Factors that affect the bistability of programmable CRISPR-based toggle switches in Escherichia coli

Genetic toggle switches, as one of most basic genetic circuits, is the building block for more advanced circuits with many practical applications in various fields. However, currently widely used version that based on limited pairs of promoter-repressor pairs suffers from low orthogonality and programmability. Recent progress in CRISPR-Cas systems have shown its potential as a new generation of genetic editing tool, especially, a catalytically ‘dead’ version of Cas proteins that lack nuclease activity can essentially function as a logic NOT gate by programing the complex binding to a promoter to interfere transcription. This work aims to provide an systematical workflow that simulate, assemble and sort out bistable CRISPRi based toggle switches from thousands of potential constructs.We first developed a thermodynamic model to investigate parameters,such as binding sites affinity and availability, and targeted promoter activity on that affects the bistability of a toggle switch. Next, a versatile X-Seq assay is used to investigate each of these parameters by characterizing many potential constructs in parallel. We found that by carefully matching promoter strength and the copy number of toggle swtich construct, we are able to see bistabilty in the cell.