Programmable and bistable CRISPR-based toggle switches in Escherichia coli

Recent developments and advances in CRISPR-Cas (Clustered regularly interspaced short palindromic repeats and CIRSPR-associated proteins) systems have ushered a new generation of powerful genetic engineering tools in synthetic biology. In particular, a catalytically ‘dead’ version of CRISPR-Cas proteins that lack nuclease activity can essentially function as a logic NOR gate by selectively binding to a promoter sequence and preventing initiation of transcription by RNA polymerase. In this work, we create programmable, bistable genetic toggle switches using pairs of mutually repressible orthogonal CRISPR-based NOR gates. Another pair of convergent positioned aTc-inducible and IPTG-inducible promoters that can program/reset the state of each toggle switch are wired into a single plasmid and transformed into E. coli cells. We show that CRISPR-based toggle switches can remain into a fixed state for many generations, and a statistical mechanical model is developed to predict a bistability criterion for CRISPR-based toggle switches. We experimentally test these predictions and we outline an experimental framework to tailor the sequence in the promoter to produce numerous pairs of toggle switches that can work in parallel and with limited cross-talk.