRISPR interference (CRISPRi) in dual self-activation (DSA) circuits. We describe a detailed workflow for chromosomal integration of a tunable dCas9 expression cassette, as well as the design of self-activation modules and module-specific guide RNAs that induce self-repression through competition for limiting dCas9. This architecture introduces effective negative feedback to the more active module while reallocating resources to the less active one, thereby promoting balanced module activity. Finally, we provide guidelines for quantitatively assessing the regulation of resource competition between DSA modules using the NCR strategy. Overall, these guidelines demonstrate how CRISPRi can be leveraged to implement NCR strategy in gene circuits, thereby enhancing circuit modularity and predictability.

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Using CRISPRi Competition to Build Multi-Part Controllers for Managing Resources in Genetic Circuits

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