Factors That Affect the Bistability of Programmable Crispr-Based Toggle Switches in Escherichia Coli

Research from Cornell University aims to provide a systematic workflow that simulates, assembles and sorts out bistable CRISPRi-based toggle switches from thousands of potential constructs.

The research, Factors That Affect the Bistability of Programmable CRISPR-based Toggle Switches in Escherichia Coli, is being presented at the American Physical Society’s March Meeting, March 14-18, in Chicago, by Yasu Xu, a doctoral student in the Lambert Lab.

Genetic toggle switches, as one of the most basic genetic circuits, is the building block for more advanced circuits with many practical applications in various fields. However, a widely-used version based on limited pairs of promoter-repressor pairs suffers from low orthogonality and programmability. Recent progress in CRISPR-Cas systems has shown its potential as a new generation of genetic editing tool, especially a catalytically ‘dead’ version of Cas proteins that lack nuclease activity and can essentially function as a logic NOT gate by programing the complex binding to a promoter to interfere transcription.

Xu and colleagues 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.

“Our thermodynamic model simplifies the process and kinetics of all protein binding interactions,” Xu said, “as long as the binding site is occupied by the RNAP or dCas complex, activity or repression on the promoter is achieved. This assumption helps us simulate how different features like promoter strength, targeting affinity and construct copy number contribute to bistability by changing only one feature at a steady state.”

Next, a versatile X-Seq assay was used to investigate each of these parameters by characterizing many potential constructs in parallel. Xu found that by carefully matching promoter strength and the copy number of toggle switch construct, bistabilty in the cell is able to be seen.

“On the one hand, this research provides an efficient workflow to generate orthogonal TSs that can function in parallel to create more advanced genetic circuits that increase the layer of complexity of genetic network,” Xu said. “On the other hand, it enables researchers to program TS for different purposes, which has many practical applications in various fields. For example, it can be used to report the existence or concentration of some harmful molecule or content from the environment.”

Provided by Cornell University College of Engineering
Contact: Syl Kacapyr, (607) 255-3981, vpk6@cornell.edu

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Schematic of CRISPRi/dCas12a based dual plasmid genetic toggle switchCREDIT: Yasu Xu

Schematic of CRISPRi/dCas12a based dual plasmid genetic toggle switch.