Design of CRISPRi-Based Synthetic Gene Circuits in Bacteria

Synthetic gene circuits are key elements of engineered biological systems that allow us to control and program cellular behavior. Yet, circuit design can be challenging to newcomers due to the numerous design choices and the abundance and variety of parameters that can influence circuit performance. While transcription factors have dominated the circuit construction toolbox for two decades, CRISPRi-based tools offer important benefits-especially for large circuits-but also require unique design considerations. Here I provide a detailed guide for designing CRISPRi circuits in bacteria, using the CRISPRlator, the first CRISPRi oscillator, as an example that illustrates the design process.