Characterization of programmable CRISPR-based toggle switches in Escherichia coli

Recent developments in CRISPR-Cas systems have ushered a new generation of powerful genetic engineering tools in synthetic biology. In particular, a catalytically ‘dead’ version of Cas proteins that lack nuclease activity can essentially function as a logic NOT gate by selectively binding to a promoter and preventing transcription initiation. This work aims to provide an efficient and systematic workflow to design, assemble and fine-tune CRISPRi based genetic toggle switches from many potential constructs. By assuming that CRISPRi turns off transcription shares the same mechanism as simple repression motif, a thermodynamic model is developed to investigate the interaction of two mutually repressed CRISPRi NOT gate under various cellular background by tuning the availability and affinity of specific binding sites, competing sites, and the activity of targeted promoter. Next, a rapid and versatile characterization platform, X-seq, was used to assemble and compare hundreds and thousands of potential constructs in parallel, from which a few candidates can be picked for further fine-tuning. In the end, the cooperativity of chosen toggle switch candidates can be optimized through pairing with a proper competing sites number that is loaded on a helper plasmid with a tunable copy number.