Abstract
Genetic networks that generate oscillations in gene expression activity are found in a wide range of organisms throughout all kingdoms of life. Oscillatory dynamics facilitates the temporal orchestration of metabolic and growth processes inside cells and organisms, as well as the synchronization of such processes with periodically occurring changes in the environment. Synthetic oscillator gene circuits such as the "repressilator" can perform similar functions in bacteria. Until recently, such circuits were mainly based on a relatively small set of well-characterized transcriptional repressors and activators. A promising, sequence-programmable alternative for gene regulation is given by CRISPR interference (CRISPRi), which enables transcriptional repression of nearly arbitrary gene targets directed by short guide RNA molecules. In order to demonstrate the use of CRISPRi in the context of dynamic gene circuits, we here replaced one of the nodes of a repressilator circuit by the RNA-guided dCas9 protein. Using single cell experiments in microfluidic reactors we show that this system displays robust relaxation oscillations over multiple periods and over several days. With a period of ≈14 bacterial generations, our oscillator is similar in speed as previously reported oscillators. Using an information-theoretic approach for the analysis of the single cell data, the potential of the circuit to act as a synthetic pacemaker for cellular processes is evaluated. We also observe that the oscillator appears to affect cellular growth, leading to variations in growth rate with the oscillator's frequency.
Highlights
Genetic networks that generate oscillations in gene expression activity are found in a wide range of organisms throughout all kingdoms of life
We replaced LacI as this is the only endogenous protein from E. coli and might interfere with the circuit
We have integrated transcriptional repression via CRISPR interference into a three-node repressilator network, which resulted in stable genetic oscillations at the single cell level
Summary
Genetic networks that generate oscillations in gene expression activity are found in a wide range of organisms throughout all kingdoms of life. In order to demonstrate the use of CRISPRi in the context of dynamic gene circuits, we here replaced one of the nodes of a repressilator circuit by the RNA-guided dCas protein. Synthetic genetic oscillators are interesting, as they do require control over gene expression levels, and over expression dynamics In their seminal work, Elowitz and Leibler [3] created a genetic oscillator termed ‘repressilator’, whose negative feedback loop was composed of three genetic repressors (LacI, TetR, and λ-cI), which cyclically repressed each other’s expression. We took a hybrid approach - which we termed ‘Rock Paper CRISPR’ (RPC) oscillator-, in which only one of the transcriptional repressor links of a repressilator network was replaced by CRISPRi. Even though CRISPRi is not expected to provide highly non-linear repression, we demonstrate, both experimentally and via computational modeling, that our system is still capable of sustained temporal oscillations
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