Multistable and dynamic CRISPRi-based synthetic circuits

Gene expression control based on CRISPRi (clustered regularly interspaced short palindromic repeats interference) has emerged as a powerful tool for creating synthetic gene circuits, both in prokaryotes and in eukaryotes; yet, its lack of cooperativity has been pointed out as a potential obstacle for dynamic or multistable synthetic circuit construction. Here we use CRISPRi to build a synthetic oscillator (“CRISPRlator”), bistable network (toggle switch) and stripe pattern-forming incoherent feed-forward loop (IFFL). Our circuit designs, conceived to feature high predictability and orthogonality, as well as low metabolic burden and context-dependency, allow us to achieve robust circuit behaviors in Escherichia coli populations. Mathematical modeling suggests that unspecific binding in CRISPRi is essential to establish multistability. Our work demonstrates the wide applicability of CRISPRi in synthetic circuits and paves the way for future efforts towards engineering more complex synthetic networks, boosted by the advantages of CRISPR technology.

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Single colonies were used to inoculate 2 ml of selective EZ medium (Teknova) containing 0.2% Ara or 10 µM AHL and incubated at 37°C for 10 h in a tilted 200 rpm shaker. Next, cells were pelleted at 5000 rcf for 10 min and resuspended in selective EZ medium free of inducers; 1 µl of the resuspension was used to inoculate 120 µl of inducer-free selective EZ in a 96-well plate, which was incubated at 37°C for 12 h with double-orbital shaking in a Synergy H1 microplate reader (Biotek). Cells were then diluted 1:120 in 120 µl of selective EZ containing the opposed inducer (10 µM AHL or 0.2% Ara) and grown for 10 h more in the microplate reader under identical conditions. Cells were then diluted into an inducer-free medium as before (resuspension in inducer-free selective EZ followed by 1:120 dilution in the same medium) and incubated for 12 h more in the microplate reader under identical conditions. At the end of each induction period (inducer 1, no inducer, inducer 2, no inducer) a sample was taken, diluted 1:100 in phosphate-buffered saline (PBS) and analyzed with a BD LSRFortessa flow cytometer using a 488 nm laser in combination with FITC filter for sfGFP fluorescence determination.