In Silico Characterization of CRISPRi Circuit Bandwidth: Resource-Driven Retroactivity Shifts Cut-off Frequencies in Feed-Forward and Feedback Topologies.

The assumption of modularity in synthetic genetic circuits is often challenged in vivo due to competition for shared cellular resources. When multiple circuits compete for finite components—such as ribosomes or the CRISPR interference (CRISPRi) actuator dCas9—the system experiences retroactivity, where downstream loading impairs upstream circuit function. This study utilizes a deterministic mass-action modeling framework to evaluate how dCas9 competition impacts the signal processing bandwidth of canonical genetic motifs. Our simulations indicate that unregulated, open-loop circuits experience significant signal attenuation (up to 75.9% reduction) under competitive resource loading. To explore potential mitigation strategies, we compared the Coherent Type-1 Feed-Forward Loop (C1-FFL) and the Negative Feedback Loop (NFL). Time-domain simulations support the C1-FFL's function as a low-pass filter for transient inputs (<20 min). However, in silico Bode plot analysis suggests that under simulated dCas9 starvation, the C1-FFL’s operational bandwidth is severely restricted, compromising its filtering capacity. In contrast, the NFL demonstrates faster response times (>85% improvement) and maintains approximately 80% of its steady-state output even when production capacity is reduced by 70%. While negative feedback provides notable dynamic stability under these conditions, the model indicates it requires a substantially high basal production rate. This highlights a standard design trade-off: mitigating the dynamic sensitivity of feed-forward filters may come at the cost of increased metabolic burden associated with feedback-driven stability.